WorldWideScience

Sample records for fractions protein degradability

  1. The N-end rule pathway catalyzes a major fraction of the protein degradation in skeletal muscle

    Science.gov (United States)

    Solomon, V.; Lecker, S. H.; Goldberg, A. L.

    1998-01-01

    In skeletal muscle, overall protein degradation involves the ubiquitin-proteasome system. One property of a protein that leads to rapid ubiquitin-dependent degradation is the presence of a basic, acidic, or bulky hydrophobic residue at its N terminus. However, in normal cells, substrates for this N-end rule pathway, which involves ubiquitin carrier protein (E2) E214k and ubiquitin-protein ligase (E3) E3alpha, have remained unclear. Surprisingly, in soluble extracts of rabbit muscle, we found that competitive inhibitors of E3alpha markedly inhibited the 125I-ubiquitin conjugation and ATP-dependent degradation of endogenous proteins. These inhibitors appear to selectively inhibit E3alpha, since they blocked degradation of 125I-lysozyme, a model N-end rule substrate, but did not affect the degradation of proteins whose ubiquitination involved other E3s. The addition of several E2s or E3alpha to the muscle extracts stimulated overall proteolysis and ubiquitination, but only the stimulation by E3alpha or E214k was sensitive to these inhibitors. A similar general inhibition of ubiquitin conjugation to endogenous proteins was observed with a dominant negative inhibitor of E214k. Certain substrates of the N-end rule pathway are degraded after their tRNA-dependent arginylation. We found that adding RNase A to muscle extracts reduced the ATP-dependent proteolysis of endogenous proteins, and supplying tRNA partially restored this process. Finally, although in muscle extracts the N-end rule pathway catalyzes most ubiquitin conjugation, it makes only a minor contribution to overall protein ubiquitination in HeLa cell extracts.

  2. Protein Secondary Structures (α-helix and β-sheet) at a Cellular Level and Protein Fractions in Relation to Rumen Degradation Behaviours of Protein: A New Approach

    International Nuclear Information System (INIS)

    Yu, P.

    2007-01-01

    Studying the secondary structure of proteins leads to an understanding of the components that make up a whole protein, and such an understanding of the structure of the whole protein is often vital to understanding its digestive behaviour and nutritive value in animals. The main protein secondary structures are the α-helix and β-sheet. The percentage of these two structures in protein secondary structures influences protein nutritive value, quality and digestive behaviour. A high percentage of β-sheet structure may partly cause a low access to gastrointestinal digestive enzymes, which results in a low protein value. The objectives of the present study were to use advanced synchrotron-based Fourier transform IR (S-FTIR) microspectroscopy as a new approach to reveal the molecular chemistry of the protein secondary structures of feed tissues affected by heat-processing within intact tissue at a cellular level, and to quantify protein secondary structures using multicomponent peak modelling Gaussian and Lorentzian methods, in relation to protein digestive behaviours and nutritive value in the rumen, which was determined using the Cornell Net Carbohydrate Protein System. The synchrotron-based molecular chemistry research experiment was performed at the National Synchrotron Light Source at Brookhaven National Laboratory, US Department of Energy. The results showed that, with S-FTIR microspectroscopy, the molecular chemistry, ultrastructural chemical make-up and nutritive characteristics could be revealed at a high ultraspatial resolution (∼10 μm). S-FTIR microspectroscopy revealed that the secondary structure of protein differed between raw and roasted golden flaxseeds in terms of the percentages and ratio of α-helixes and β-sheets in the mid-IR range at the cellular level. By using multicomponent peak modelling, the results show that the roasting reduced (P <0.05) the percentage of α-helixes (from 47.1% to 36.1%: S-FTIR absorption intensity), increased the

  3. Protein Secondary Structures (alpha-helix and beta-sheet) at a Cellular Levle and Protein Fractions in Relation to Rumen Degradation Behaviours of Protein: A New Approach

    Energy Technology Data Exchange (ETDEWEB)

    Yu,P.

    2007-01-01

    Studying the secondary structure of proteins leads to an understanding of the components that make up a whole protein, and such an understanding of the structure of the whole protein is often vital to understanding its digestive behaviour and nutritive value in animals. The main protein secondary structures are the {alpha}-helix and {beta}-sheet. The percentage of these two structures in protein secondary structures influences protein nutritive value, quality and digestive behaviour. A high percentage of {beta}-sheet structure may partly cause a low access to gastrointestinal digestive enzymes, which results in a low protein value. The objectives of the present study were to use advanced synchrotron-based Fourier transform IR (S-FTIR) microspectroscopy as a new approach to reveal the molecular chemistry of the protein secondary structures of feed tissues affected by heat-processing within intact tissue at a cellular level, and to quantify protein secondary structures using multicomponent peak modelling Gaussian and Lorentzian methods, in relation to protein digestive behaviours and nutritive value in the rumen, which was determined using the Cornell Net Carbohydrate Protein System. The synchrotron-based molecular chemistry research experiment was performed at the National Synchrotron Light Source at Brookhaven National Laboratory, US Department of Energy. The results showed that, with S-FTIR microspectroscopy, the molecular chemistry, ultrastructural chemical make-up and nutritive characteristics could be revealed at a high ultraspatial resolution ({approx}10 {mu}m). S-FTIR microspectroscopy revealed that the secondary structure of protein differed between raw and roasted golden flaxseeds in terms of the percentages and ratio of {alpha}-helixes and {beta}-sheets in the mid-IR range at the cellular level. By using multicomponent peak modelling, the results show that the roasting reduced (P <0.05) the percentage of {alpha}-helixes (from 47.1% to 36.1%: S

  4. Investigation on the Protein Degradation, Free Fatty Acid Content and Area Fraction of Poosti Cheese, Iranian Traditional Cheese Ripened in Skin

    Directory of Open Access Journals (Sweden)

    Mojgan Hemmatian

    2015-03-01

    Full Text Available Background and Objectives: In this study, the proteolysis and lipolysis of Poosti cheese produced from raw sheep milk in mountainous eastern regions of Iran were investigated during 90 days of ripening. Materials and Methods: Sodium dodecyl sulfate polyacrylamide gel electrophoresis for proteolysis (SDS-PAGE and gas chromatography (GC for free fatty acids (FFAs were applied to investigate the intensity of lipid degradation. To evaluate the Poosti cheese microstructural changes, the area fraction parameter of the scanning electron microscopy (SEM micrographs was also calculated by the Image J software. Results: The most alteration in protein profile was occurred in the first month of aging for high activity of the proteolytic microorganisms in this period. The amount of free fatty acids was depended on their length due to the variety of involved mechanisms. In addition, the microstructural parameter was considerably affected by the aging as a consequence of the effect of salt on the activity of raw milk and skin micro flora. Conclusions: The decline in proteolysis rate during the last stage of aging could be correlated with the inhibitory effects of salt on the engaged microorganisms, and increase in the pore fraction of the microstructure during the first month of Poosti cheese aging could be due to casein rearrangement and gas release by the fermentative activity of microorganisms. Keywords: Proteolysis, Lipolysis, Poosti cheese, Raw sheep milk.

  5. Ruminal degradability and carbohydrates and proteins fractioning of triticale silages in singular culture or in mixtures with oat and/or legumes

    OpenAIRE

    Bumbieris Junior, Valter Harry; Jobim, Cloves Cabreira; Emile, Jean Claude; Rossi, Robson; Calixto Junior, Moyses; Branco, Antonio Ferriani

    2011-01-01

    It was aimed to evaluate the ruminal degradability, and the fractioning of carbohydrates, as well as of the nitrogen fractions of triticale silages in singular culture or in mixtures with oats and/or legumes. The treatments had been: triticale silage (X. Triticosecale Wittimack) (ST); triticale silage + forage pea (Pisum arvense) (STE); triticale silage + oats (Avena strigosa Scheb) + forage pea + vetch (Vicia sativa) (STAE). Three castrated bovine Prim’Holstein males had been used, with aver...

  6. Anaerobic degradation of the various fractions of slaughterhouse wastewater

    Energy Technology Data Exchange (ETDEWEB)

    Sayed, S.; Zanden, J. van der; Wijffels, R.; Lettinga, G.

    1988-01-01

    The main objectives of the present investigations were to determine the maximum extent of anaerobic biological degradation of the soluble, colloidal and Coarse Suspended Solids fractions of slaughterhouse wastewater, in order to elucidate the mechanisms involved in the removal of these organic pollutant fractions and to determine the rate-limiting steps in the degradation of each fraction of the wastewater, and to assess the effects of the fractions on the methanogenic activity of the sludge.

  7. Identification of novel autophagic Radix Polygalae fraction by cell membrane chromatography and UHPLC-(Q)TOF-MS for degradation of neurodegenerative disease proteins.

    Science.gov (United States)

    Wu, An-Guo; Wong, Vincent Kam-Wai; Zeng, Wu; Liu, Liang; Law, Betty Yuen-Kwan

    2015-11-24

    With its traditional use in relieving insomnia and anxiety, our previous study has identified onjisaponin B from Radix Polygalae (RP), as a novel autophagic enhancer with potential neuroprotective effects. In current study, we have further identified a novel active fraction from RP, contains 17 major triterpenoid saponins including the onjisaponin B, by the combinational use of cell membrane chromatography (CMC) and ultra-performance liquid chromatography coupled to (quadrupole) time-of-flight mass spectrometry {UHPLC-(Q)TOF-MS}. By exhibiting more potent autophagic effect in cells, the active fraction enhances the clearance of mutant huntingtin, and reduces protein level and aggregation of α-synuclein in a higher extent when compared with onjisaponin B. Here, we have reported for the first time the new application of cell-based CMC and UHPLC-(Q)TOF-MS analysis in identifying new autophagy inducers with neuroprotective effects from Chinese medicinal herb. This result has provided novel insights into the possible pharmacological actions of the active components present in the newly identified active fraction of RP, which may help to improve the efficacy of the traditional way of prescribing RP, and also provide new standard for the quality control of decoction of RP or its medicinal products in the future.

  8. Radiation degradation of silk protein

    International Nuclear Information System (INIS)

    Pewlong, W.; Sudatis, B.; Takeshita, Hidefumi; Yoshii, Fumio; Kume, Tamikazu

    2000-01-01

    Silk fibroin fiber from the domesticated silkworm Bombyx mori was irradiated using an electron beam accelerator to investigate the application of the radiation degradation technique as a means to solubilize fibroin. The irradiation caused a significant degradation of the fiber. The tensile strength of fibroin fiber irradiated up to 2500 kGy decreased rapidly with increasing dose. The presence of oxygen in the irradiation atmosphere enhanced degradation of the tensile strength. The solubilization of irradiated fibroin fiber was evaluated using the following three kinds of solutions: a calcium chloride solution(CaCl 2 /C 2 H 5 OH/H 2 O=1:2:8 in mole ratio), a hydrochloric acid (0.5 N) and a distilled water. Dissolution of fibroin fiber into these solutions was significantly enhanced by irradiation. Especially, an appreciable amount of water soluble proteins was extracted by a distilled water. (author)

  9. Radiation degradation of silk protein

    Energy Technology Data Exchange (ETDEWEB)

    Pewlong, W; Sudatis, B [Office of Atomic Energy for Peace, Bangkok (Thailand); Takeshita, Hidefumi; Yoshii, Fumio; Kume, Tamikazu [Japan Atomic Energy Research Inst., Takasaki, Gunma (Japan). Takasaki Radiation Chemistry Research Establishment

    2000-03-01

    Silk fibroin fiber from the domesticated silkworm Bombyx mori was irradiated using an electron beam accelerator to investigate the application of the radiation degradation technique as a means to solubilize fibroin. The irradiation caused a significant degradation of the fiber. The tensile strength of fibroin fiber irradiated up to 2500 kGy decreased rapidly with increasing dose. The presence of oxygen in the irradiation atmosphere enhanced degradation of the tensile strength. The solubilization of irradiated fibroin fiber was evaluated using the following three kinds of solutions: a calcium chloride solution(CaCl{sub 2}/C{sub 2}H{sub 5}OH/H{sub 2}O=1:2:8 in mole ratio), a hydrochloric acid (0.5 N) and a distilled water. Dissolution of fibroin fiber into these solutions was significantly enhanced by irradiation. Especially, an appreciable amount of water soluble proteins was extracted by a distilled water. (author)

  10. Radiation degradation of silk protein

    Energy Technology Data Exchange (ETDEWEB)

    Wachiraporn Pewlong; Boonya Sudatis [Office of Atomic Energy for Peace, Bangkok (Thailand); Takeshita, Hidefumi; Yoshii, Fumio; Kume, Tamikazu [Japan Atomic Energy Research Inst., Takasaki, Gunma (Japan). Takasaki Radiation Chemistry Research Establishment

    2000-09-01

    Silk fibroin fiber from the domesticated silkworm Bombyx mori was irradiated in the dose range up to 2500 kGy using an electron beam accelerator to apply the radiation degradation technique as a means to solubilize fibroin. The tensile strength of irradiated fibroin fiber decreased with increasing dose and the presence of oxygen in the irradiation atmosphere enhanced the degradation. The solubilization of irradiated fibroin fiber was evaluated using the following three kinds of solutions: calcium chloride solution (CaCl{sub 2}/C{sub 2}H{sub 5}OH/H{sub 2}O = 1 : 2 : 8 in mole ratio), hydrochloric acid (0.5N) and distilled water. Dissolution of fibroin fiber into these solutions was significantly enhanced by irradiation. Especially, an appreciable amount of water-soluble protein was extracted by distilled water. (author)

  11. Safety of protein hydrolysates, fractions thereof and

    NARCIS (Netherlands)

    Gertjan Schaafsma

    2009-01-01

    This paper evaluates the safety for humans with regard to consumption of protein hydrolysates and fractions thereof, including bioactive peptides. The available literature on the safety of protein, protein hydrolysates, fractions thereof and free amino acids on relevant food legislation is reviewed

  12. Degradabilidade ruminal da matéria seca, da fração fibrosa e da proteína bruta de forrageiras Forages dry matter, fibrous fraction and crude protein ruminal degradability

    Directory of Open Access Journals (Sweden)

    Aureliano José Vieira Pires

    2006-04-01

    Full Text Available O objetivo deste trabalho foi avaliar a degradabilidade ruminal da matéria seca, da fibra em detergente neutro, da fibra em detergente ácido e da proteína bruta da alfafa (Medicago sativa, aveia-preta (Avena strigosa, leucena (Leucaena leucocephala e guandu (Cajanus cajan. Amostras de 3 g das forragens foram incubadas no rúmen de três novilhos por períodos de 0, 6, 12, 24, 36, 48 e 72 horas. As degradabilidades efetivas da matéria seca da alfafa e da aveia, para a taxa de passagem de 5% por hora, foram elevadas (acima de 60%. A leucena e o guandu apresentaram valores inferiores, 50,9 e 56,0%, respectivamente. A partir de 24 horas de incubação, a aveia se destacou com maior desaparecimento da fibra em detergente neutro e da fibra em detergente ácido, e ainda apresentou as mais elevadas taxas de degradação efetiva destas frações. A aveia foi a forragem que apresentou maior degradabilidade da matéria seca, da fibra em detergente neutro, da fibra em detergente ácido e da proteína bruta no rúmen. O guandu, entretanto, foi a forragem com as piores taxas de degradação.The objective of this work was to evaluate ruminal degradability of dry matter, neutral detergent fiber, acid detergent fiber and crude protein of alfalfa (Medicago sativa, black oat (Avena strigosa, leucaena (Leucaena leucocephala and pigeon pea (Cajanus cajan. Samples of 3 g of forages were incubated in the rumen of three steers for 0, 6, 12, 24, 36, 48 and 72 hours periods. The dry matter effective degradabilities of alfalfa and oat, for a passage rate of 5%/hour, were high (over 60%. However, leucaena and pigeon pea showed lower values, 50.9 and 56.0%, respectively. From 24-hour incubation period on, the oat presented the highest neutral detergent fiber and acid detergent fiber disappearance and showed the greatest effective degradation rates of these fractions. The oat was the forage with the highest dry matter, neutral detergent fiber, acid detergent fiber and

  13. Identification of novel autophagic Radix Polygalae fraction by cell membrane chromatography and UHPLC-(Q)TOF-MS for degradation of neurodegenerative disease proteins

    OpenAIRE

    An-Guo Wu; Vincent Kam-Wai Wong; Wu Zeng; Liang Liu; Betty Yuen-Kwan Law

    2015-01-01

    With its traditional use in relieving insomnia and anxiety, our previous study has identified onjisaponin B from Radix Polygalae (RP), as a novel autophagic enhancer with potential neuroprotective effects. In current study, we have further identified a novel active fraction from RP, contains 17 major triterpenoid saponins including the onjisaponin B, by the combinational use of cell membrane chromatography (CMC) and ultra-performance liquid chromatography coupled to (quadrupole) time-of-fligh...

  14. Dissection of membrane protein degradation mechanisms by reversible inhibitors

    International Nuclear Information System (INIS)

    Hare, J.F.

    1988-01-01

    The degradation of slowly turning over 125I-lactoperoxidase-labeled plasma membrane polypeptides in response to reversible temperature and lysosomotropic inhibitors was studied in rat hepatoma cultures. Cells were radiolabeled and left for 24 h to allow the removal of rapidly degraded proteins. Remaining trichloroacetic acid-precipitable protein was degraded (t 1/2 = 40-68 h) by an apparent first order process 60-86% sensitive to 10 mM NH4Cl or 5 mM methylamine and greater than 95% inhibited by temperature reduction to 18 degrees C. Thus, membrane proteins are selected for degradation in a time-dependent manner by a system which is sensitive to both 18 degrees C and to lysosomotropic amines. When inhibitory conditions were removed after 40-48 h, degradation of 125I-labeled protein resumed at the same rate as that seen in their absence. Since membrane proteins do not exhibit accelerated degradation after removal of inhibitory conditions, there can be no marking or sorting of those proteins destined for degradation during the 40-h exposure to inhibitory conditions. Exposure to amines or 18 degrees C did not affect the position of two-dimensionally resolved labeled polypeptides. Fractionation of labeled cells on Percoll gradients after 40 h of exposure to low temperature or amines showed that labeled protein remained in the plasma membrane fractions of the gradient although shifted to a slightly lower buoyant density in the presence of amines. These results support the notion that selection of plasma membrane proteins for degradation requires their internalization into acidic vesicles. Lysosomotropic amines and reduced temperature interfere with the selection process by preventing membrane fusion events

  15. Microbial degradation of resins fractionated from Arabian light crude oil

    International Nuclear Information System (INIS)

    Venkateswaran, K.; Hoaki, T.; Kato, M.; Maruyama, T.

    1995-01-01

    Sediment samples from the Japanese coasts were screened for microorganisms able to degrade resin components of crude oil. A mixed population that could degrade 35% of 5000 ppm resin in 15 days was obtained. This population also metabolized 50% of saturates and aromatics present in crude oil (5000 ppm) in 7 days. A Pseudomonas sp., isolated from the mixed population, emulsified and degraded 30% of resins. It also degraded saturates and aromatics (30%) present in crude oil (5000 ppm). These results were obtained from Iatroscan analysis. Degradation of crude oil was also analyzed by gas chromatography (GC). The peaks corresponding to known aliphatic hydrocarbons in crude oil greatly decreased within the first two days of incubation in the cultures of the RY-mixed population and of Pseudomonas strain UN3. Aromatic compounds detected as a broad peak by GC were significantly degraded at day 7 by Pseudomonas strain UN3, and at day 15 by the RY-mixed population. Investigations are ongoing to determine the genetic basis for the ability of these organisms to grow on the resin fractions of crude oil as a sole source of carbon and energy. 28 refs., 4 figs., 1 tab

  16. Selectivity in protein degradation during sporulation of Bacillus subtilis

    International Nuclear Information System (INIS)

    Mitani, Takahiko; Kadota, Hajime

    1976-01-01

    The breakdown of cellular protein was investigated in Bacillus subtilis ATCC 6051 labeled with glycine-2- 3 H or L-phenylalanine-U- 14 C at the different stages of vegetative growth and sporulation. The growth of the culture was determined by measuring optical density at 660 nm. The heat-resistant spores were scored by plating after heating at 80 deg C for 10 minutes. A question whether the turnover of glycine-labeled protein is similar to that of phenylalanine-labeled protein was experimentally studied. The patterns obtained with the glycine-labeled protein were different from those of phenylalanine-labeled protein. This was not multiple turnover. The cellular protein which was labeled with glycine at an early stage of sporulation showed rapid degradation, but the degradation of the protein labeled with glycine at later stages did not occur at all. Another question whether the labeled glycine incorporated into cells at the different stages of growth and sporulation was present in the spore coat fraction of matured spores was studied. Experiment demonstrated that the glycine incorporated into cells at the late sporulation stage was mainly utilized for the biosynthesis of the spore coat protein. These data suggest that the spore coat protein which contains relatively large amount of glycine is rarely subject to further degradation. (Iwakiri, K.)

  17. 21 CFR 862.1630 - Protein (fractionation) test system.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Protein (fractionation) test system. 862.1630... Systems § 862.1630 Protein (fractionation) test system. (a) Identification. A protein (fractionation) test system is a device intended to measure protein fractions in blood, urine, cerebrospinal fluid, and other...

  18. Improved feed protein fractionation schemes for formulating rations with the cornell net carbohydrate and protein system.

    Science.gov (United States)

    Lanzas, C; Broderick, G A; Fox, D G

    2008-12-01

    Adequate predictions of rumen-degradable protein (RDP) and rumen-undegradable protein (RUP) supplies are necessary to optimize performance while minimizing losses of excess nitrogen (N). The objectives of this study were to evaluate the original Cornell Net Carbohydrate Protein System (CNCPS) protein fractionation scheme and to develop and evaluate alternatives designed to improve its adequacy in predicting RDP and RUP. The CNCPS version 5 fractionates CP into 5 fractions based on solubility in protein precipitant agents, buffers, and detergent solutions: A represents the soluble nonprotein N, B1 is the soluble true protein, B2 represents protein with intermediate rates of degradation, B3 is the CP insoluble in neutral detergent solution but soluble in acid detergent solution, and C is the unavailable N. Model predictions were evaluated with studies that measured N flow data at the omasum. The N fractionation scheme in version 5 of the CNCPS explained 78% of the variation in RDP with a root mean square prediction error (RMSPE) of 275 g/d, and 51% of the RUP variation with RMSPE of 248 g/d. Neutral detergent insoluble CP flows were overpredicted with a mean bias of 128 g/d (40% of the observed mean). The greatest improvements in the accuracy of RDP and RUP predictions were obtained with the following 2 alternative schemes. Alternative 1 used the inhibitory in vitro system to measure the fractional rate of degradation for the insoluble protein fraction in which A = nonprotein N, B1 = true soluble protein, B2 = insoluble protein, C = unavailable protein (RDP: R(2) = 0.84 and RMSPE = 167 g/d; RUP: R(2) = 0.61 and RMSPE = 209 g/d), whereas alternative 2 redefined A and B1 fractions as the non-amino-N and amino-N in the soluble fraction respectively (RDP: R(2) = 0.79 with RMSPE = 195 g/d and RUP: R(2) = 0.54 with RMSPE = 225 g/d). We concluded that implementing alternative 1 or 2 will improve the accuracy of predicting RDP and RUP within the CNCPS framework.

  19. Fractionation of carbohydrate and protein content of some forage feeds of ruminants for nutritive evaluation.

    Science.gov (United States)

    Das, Lalatendu Keshary; Kundu, S S; Kumar, Dinesh; Datt, Chander

    2015-02-01

    To evaluate some forage feeds of ruminants in terms of their carbohydrate (CHO) and protein fractions using Cornell Net Carbohydrate and Protein System (CNCPS). Eleven ruminant feeds (six green fodders - maize, oat, sorghum, bajra, cowpea, berseem and five range herbages - para grass, guinea grass, hedge lucerne, setaria grass and hybrid napier) were selected for this study. Each feed was chemically analyzed for proximate principles (dry matter, crude protein [CP], ether extract, organic matter and ash), fiber fractions (neutral detergent fiber, acid detergent fiber, acid detergent lignin, cellulose and hemicellulose), primary CHO fractions (CHO, non-structural CHO, structural CHO and starch) and primary protein fractions (neutral detergent insoluble CP, acid detergent insoluble CP, non-protein nitrogen and soluble protein). The results were fitted to the equations of CNCPS to arrive at various CHO (CA - fast degrading, CB1 - intermediate degrading, CB2 - slow degrading and CC - non-degrading or unavailable) and protein (PA - instantaneously degrading, PB1 - fast degrading, PB2 - intermediate degrading, PB3 - slow degrading and PC - non-degrading or unavailable) fractions of test feeds. Among green fodders, cowpea and berseem had higher CA content while except hedge lucerne all range herbages had lower CA values. CB1 content of all feeds was low but similar. All feeds except cowpea, berseem, and hedge lucerne contained higher CB2 values. Oat among green fodders and hybrid napier among range herbages had lower CC fraction. Feeds such as bajra, cowpea, berseem and the setaria grass contained lower PA fraction. All green fodders had higher PB1 content except maize and cowpea while all range herbages had lower PB1 values except hedge lucerne. Para grass and hybrid napier contained exceptionally low PB2 fraction among all feeds. Low PC contents were reported in oat and berseem fodders. Based on our findings, it was concluded that feeds with similar CP and CHO content

  20. AAA-ATPases in Protein Degradation

    Directory of Open Access Journals (Sweden)

    Ravikiran S. Yedidi

    2017-06-01

    Full Text Available Proteolytic machineries containing multisubunit protease complexes and AAA-ATPases play a key role in protein quality control and the regulation of protein homeostasis. In these protein degradation machineries, the proteolytically active sites are formed by either threonines or serines which are buried inside interior cavities of cylinder-shaped complexes. In eukaryotic cells, the proteasome is the most prominent protease complex harboring AAA-ATPases. To degrade protein substrates, the gates of the axial entry ports of the protease need to be open. Gate opening is accomplished by AAA-ATPases, which form a hexameric ring flanking the entry ports of the protease. Protein substrates with unstructured domains can loop into the entry ports without the assistance of AAA-ATPases. However, folded proteins require the action of AAA-ATPases to unveil an unstructured terminus or domain. Cycles of ATP binding/hydrolysis fuel the unfolding of protein substrates which are gripped by loops lining up the central pore of the AAA-ATPase ring. The AAA-ATPases pull on the unfolded polypeptide chain for translocation into the proteolytic cavity of the protease. Conformational changes within the AAA-ATPase ring and the adjacent protease chamber create a peristaltic movement for substrate degradation. The review focuses on new technologies toward the understanding of the function and structure of AAA-ATPases to achieve substrate recognition, unfolding and translocation into proteasomes in yeast and mammalian cells and into proteasome-equivalent proteases in bacteria and archaea.

  1. AAA-ATPases in Protein Degradation.

    Science.gov (United States)

    Yedidi, Ravikiran S; Wendler, Petra; Enenkel, Cordula

    2017-01-01

    Proteolytic machineries containing multisubunit protease complexes and AAA-ATPases play a key role in protein quality control and the regulation of protein homeostasis. In these protein degradation machineries, the proteolytically active sites are formed by either threonines or serines which are buried inside interior cavities of cylinder-shaped complexes. In eukaryotic cells, the proteasome is the most prominent protease complex harboring AAA-ATPases. To degrade protein substrates, the gates of the axial entry ports of the protease need to be open. Gate opening is accomplished by AAA-ATPases, which form a hexameric ring flanking the entry ports of the protease. Protein substrates with unstructured domains can loop into the entry ports without the assistance of AAA-ATPases. However, folded proteins require the action of AAA-ATPases to unveil an unstructured terminus or domain. Cycles of ATP binding/hydrolysis fuel the unfolding of protein substrates which are gripped by loops lining up the central pore of the AAA-ATPase ring. The AAA-ATPases pull on the unfolded polypeptide chain for translocation into the proteolytic cavity of the protease. Conformational changes within the AAA-ATPase ring and the adjacent protease chamber create a peristaltic movement for substrate degradation. The review focuses on new technologies toward the understanding of the function and structure of AAA-ATPases to achieve substrate recognition, unfolding and translocation into proteasomes in yeast and mammalian cells and into proteasome-equivalent proteases in bacteria and archaea.

  2. Radiation degradation of molasses pigment. 2. Molecular weight fraction

    International Nuclear Information System (INIS)

    Sawai, Teruko; Sekiguchi, Masayuki; Tanabe, Hiroko

    1996-01-01

    Water demand in Tokyo has increased rapidly. Because of the scarcity of water sources within the city, Tokyo is dependent on water from other prefectures. Recycling of municipal effluent is an effective means of coping with water shortage in Tokyo. We have studied the radiation treatment of waste water for recycling. The degradation of molasses pigments in waste water from yeast factory by radiation was investigated. The dialyzed molasses pigments and non-dialyzed samples in waste waters were compared in chromaticity, UV absorption, color different and COD. The dialysis and fractionation by permeable membrane were carried out with Seamless Cellulose tubing (Union Carbide Corporation) and spectra/Por membrane (Spectrum Medical Industries INC.) The TOC values decreased and the dark brown color faded with increasing dose. The high molecular weight components of molasses pigment were degraded to lower molecular weight substances and decomposed to carbon dioxide. The relationships between the value of chromaticity/TOC and molecular weight of molasses pigments were obtained by radiation. (author)

  3. Uptake and degradation of circulating proteins by the liver.

    NARCIS (Netherlands)

    Buys, Carolus Henricus Cornelis Maria

    1976-01-01

    Circulating proteins, like all proteins in a living animal, are subject to continual replacement or turnover. This process implies both synthesis and degradation, This thesis deals with the degradative part of turnover of circulating proteins. ... Summary

  4. Fractionation of carbohydrate and protein content of some forage feeds of ruminants for nutritive evaluation

    Directory of Open Access Journals (Sweden)

    Lalatendu Keshary Das

    2015-02-01

    Full Text Available Aim: To evaluate some forage feeds of ruminants in terms of their carbohydrate (CHO and protein fractions using Cornell Net Carbohydrate and Protein System (CNCPS. Materials and Methods: Eleven ruminant feeds (six green fodders - maize, oat, sorghum, bajra, cowpea, berseem and five range herbages - para grass, guinea grass, hedge lucerne, setaria grass and hybrid napier were selected for this study. Each feed was chemically analyzed for proximate principles (dry matter, crude protein [CP], ether extract, organic matter and ash, fiber fractions (neutral detergent fiber, acid detergent fiber, acid detergent lignin, cellulose and hemicellulose, primary CHO fractions (CHO, non-structural CHO, structural CHO and starch and primary protein fractions (neutral detergent insoluble CP, acid detergent insoluble CP, non-protein nitrogen and soluble protein. The results were fitted to the equations of CNCPS to arrive at various CHO (CA - fast degrading, CB1 - intermediate degrading, CB2 - slow degrading and CC - nondegrading or unavailable and protein (PA - instantaneously degrading, PB1 - fast degrading, PB2 - intermediate degrading, PB3 - slow degrading and PC - non-degrading or unavailable fractions of test feeds. Results: Among green fodders, cowpea and berseem had higher CA content while except hedge lucerne all range herbages had lower CA values. CB1 content of all feeds was low but similar. All feeds except cowpea, berseem, and hedge lucerne contained higher CB2 values. Oat among green fodders and hybrid napier among range herbages had lower CC fraction. Feeds such as bajra, cowpea, berseem and the setaria grass contained lower PA fraction. All green fodders had higher PB1 content except maize and cowpea while all range herbages had lower PB1 values except hedge lucerne. Para grass and hybrid napier contained exceptionally low PB2 fraction among all feeds. Low PC contents were reported in oat and berseem fodders. Conclusion: Based on our findings, it

  5. Microbial degradation of dissolved proteins in seawater

    International Nuclear Information System (INIS)

    Hollibaugh, J.T.; Azam, F.

    1983-01-01

    An experimental protocol using radiolabeled proteins was developed to investigate the rates and mechanisms whereby dissolved proteins are degraded in natural marine plankton communities. The results of field observations and laboratory experiments indicate that proteins are degraded by a particle-bound, thermolabile system, presumably bacteria-associated enzymes, with an apparent half-saturation constant of ca. 25 μg bovine serum albumin (BSA) per liter. Gel permeation chromatography indicated that peptides of chain length intermediate between BSA and the final products of degradation (MW<700) do not accumulate in the medium. Competition experiments indicate that the system is relatively nonspecific. Turnover rates for the protein pool in samples collected in the Southern California Bight were of the same order of magnitude as the turnover rate of the L-leucine pool and were correlated with primary productivity, chlorophyll a concentrations, bacterial abundance and biomass, and L-leucine turnover rate. These data suggest that amino acids derived from proteins are utilized preferentially and do not completely mix with the amino acids in the bulk phase

  6. Antiviral and antitumor activities of the protein fractions from the ...

    African Journals Online (AJOL)

    In this study, we present the extraction and purification of protein fractions from the larvae of the housefly, Musca domestica. The bioactivities of the protein fractions were indicated by pseudorabies virus (PRV) and human lung cancer cell line A 549. The crude protein fractions had no toxicity to chick embryo fibroblast-like ...

  7. Protein synthesis and degradation during starvation-induced cardiac atrophy in rabbits

    International Nuclear Information System (INIS)

    Samarel, A.M.; Parmacek, M.S.; Magid, N.M.; Decker, R.S.; Lesch, M.

    1987-01-01

    To determine the relative importance of protein degradation in the development of starvation-induced cardiac atrophy, in vivo fractional synthetic rates of total cardiac protein, myosin heavy chain, actin, light chain 1, and light chain 2 were measured in fed and fasted rabbits by continuous infusion of [ 3 H] leucine. In addition, the rate of left ventricular protein accumulation and loss were assessed in weight-matched control and fasted rabbits. Rates of total cardiac protein degradation were then estimated as the difference between rates of synthesis and growth. Fasting produced left ventricular atrophy by decreasing the rate of left ventricular protein synthesis (34.8 +/- 1.4, 27.3 +/- 3.0, and 19.3 +/- 1.2 mg/day of left ventricular protein synthesized for 0-, 3-, and 7-day fasted rabbits, respectively). Inhibition of contractile protein synthesis was evident by significant reductions in the fractional synthetic rates of all myofibrillar protein subunits. Although fractional rates of protein degradation increased significantly within 7 days of fasting, actual amounts of left ventricular protein degraded per day were unaffected. Thus, prolonged fasting profoundly inhibits the synthesis of new cardiac protein, including the major protein constituents of the myofibril. Both this inhibition in new protein synthesis as well as a smaller but significant reduction in the average half-lives of cardiac proteins are responsible for atrophy of the heart in response to fasting

  8. Protein degradation and protection against misfolded or damaged proteins

    Science.gov (United States)

    Goldberg, Alfred L.

    2003-12-01

    The ultimate mechanism that cells use to ensure the quality of intracellular proteins is the selective destruction of misfolded or damaged polypeptides. In eukaryotic cells, the large ATP-dependent proteolytic machine, the 26S proteasome, prevents the accumulation of non-functional, potentially toxic proteins. This process is of particular importance in protecting cells against harsh conditions (for example, heat shock or oxidative stress) and in a variety of diseases (for example, cystic fibrosis and the major neurodegenerative diseases). A full understanding of the pathogenesis of the protein-folding diseases will require greater knowledge of how misfolded proteins are recognized and selectively degraded.

  9. Non-degradative Ubiquitination of Protein Kinases.

    Directory of Open Access Journals (Sweden)

    K Aurelia Ball

    2016-06-01

    Full Text Available Growing evidence supports other regulatory roles for protein ubiquitination in addition to serving as a tag for proteasomal degradation. In contrast to other common post-translational modifications, such as phosphorylation, little is known about how non-degradative ubiquitination modulates protein structure, dynamics, and function. Due to the wealth of knowledge concerning protein kinase structure and regulation, we examined kinase ubiquitination using ubiquitin remnant immunoaffinity enrichment and quantitative mass spectrometry to identify ubiquitinated kinases and the sites of ubiquitination in Jurkat and HEK293 cells. We find that, unlike phosphorylation, ubiquitination most commonly occurs in structured domains, and on the kinase domain, ubiquitination is concentrated in regions known to be important for regulating activity. We hypothesized that ubiquitination, like other post-translational modifications, may alter the conformational equilibrium of the modified protein. We chose one human kinase, ZAP-70, to simulate using molecular dynamics with and without a monoubiquitin modification. In Jurkat cells, ZAP-70 is ubiquitinated at several sites that are not sensitive to proteasome inhibition and thus may have other regulatory roles. Our simulations show that ubiquitination influences the conformational ensemble of ZAP-70 in a site-dependent manner. When monoubiquitinated at K377, near the C-helix, the active conformation of the ZAP-70 C-helix is disrupted. In contrast, when monoubiquitinated at K476, near the kinase hinge region, an active-like ZAP-70 C-helix conformation is stabilized. These results lead to testable hypotheses that ubiquitination directly modulates kinase activity, and that ubiquitination is likely to alter structure, dynamics, and function in other protein classes as well.

  10. Chemical degradation of proteins in the solid state with a focus on photochemical reactions.

    Science.gov (United States)

    Mozziconacci, Olivier; Schöneich, Christian

    2015-10-01

    Protein pharmaceuticals comprise an increasing fraction of marketed products but the limited solution stability of proteins requires considerable research effort to prepare stable formulations. An alternative is solid formulation, as proteins in the solid state are thermodynamically less susceptible to degradation. Nevertheless, within the time of storage a large panel of kinetically controlled degradation reactions can occur such as, e.g., hydrolysis reactions, the formation of diketopiperazine, condensation and aggregation reactions. These mechanisms of degradation in protein solids are relatively well covered by the literature. Considerably less is known about oxidative and photochemical reactions of solid proteins. This review will provide an overview over photolytic and non-photolytic degradation reactions, and specially emphasize mechanistic details on how solid structure may affect the interaction of protein solids with light. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Effect of protein degradability on milk production of dairy ewes.

    Science.gov (United States)

    Mikolayunas-Sandrock, C; Armentano, L E; Thomas, D L; Berger, Y M

    2009-09-01

    The objective of this experiment was to determine the effect of protein degradability of dairy sheep diets on milk yield and protein utilization across 2 levels of milk production. Three diets were formulated to provide similar energy concentrations and varying concentrations of rumen-degradable protein (RDP) and rumen-undegradable protein (RUP): 12% RDP and 4% RUP (12-4) included basal levels of RDP and RUP, 12% RDP and 6% RUP (12-6) included additional RUP, and 14% RDP and 4% RUP (14-4) included additional RDP. Diets were composed of alfalfa-timothy cubes, whole and ground corn, whole oats, dehulled soybean meal, and expeller soybean meal (SoyPlus, West Central, Ralston, IA). Estimates of RDP and RUP were based on the Small Ruminant Nutrition System model (2008) and feed and orts were analyzed for Cornell N fractions. Eighteen multiparous dairy ewes in midlactation were divided by milk yield (low and high) into 2 blocks of 9 ewes each and were randomly assigned within block (low and high) to 3 pens of 3 ewes each. Dietary treatments were arranged in a 3 x 3 Latin square within each block and applied to pens for 14-d periods. We hypothesized that pens consuming high-RUP diets (12-6) would produce more milk and milk protein than the basal diet (12-4) and pens consuming high-RDP diets (14-4) would not produce more milk than the basal diet (12-4). Ewes in the high-milk-yield square consumed more dry matter and produced more milk, milk fat, and milk protein than ewes in the low-milk-yield square. There was no effect of dietary treatment on dry matter intake. Across both levels of milk production, the 12-6 diet increased milk yield by 14%, increased milk fat yield by 14%, and increased milk protein yield by 13% compared with the 14-4 and 12-4 diets. Gross N efficiency (milk protein N/intake protein N) was 11 and 15% greater in the 12-6 and 12-4 diets, respectively, compared with the 14-4 diet. Milk urea N concentration was greater in the 12-6 diet and tended to be

  12. Carbon and Hydrogen Stable Isotope Fractionation during Aerobic Bacterial Degradation of Aromatic Hydrocarbons†

    Science.gov (United States)

    Morasch, Barbara; Richnow, Hans H.; Schink, Bernhard; Vieth, Andrea; Meckenstock, Rainer U.

    2002-01-01

    13C/12C and D/H stable isotope fractionation during aerobic degradation was determined for Pseudomonas putida strain mt-2, Pseudomonas putida strain F1, Ralstonia pickettii strain PKO1, and Pseudomonas putida strain NCIB 9816 grown with toluene, xylenes, and naphthalene. Different types of initial reactions used by the respective bacterial strains could be linked with certain extents of stable isotope fractionation during substrate degradation. PMID:12324375

  13. Dry fractionation for sustainable production of functional legume protein concentrates

    NARCIS (Netherlands)

    Schutyser, M.A.I.; Pelgrom, P.J.M.; Goot, van der A.J.; Boom, R.M.

    2015-01-01

    Plant proteins gain increasing interest as part of a sustainable diet. Because plant materials not only contain protein, they are generally isolated via an energy intensive wet fractionation. This review discusses dry fractionation as an alternative and more sustainable route for producing

  14. Stable Isotope Fractionation Caused by Glycyl Radical Enzymes during Bacterial Degradation of Aromatic Compounds

    Science.gov (United States)

    Morasch, Barbara; Richnow, Hans H.; Vieth, Andrea; Schink, Bernhard; Meckenstock, Rainer U.

    2004-01-01

    Stable isotope fractionation was studied during the degradation of m-xylene, o-xylene, m-cresol, and p-cresol with two pure cultures of sulfate-reducing bacteria. Degradation of all four compounds is initiated by a fumarate addition reaction by a glycyl radical enzyme, analogous to the well-studied benzylsuccinate synthase reaction in toluene degradation. The extent of stable carbon isotope fractionation caused by these radical-type reactions was between enrichment factors (ɛ) of −1.5 and −3.9‰, which is in the same order of magnitude as data provided before for anaerobic toluene degradation. Based on our results, an analysis of isotope fractionation should be applicable for the evaluation of in situ bioremediation of all contaminants degraded by glycyl radical enzyme mechanisms that are smaller than 14 carbon atoms. In order to compare carbon isotope fractionations upon the degradation of various substrates whose numbers of carbon atoms differ, intrinsic ɛ (ɛintrinsic) were calculated. A comparison of ɛintrinsic at the single carbon atoms of the molecule where the benzylsuccinate synthase reaction took place with compound-specific ɛ elucidated that both varied on average to the same extent. Despite variations during the degradation of different substrates, the range of ɛ found for glycyl radical reactions was reasonably narrow to propose that rough estimates of biodegradation in situ might be given by using an average ɛ if no fractionation factor is available for single compounds. PMID:15128554

  15. Bovine plasma protein fractionation by ion exchange chromatography.

    Science.gov (United States)

    Moure, F; Rendueles, M; Díaz, M

    2004-12-01

    An ion exchange chromatography process was developed to separate the main protein fractions of bovine blood plasma using a composite material, Q-HyperD resin, and a gel material, DEAE-Sepharose. The experiments were carried out at semipreparative scale. It was necessary to establish analytical methods of electrophoresis and HPLC to identify the fractionated proteins. Results show that these materials are able to adequately fractionate different protein groups from the raw blood plasma. This method may be used to avoid chemical fractionation using agents such as ethanol or PEG and, thus, decrease protein denaturation of the different fractions to be used for research or pharmaceutical purposes. The Q-HyperD resin presents a better retention capacity for plasma protein than DEAE-Sepharose under the experimental conditions employed.

  16. Caracterização, fracionamento protéico, degradabilidade ruminal e digestibilidade in vitro da matéria seca e proteína bruta do resíduo de cervejaria úmido e fermentado = Characterization, protein fractioning, dry matter and crude protein rumen degradability and in vitro digestibility of wet and fermented brewer’s grain

    Directory of Open Access Journals (Sweden)

    Luiz Juliano Valério Geron

    2007-07-01

    Full Text Available Foram avaliadas as frações da proteína e dos carboidratos, a degradabilidade ruminal efetiva (DE da matéria seca (MS e proteína bruta (PB, a digestibilidade ruminal in vitro (DRIV da MS e PB, a digestibilidade intestinal in vitro (DIIV da proteína nãodegradadano rúmen (PNDR e os perfis de aminoácidos (AA e de ácidos graxos (AG do resíduo de cervejaria úmido (RCU e fermentado (RCF. O RCF foi obtido pelo processo de fermentação microbiana do RCU. Para determinar a DE da MS e PB do RCU e RCF, foram utilizados três novilhos da raça Holandesa, portadores de cânula ruminal. A DIIV daPNDR foi obtida pelo método de três estágios. Os dados obtidos para DE da MS e PB foram submetidos à análise de variância, em elineamento inteiramente casualizado. A fração A da PB do RCU foi de 7,9% e do RCF de 13,1% da PB. A DE da PB a 5% h-1 não diferiu (p The study evaluated the protein and carbohydrate fraction, dry matter (DM and crude protein (CP effective rumen degradability (ED, DM and CP in vitro ruminal digestibility (RDIV, rumen-undegradable protein (RUDP in vitro intestinal digestion (IDIV and amino-acid (AA and fatty acid (FA profile of the wet brewer’s grain (WBG, andfermented brewer’s grain (FBG. FBG was obtained from WBG fermentation. The DM and CP ED of WBG and FBG were determined in three Holstein steers with ruminal cannula. The IDIV of RUDP was obtained by the three-stage method. The values obtained for DM and CP ED were submitted to variance analysis, in a randomized design. The Afraction of WBG CP was 7.9%, and for FBG 13.1% of CP. The CP RD in a rate of 5% h-1 did not differ (p > 0.05 between WBG and FBG. The crude protein RDIV of FBG was 8.7% and IDIV of RUDP of WBG and FBG were of 70.5% and 72.5%, respectively. The AA and FA profile of WBG and FBG were similar. The anaerobic fermentation process did not change the nutritional characteristics of the WBG.

  17. Lysine Rich Proteins in the Salt-Soluble Protein Fraction of Barley

    DEFF Research Database (Denmark)

    Ingversen, J.; Køie, B.

    1973-01-01

    Fractionation of the protein complex from Emir barley showed that the salt-soluble fraction accounts for 44% of the total lysine content but only for 2.......Fractionation of the protein complex from Emir barley showed that the salt-soluble fraction accounts for 44% of the total lysine content but only for 2....

  18. Ruminal degradability and carbohydrates and proteins fractioning of triticale silages in singular culture or in mixtures with oat and/or legumesDegradabilidade ruminal e fracionamento de carboidratos e proteínas em silagens de triticale em cultivo singular ou em misturas com aveia e/ou leguminosas

    Directory of Open Access Journals (Sweden)

    Moysés Calixto Junior

    2011-07-01

    Full Text Available It was aimed to evaluate the ruminal degradability, and the fractioning of carbohydrates, as well as of the nitrogen fractions of triticale silages in singular culture or in mixtures with oats and/or legumes. The treatments had been: triticale silage (X. Triticosecale Wittimack (ST; triticale silage + forage pea (Pisum arvense (STE; triticale silage + oats (Avena strigosa Scheb + forage pea + vetch (Vicia sativa (STAE. Three castrated bovine Prim’Holstein males had been used, with average live weight of 300 kg, having ruminal cannulas. The incubation times had duration of 0, 6, 12, 24, 48, 72 and 96 hours. Fractions, a, c and ED of the DM of STAE silage was higher (30,33; 0,026 and 45,45% than others silages. Fraction b of the DM of ST silage was higher (58,45% than STE silages (45,36% and STAE (44,37%. In CP the ST silage presented higher fraction (72.12%. For the potentially degradability fraction (b of CP it was not observed difference among treatments. The degradation rate (c of the CP was higher for STE silage (0.063% than ST ensilage (0,012%, however this was similar to the STAE (0,045%. ED of CP was better for STE silage (77,71%. ST silage presented fraction a and b for NDF highest (8,62 and 81,99% than others silages. The degradation rate (c of NDF was higher for STAE (0,027%. ED for NDF did not present difference among treatments. ST silage presented the best values for the total carbohydrates (83,97% than STE (79,87% and STAE silages (76,77%. STE and STAE silages presented better DM degradability and suggest to be a potential source of non degradable protein in the rumen. The exclusive triticale silage revealed superior with regard to the degradability of fiber fraction, also presenting higher amount of total carbohydrates potentially degraded.O objetivo desse trabalho foi avaliar a degradabilidade ruminal, e o fracionamento de carboidratos, assim como das frações nitrogenadas das silagens de triticale em plantio singular ou em

  19. Characterization of pea (Pisum sativum) seed protein fractions.

    Science.gov (United States)

    Rubio, Luis A; Pérez, Alicia; Ruiz, Raquel; Guzmán, M Ángeles; Aranda-Olmedo, Isabel; Clemente, Alfonso

    2014-01-30

    Legume seed proteins have to be chemically characterized in order to properly link their nutritional effects with their chemical structure. Vicilin and albumin fractions devoid of cross-contamination, as assessed by mass peptide fingerprinting analysis, were obtained from defatted pea (Pisum sativum cv. Bilbo) meal. The extracted protein fractions contained 56.7-67.7 g non-starch polysaccharides kg⁻¹. The vicilin fraction was higher than legumins in arginine, isoleucine, leucine, phenylalanine and lysine. The most abundant amino acids in the albumin fraction were aspartic acid, glutamic acid, lysine and arginine, and the amounts of methionine were more than double than those in legumins and vicilins. The pea albumin fraction showed a clear enrichment of protease inhibitory activity when compared with the seed meal. In vitro digestibility values for pea proteins were 0.63 ±  0.04, 0.88 ±  0.04 and 0.41 ±  0.23 for legumins, vicilins and albumins respectively. Vicilin and albumin fractions devoid of cross-contamination with other proteins were obtained from pea seed meal. The vicilin fraction also contained low amounts of soluble non-starch polysaccharides and was enriched in isoleucine, leucine, phenylalanine and lysine. In vitro digestibility values for pea proteins were similar or even numerically higher than those for control proteins. © 2013 Society of Chemical Industry.

  20. Antioxidant Activity of Coconut (Cocos nucifera L.) Protein Fractions.

    Science.gov (United States)

    Li, Yan; Zheng, Yajun; Zhang, Yufeng; Xu, Jianguo; Gao, Gang

    2018-03-20

    Coconut cake is an abundant and good potential edible protein source. However, until now it has not been extensively used in the food industry. To promote its usage, the characterization, nutrition value and antioxidant activity of coconut cake protein fractions (albumin, globulin, prolamine, glutelin-1 and glutelin-2) were studied. Results revealed that all the albumin, globulin, glutelin-1 and glutelin-2 fractions showed a high nutrition value. The prolamine, glutelin-1 and glutelin-2 all exhibited good radical scavenging activity and reducing power, and the globulin and prolamine showed high ion chelating ability (89.14-80.38%). Moreover, all the fractions except glutelin-2 could effectively protect DNA against oxidative damage. Several peptides containing five to eight amino acids with antioxidant activity were also identified by LC-MS/MS from the globulin and glutelin-2 fractions. The results demonstrated that the coconut cake protein fractions have potential usages in functional foods.

  1. THE SERUM PROTEIN FRACTIONS IN THYMOQUINONE TREATED RATS.

    Science.gov (United States)

    A, Güllü; S, Dede

    2016-01-01

    TQ has been used as treatment and preventive agent for many diseases over the years. The goal of this study was to investigate the effects of TQ supplement on fractions of serum proteins. Fourteen male Wistar-Albino rats (200-250 g weight) were used as material for two groups; (control (C) and thymoquinone (TQ) respectively. Each group contained seven rats. The control group had only corn oil, while the TQ group was dissolved in corn oil. 30 mg/kg/day were given by oral gavage for four weeks. The serum protein fractions were identified using cellulose acetate technique. The total protein level and albumin, α-1, α-2 fractions and A/G ratio have showed no difference between groups (p>0.05). β-globulin fractions of TQ group were higher than control's (pfractions may have originated from elevation or decline synthesis, or activities of containing proteins.

  2. A Hybrid Dry and Aqueous Fractionation Method to Obtain Protein-Rich Fractions from Quinoa (Chenopodium quinoa Willd)

    NARCIS (Netherlands)

    Avila Ruiz, Geraldine; Arts, Anke; Minor, Marcel; Schutyser, Maarten

    2016-01-01

    Combination of dry and aqueous fractionation is investigated to obtain protein-rich fractions from quinoa in a milder and more sustainable way compared to conventional wet fractionation. Dry fractionation of quinoa involved milling and subsequent air classification, generating a protein-enriched

  3. Dry fractionation for production of functional pea protein concentrates

    NARCIS (Netherlands)

    Pelgrom, P.J.M.; Vissers, A.M.; Boom, R.M.; Schutyser, M.A.I.

    2013-01-01

    Dry milling in combination with air classification was evaluated as an alternative to conventional wet extraction of protein from yellow field peas (Pisum sativum). Major advantages of dry fractionation are retention of native functionality of proteins and its lower energy and water use. Peas were

  4. Preparation of functional lupine protein fractions by dry separation

    NARCIS (Netherlands)

    Pelgrom, P.J.M.; Berghout, J.A.M.; Goot, van der A.J.; Boom, R.M.; Schutyser, M.A.I.

    2014-01-01

    Lupine protein concentrate is a promising ingredient that can be obtained by a combination of milling and air classification, generally called dry fractionation. This is a more sustainable route than conventional wet extraction and delivers a protein concentrate with native functional properties.

  5. The establishment of a protein degradability data base for dairy ...

    African Journals Online (AJOL)

    The degradability values ranged from 96,2; 91,9 and 88,7% for ... McDonald, 1979; Kristensen, et ai., 1982; Lindberg,. 1983 ... mass of 570 kg, and average production of 5000 kg ... Dicalcium phosphate'. 0,5. Commercial vitamin and trace mineral premix. 0,3 .... effects on the degradation of most protein supplements.

  6. Cellular proteostasis: degradation of misfolded proteins by lysosomes

    Science.gov (United States)

    Jackson, Matthew P.

    2016-01-01

    Proteostasis refers to the regulation of the cellular concentration, folding, interactions and localization of each of the proteins that comprise the proteome. One essential element of proteostasis is the disposal of misfolded proteins by the cellular pathways of protein degradation. Lysosomes are an important site for the degradation of misfolded proteins, which are trafficked to this organelle by the pathways of macroautophagy, chaperone-mediated autophagy and endocytosis. Conversely, amyloid diseases represent a failure in proteostasis, in which proteins misfold, forming amyloid deposits that are not degraded effectively by cells. Amyloid may then exacerbate this failure by disrupting autophagy and lysosomal proteolysis. However, targeting the pathways that regulate autophagy and the biogenesis of lysosomes may present approaches that can rescue cells from the deleterious effects of amyloidogenic proteins. PMID:27744333

  7. Fractional rate of degradation (kd) of starch in the rumen and its ...

    African Journals Online (AJOL)

    Fractional rate of degradation (kd) of fermentable nutrients in the rumen is an important parameter in modern feed evaluation systems based on mechanistic models. Estimates of kd for starch was obtained on 19 starch sources originating from barley, wheat, oat, maize and peas and treated in different ways both chemically ...

  8. Proteasomal and Lysosomal Protein Degradation and Heart Disease

    OpenAIRE

    Wang, Xuejun; Robbins, Jeffrey

    2013-01-01

    In the cell, the proteasome and lysosomes represent the most important proteolytic machineries, responsible for the protein degradation in the ubiquitin-proteasome system (UPS) and autophagy, respectively. Both the UPS and autophagy are essential to protein quality and quantity control. Alterations in cardiac proteasomal and lysosomal degradation are remarkably associated with most heart disease in humans and are implicated in the pathogenesis of congestive heart failure. Studies carried out ...

  9. Knowns and unknowns of plasma membrane protein degradation in plants.

    Science.gov (United States)

    Liu, Chuanliang; Shen, Wenjin; Yang, Chao; Zeng, Lizhang; Gao, Caiji

    2018-07-01

    Plasma membrane (PM) not only creates a physical barrier to enclose the intracellular compartments but also mediates the direct communication between plants and the ever-changing environment. A tight control of PM protein homeostasis by selective degradation is thus crucial for proper plant development and plant-environment interactions. Accumulated evidences have shown that a number of plant PM proteins undergo clathrin-dependent or membrane microdomain-associated endocytic routes to vacuole for degradation in a cargo-ubiquitination dependent or independent manner. Besides, several trans-acting determinants involved in the regulation of endocytosis, recycling and multivesicular body-mediated vacuolar sorting have been identified in plants. More interestingly, recent findings have uncovered the participation of selective autophagy in PM protein turnover in plants. Although great progresses have been made to identify the PM proteins that undergo dynamic changes in subcellular localizations and to explore the factors that control the membrane protein trafficking, several questions remain to be answered regarding the molecular mechanisms of PM protein degradation in plants. In this short review article, we briefly summarize recent progress in our understanding of the internalization, sorting and degradation of plant PM proteins. More specifically, we focus on discussing the elusive aspects underlying the pathways of PM protein degradation in plants. Copyright © 2018 Elsevier B.V. All rights reserved.

  10. Protein degradation pathways in Parkinson's disease: curse or blessing.

    Science.gov (United States)

    Ebrahimi-Fakhari, Darius; Wahlster, Lara; McLean, Pamela J

    2012-08-01

    Protein misfolding, aggregation and deposition are common disease mechanisms in many neurodegenerative diseases including Parkinson's disease (PD). Accumulation of damaged or abnormally modified proteins may lead to perturbed cellular function and eventually to cell death. Thus, neurons rely on elaborated pathways of protein quality control and removal to maintain intracellular protein homeostasis. Molecular chaperones, the ubiquitin-proteasome system (UPS) and the autophagy-lysosomal pathway (ALP) are critical pathways that mediate the refolding or removal of abnormal proteins. The successive failure of these protein degradation pathways, as a cause or consequence of early pathological alterations in vulnerable neurons at risk, may present a key step in the pathological cascade that leads to spreading neurodegeneration. A growing number of studies in disease models and patients have implicated dysfunction of the UPS and ALP in the pathogenesis of Parkinson's disease and related disorders. Deciphering the exact mechanism by which the different proteolytic systems contribute to the elimination of pathogenic proteins, like α-synuclein, is therefore of paramount importance. We herein review the role of protein degradation pathways in Parkinson's disease and elaborate on the different contributions of the UPS and the ALP to the clearance of altered proteins. We examine the interplay between different degradation pathways and provide a model for the role of the UPS and ALP in the evolution and progression of α-synuclein pathology. With regards to exciting recent studies we also discuss the putative potential of using protein degradation pathways as novel therapeutic targets in Parkinson's disease.

  11. Human Serum Protein-Bound iodine and Protein Fractions at ...

    African Journals Online (AJOL)

    Iodine profile of Nigerians at different ages in both sexes and in pregnant women, and under narcotic influence, such as alcoholism, cigarette smoking and marijuana addiction were studied. Their serum total protein, albumin and globulin concentrations were also determined. Results of the study showed that serum protein ...

  12. Degradation kinetics and assessment of the prediction equation of indigestible fraction of neutral detergent fiber from agroindustrial byproducts

    Directory of Open Access Journals (Sweden)

    José Gilson Louzada Regadas Filho

    2011-09-01

    Full Text Available This study aimed at estimating the kinetic parameters of ruminal degradation of neutral detergent fiber from agroindustrial byproducts of cashew (pulp and cashew nut, passion fruit, melon, pineapple, West Indian cherry, grape, annatto and coconut through the gravimetric technique of nylon bag, and to evaluate the prediction equation of indigestible fraction of neutral detergent fiber suggested by the Cornell Net Carbohydrate and Protein System. Samples of feed crushed to 2 mm were placed in 7 × 14 cm nylon bags with porosity of 50 µm in a ratio of 20 g DM/cm² and incubated in duplicate in the rumen of a heifer at 0, 3, 6, 9, 12, 16, 24, 36, 48, 72, 96 and 144 hours. The incubation residues were analyzed for NDF content and evaluated by a non-linear logistic model. The evaluation process of predicting the indigestible fraction of NDF was carried out through adjustment of linear regression models between predicted and observed values. There was a wide variation in the degradation parameters of NDF among byproducts. The degradation rate of NDF ranged from 0.0267 h-1 to 0.0971 h-1 for grape and West Indian cherry, respectively. The potentially digestible fraction of NDF ranged from 4.17 to 90.67%, respectively, for melon and coconut byproducts. The CNCPS equation was sensitive to predict the indigestible fraction of neutral detergent fiber of the byproducts. However, due to the high value of the mean squared error of prediction, such estimates are very variable; hence the most suitable would be estimation by biological methods.

  13. Herp enhances ER-associated protein degradation by recruiting ubiquilins

    International Nuclear Information System (INIS)

    Kim, Tae-Yeon; Kim, Eunmin; Yoon, Sungjoo Kim; Yoon, Jong-Bok

    2008-01-01

    ER-associated protein degradation (ERAD) is a protein quality control system of ER, which eliminates misfolded proteins by proteasome-dependent degradation and ensures export of only properly folded proteins from ER. Herp, an ER membrane protein upregulated by ER stress, is implicated in regulation of ERAD. In the present study, we show that Herp interacts with members of the ubiquilin family, which function as a shuttle factor to deliver ubiquitinated substrates to the proteasome for degradation. Knockdown of ubiquilin expression by small interfering RNA stabilized the ERAD substrate CD3δ, whereas it did not alter or increased degradation of non-ERAD substrates tested. CD3δ was stabilized by overexpressed Herp mutants which were capable of binding to ubiquilins but were impaired in ER membrane targeting by deletion of the transmembrane domain. Our data suggest that Herp binding to ubiquilin proteins plays an important role in the ERAD pathway and that ubiquilins are specifically involved in degradation of only a subset of ubiquitinated targets, including Herp-dependent ERAD substrates

  14. Protein oxidation and degradation caused by particulate matter

    Science.gov (United States)

    Lai, Ching-Huang; Lee, Chun-Nin; Bai, Kuan-Jen; Yang, You-Lan; Chuang, Kai-Jen; Wu, Sheng-Ming; Chuang, Hsiao-Chi

    2016-09-01

    Particulate matter (PM) modulates the expression of autophagy; however, the role of selective autophagy by PM remains unclear. The objective of this study was to determine the underlying mechanisms in protein oxidation and degradation caused by PM. Human epithelial A549 cells were exposed to diesel exhaust particles (DEPs), urban dust (UD), and carbon black (CB; control particles). Cell survival and proliferation were significantly reduced by DEPs and UD in A549 cells. First, benzo(a)pyrene diolepoxide (BPDE) protein adduct was caused by DEPs at 150 μg/ml. Methionine oxidation (MetO) of human albumin proteins was induced by DEPs, UD, and CB; however, the protein repair mechanism that converts MetO back to methionine by methionine sulfoxide reductases A (MSRA) and B3 (MSRB3) was activated by DEPs and inhibited by UD, suggesting that oxidized protein was accumulating in cells. As to the degradation of oxidized proteins, proteasome and autophagy activation was induced by CB with ubiquitin accumulation, whereas proteasome and autophagy activation was induced by DEPs without ubiquitin accumulation. The results suggest that CB-induced protein degradation may be via an ubiquitin-dependent autophagy pathway, whereas DEP-induced protein degradation may be via an ubiquitin-independent autophagy pathway. A distinct proteotoxic effect may depend on the physicochemistry of PM.

  15. The establishment of a protein degradability data base for dairy ...

    African Journals Online (AJOL)

    The establishment of a protein degradability data base for dairy cattle using the nylon bag technique. 1. Protein sources. LJ Erasmus, J Prinsloo, HH Meissner. Abstract. No Abstract. Full Text: EMAIL FREE FULL TEXT EMAIL FREE FULL TEXT · DOWNLOAD FULL TEXT DOWNLOAD FULL TEXT · AJOL African Journals ...

  16. A new approach to estimate the in situ fractional degradation rate of organic matter and nitrogen in wheat yeast concentrates

    NARCIS (Netherlands)

    De Jonge, L. H.; Van Laar, H.; Hendriks, W. H.; Dijkstra, J.

    2015-01-01

    In the classic in situ method, small particles are removed during rinsing and hence their fractional degradation rate cannot be determined. A new approach was developed to estimate the fractional degradation rate of nutrients in small particles. This approach was based on an alternative rinsing

  17. Microbial degradation of lignocellulosic fractions during drum composting of mixed organic waste

    Directory of Open Access Journals (Sweden)

    Vempalli Sudharsan Varma

    2017-11-01

    Full Text Available The study aimed to characterize the microbial population involved in lignocellulose degradation during drum composting of mixed organic waste i.e. vegetable waste, cattle manure, saw dust and dry leaves in a 550 L rotary drum composter. Lignocellulose degradation by different microbial populations was correlated by comparing results from four trials, i.e., Trial 1 (5:4, Trial 2 (6:3, Trial 3 (7:2 and Trial 4 (8:1 of varying waste combinations during 20 days of composting period. Due to proper combination of waste materials and agitation in drum composter, a maximum of 66.5 and 61.4 °C was achieved in Trial 1 and 2 by observing a temperature level of 55 °C for 4–6 d. The study revealed that combinations of waste materials had a major effect on the microbial degradation of waste material and quality of final compost due to its physical properties. However, Trial 1 was observed to have longer thermophilic phase leading to higher degradation of lignocellulosic fractions. Furthermore, Fourier transform infrared spectrometer and fluorescent spectroscopy confirmed the decrease in aliphatic to aromatic ratio and increase in polyphenolic compounds of the compost. Heterotrophic bacteria were observed predominantly due to the readily available organic matter during the initial period of composting. However, fungi and actinomycetes were active in the degradation of lignocellulosic fractions.

  18. In situ degradability of dry matter and fibrous fraction of sorghum silage

    Directory of Open Access Journals (Sweden)

    Renê Ferreira Costa

    2016-05-01

    Full Text Available This study aimed to evaluate in situ degradability and degradation kinetics of DM, NDF and ADF of silage, with or without tannin in the grains. Two isogenic lines of grain sorghum (CMS-XS 114 with tannin and CMS-XS 165 without tannin and two sorghum hybrids (BR-700 dual purpose with tannin and BR-601 forage without tannin were ensiled; dried and ground silage samples were placed in nylon bags and introduced through the fistulas. After incubation for 6, 12, 24, 48, 72 and 96 hours, bags were taken for subsequent analysis of fibrous fractions. The experimental design was completely randomized with 4 replicates and 4 treatments and means compared by Tukey’s test at 5% probability. As for the DM degradation rate, silage of CMSXS165without tannin was superior. Silages of genotypes BR700 and CMSXS 114 with tannin showed the highest values of indigestible ADF (59.54 and 43.09%. Regarding the NDF, the potential degradation of silage of CMSXS165 line without tannin was superior. Tannin can reduce ruminal degradability of the dry matter and fibrous fractions.

  19. Human sperm degradation of zona pellucida proteins contributes to fertilization.

    Science.gov (United States)

    Saldívar-Hernández, Analilia; González-González, María E; Sánchez-Tusié, Ana; Maldonado-Rosas, Israel; López, Pablo; Treviño, Claudia L; Larrea, Fernando; Chirinos, Mayel

    2015-09-02

    The mammalian oocyte extracellular matrix known as the zona pellucida (ZP) acts as a barrier to accomplish sperm fusion with the female gamete. Although penetration of the ZP is a limiting event to achieve fertilization, this is one of the least comprehended stages of gamete interaction. Even though previous studies suggest that proteases of sperm origin contribute to facilitate the passage of sperm through the ZP, in human this process is not yet fully understood. The aim of this study was to determine the ability of human sperm to degrade recombinant human ZP (rhZPs) proteins and to characterize the proteases involved in this process. Purified rhZP2, rhZP3 and rhZP4 proteins were incubated with capacitated sperm and the proteolytic activity was determined by Western blot analysis. To further characterize the proteases involved, parallel incubations were performed in the presence of the protease inhibitors o-phenanthroline, benzamidine and MG-132 meant to block the activity of metalloproteases, serine proteases and the proteasome, respectively. Additionally, protease inhibitors effect on sperm-ZP binding was evaluated by hemizona assay. The results showed that rhZPs were hydrolyzed in the presence of capacitated sperm. O-phenanthroline inhibited the degradation of rhZP3, MG-132 inhibited the degradation of rhZP4 and benzamidine inhibited the degradation of the three proteins under investigation. Moreover, hemizona assays demonstrated that sperm proteasome inhibition impairs sperm interaction with human native ZP. This study suggests that sperm proteasomes could participate in the degradation of ZP, particularly of the ZP4 protein. Besides, metalloproteases may be involved in specific degradation of ZP3 while serine proteases may contribute to unspecific degradation of the ZP. These findings suggest that localized degradation of ZP proteins by sperm is probably involved in ZP penetration and may be of help in understanding the mechanisms of fertilization in humans.

  20. Determination of human muscle protein fractional synthesis rate

    DEFF Research Database (Denmark)

    Bornø, Andreas; Hulston, Carl J; van Hall, Gerrit

    2014-01-01

    In the present study, different MS methods for the determination of human muscle protein fractional synthesis rate (FSR) using [ring-(13)C6 ]phenylalanine as a tracer were evaluated. Because the turnover rate of human skeletal muscle is slow, only minute quantities of the stable isotopically...

  1. Antiviral and antitumor activities of the protein fractions from the ...

    African Journals Online (AJOL)

    AJL

    2012-05-15

    May 15, 2012 ... embryo fibroblast-like (CEF) cells but effective to PRV at the concentration of 100 µg/ml .... The in vitro inhibitory effect of the purified protein fractions on ... (DMSO) with 10% fetal bovine serum was plated on 96-well plates.

  2. Protein degradation: recognition of ubiquitinylated substrates

    DEFF Research Database (Denmark)

    Hartmann-Petersen, Rasmus; Gordon, Colin

    2004-01-01

    A cell-free system has been developed in budding yeast that provides direct evidence that the Dsk2/Dph1, Rad23/Rhp23 and Rpn10/Pus1 multi-ubiquitin-binding proteins, long implicated in substrate recognition and presentation to the 26S proteasome, actually fulfil such a role.......A cell-free system has been developed in budding yeast that provides direct evidence that the Dsk2/Dph1, Rad23/Rhp23 and Rpn10/Pus1 multi-ubiquitin-binding proteins, long implicated in substrate recognition and presentation to the 26S proteasome, actually fulfil such a role....

  3. Lignocellulose degradation, enzyme production and protein ...

    African Journals Online (AJOL)

    Microbial conversion of corn stover by white rot fungi has the potential to increase its ligninolysis and nutritional value, thereby transforming it into protein-enriched animal feed. Response surface methodology was applied to optimize conditions for the production of lignocellulolytic enzymes by Trametes versicolor during ...

  4. Prion protein degradation by lichens of the genus Cladonia

    Science.gov (United States)

    Bennett, James P.; Rodriguez, Cynthia M.; Johnson, Christopher J.

    2012-01-01

    It has recently been discovered that lichens contain a serine protease capable of degrading the pathogenic prion protein, the etiological agent of prion diseases such as sheep scrapie and cervid chronic wasting disease. Limited methods are available to degrade or inactivate prion disease agents, especially in the environment, and lichens or their serine protease could prove important for management of these diseases. Scant information is available regarding the presence or absence of the protease responsible for degrading prion protein (PrP) in lichen species and, in this study, we tested the hypothesis that PrP degradation activity in lichens is phylogenetically-based by testing 44 species of Cladonia lichens, a genus for which a significant portion of the phylogeny is well established. We categorized PrP degradation activity among the 44 species (high, moderate, low or none) and found that activity in Cladonia species did not correspond with phylogenetic position of the species. Degradation of PrP did correspond, however, with three classical taxonomic characters within the genus: species with brown apothecia, no usnic acid, and the presence of a cortex. Of the 44 species studied, 18 (41%) had either high or moderate PrP degradation activity, suggesting the protease may be frequent in this genus of lichens.

  5. Protein degradation during reconsolidation as a mechanism for memory reorganization

    Directory of Open Access Journals (Sweden)

    Bong-Kiun Kaang

    2011-02-01

    Full Text Available Memory is a reference formed from a past experience that is used to respond to present situations. However, the world is dynamic and situations change, so it is important to update the memory with new information each time it is reactivated in order to adjust the response in the future. Recent researches indicate that memory may undergo a dynamic process that could work as an updating mechanism. This process which is called reconsolidation involves destabilization of the memory after it is reactivated, followed by restabilization. Recently, it has been demonstrated that the initial destabilization process of reconsolidation requires protein degradation. Using protein degradation inhibition as a method to block reconsolidation, recent researches suggest that reconsolidation, especially the protein degradation-dependent destabilization process is necessary for memory reorganization.

  6. Proteolysis targeting peptide (PROTAP) strategy for protein ubiquitination and degradation.

    Science.gov (United States)

    Zheng, Jing; Tan, Chunyan; Xue, Pengcheng; Cao, Jiakun; Liu, Feng; Tan, Ying; Jiang, Yuyang

    2016-02-19

    Ubiquitination proteasome pathway (UPP) is the most important and selective way to degrade proteins in vivo. Here, a novel proteolysis targeting peptide (PROTAP) strategy, composed of a target protein binding peptide, a linker and a ubiquitin E3 ligase recognition peptide, was designed to recruit both target protein and E3 ligase and then induce polyubiquitination and degradation of the target protein through UPP. In our study, the PROTAP strategy was proved to be a general method with high specificity using Bcl-xL protein as model target in vitro and in cells, which indicates that the strategy has great potential for in vivo application. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Are animal models predictive for human postmortem muscle protein degradation?

    Science.gov (United States)

    Ehrenfellner, Bianca; Zissler, Angela; Steinbacher, Peter; Monticelli, Fabio C; Pittner, Stefan

    2017-11-01

    A most precise determination of the postmortem interval (PMI) is a crucial aspect in forensic casework. Although there are diverse approaches available to date, the high heterogeneity of cases together with the respective postmortal changes often limit the validity and sufficiency of many methods. Recently, a novel approach for time since death estimation by the analysis of postmortal changes of muscle proteins was proposed. It is however necessary to improve the reliability and accuracy, especially by analysis of possible influencing factors on protein degradation. This is ideally investigated on standardized animal models that, however, require legitimization by a comparison of human and animal tissue, and in this specific case of protein degradation profiles. Only if protein degradation events occur in comparable fashion within different species, respective findings can sufficiently be transferred from the animal model to application in humans. Therefor samples from two frequently used animal models (mouse and pig), as well as forensic cases with representative protein profiles of highly differing PMIs were analyzed. Despite physical and physiological differences between species, western blot analysis revealed similar patterns in most of the investigated proteins. Even most degradation events occurred in comparable fashion. In some other aspects, however, human and animal profiles depicted distinct differences. The results of this experimental series clearly indicate the huge importance of comparative studies, whenever animal models are considered. Although animal models could be shown to reflect the basic principles of protein degradation processes in humans, we also gained insight in the difficulties and limitations of the applicability of the developed methodology in different mammalian species regarding protein specificity and methodic functionality.

  8. Protein degradation and protein synthesis in long-term memory formation

    Directory of Open Access Journals (Sweden)

    Timothy J Jarome

    2014-06-01

    Full Text Available Long-term memory (LTM formation requires transient changes in the activity of intracellular signaling cascades that are thought to regulate new gene transcription and de novo protein synthesis in the brain. Consistent with this, protein synthesis inhibitors impair LTM for a variety of behavioral tasks when infused into the brain around the time of training or following memory retrieval, suggesting that protein synthesis is a critical step in LTM storage in the brain. However, evidence suggests that protein degradation mediated by the ubiquitin-proteasome system may also be a critical regulator of LTM formation and stability following retrieval. This requirement for increased protein degradation has been shown in the same brain regions in which protein synthesis is required for LTM storage. Additionally, increases in the phosphorylation of proteins involved in translational control parallel increases in protein polyubiquitination and the increased demand for protein degradation is regulated by intracellular signaling molecules thought to regulate protein synthesis during LTM formation. In some cases inhibiting proteasome activity can rescue memory impairments that result from pharmacological blockade of protein synthesis, suggesting that protein degradation may control the requirement for protein synthesis during the memory storage process. Results such as these suggest that protein degradation and synthesis are both critical for LTM formation and may interact to properly consolidate and store memories in the brain. Here, we review the evidence implicating protein synthesis and degradation in LTM storage and highlight the areas of overlap between these two opposing processes. We also discuss evidence suggesting these two processes may interact to properly form and store memories. LTM storage likely requires a coordinated regulation between protein degradation and synthesis at multiple sites in the mammalian brain.

  9. Effects of inorganic anions on carbon isotope fractionation during Fenton-like degradation of trichloroethene

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Yunde [State Key Laboratory of Biogeology and Environmental Geology, China University of Geosciences, Wuhan 430074 (China); School of Environmental Studies, China University of Geosciences, Wuhan 430074 (China); Laboratory of Basin Hydrology and Wetland Eco-restoration, China University of Geosciences, Wuhan 430074 (China); Zhou, Aiguo, E-mail: aiguozhou@cug.edu.cn [State Key Laboratory of Biogeology and Environmental Geology, China University of Geosciences, Wuhan 430074 (China); School of Environmental Studies, China University of Geosciences, Wuhan 430074 (China); Gan, Yiqun; Li, Xiaoqian [State Key Laboratory of Biogeology and Environmental Geology, China University of Geosciences, Wuhan 430074 (China); School of Environmental Studies, China University of Geosciences, Wuhan 430074 (China)

    2016-05-05

    Highlights: • The effect of inorganic anions on carbon isotope fractionation was evaluated. • The enrichment factors was independent concentration of NO{sub 3}{sup −}, or SO{sub 4}{sup 2−}. • Cl{sup −} significantly influenced the carbon isotope fractionation. - Abstract: Understanding the magnitude and variability in isotope fractionation with respect to specific processes is crucial to the application of stable isotopic analysis as a tool to infer and quantify transformation processes. The variability of carbon isotope fractionation during Fenton-like degradation of trichloroethene (TCE) in the presence of different inorganic ions (nitrate, sulfate, and chloride), was investigated to evaluate the potential effects of inorganic anions on carbon isotope enrichment factor (ε value). A comparison of ε values obtained in deionized water, nitrate solution, and sulfate solution demonstrated that the ε values were identical and not affected by the presence of nitrate and sulfate. In the presence of chloride, however, the ε values (ranging from −6.3 ± 0.8 to 10 ± 1.3‰) were variable and depended on the chloride concentration, indicating that chloride could significantly affect carbon isotope fractionation during Fenton-like degradation of TCE. Thus, caution should be exercised in selecting appropriate ε values for the field application of stable isotope analysis, as various chloride concentrations may be present due to naturally present or introduced with pH adjustment and iron salts during Fenton-like remediation. Furthermore, the effects of chloride on carbon isotope fractionation may be able to provide new insights about reaction mechanisms of Fenton-like processes.

  10. Effects of inorganic anions on carbon isotope fractionation during Fenton-like degradation of trichloroethene

    International Nuclear Information System (INIS)

    Liu, Yunde; Zhou, Aiguo; Gan, Yiqun; Li, Xiaoqian

    2016-01-01

    Highlights: • The effect of inorganic anions on carbon isotope fractionation was evaluated. • The enrichment factors was independent concentration of NO_3"−, or SO_4"2"−. • Cl"− significantly influenced the carbon isotope fractionation. - Abstract: Understanding the magnitude and variability in isotope fractionation with respect to specific processes is crucial to the application of stable isotopic analysis as a tool to infer and quantify transformation processes. The variability of carbon isotope fractionation during Fenton-like degradation of trichloroethene (TCE) in the presence of different inorganic ions (nitrate, sulfate, and chloride), was investigated to evaluate the potential effects of inorganic anions on carbon isotope enrichment factor (ε value). A comparison of ε values obtained in deionized water, nitrate solution, and sulfate solution demonstrated that the ε values were identical and not affected by the presence of nitrate and sulfate. In the presence of chloride, however, the ε values (ranging from −6.3 ± 0.8 to 10 ± 1.3‰) were variable and depended on the chloride concentration, indicating that chloride could significantly affect carbon isotope fractionation during Fenton-like degradation of TCE. Thus, caution should be exercised in selecting appropriate ε values for the field application of stable isotope analysis, as various chloride concentrations may be present due to naturally present or introduced with pH adjustment and iron salts during Fenton-like remediation. Furthermore, the effects of chloride on carbon isotope fractionation may be able to provide new insights about reaction mechanisms of Fenton-like processes.

  11. Degradation of Akt using protein-catalyzed capture agents.

    Science.gov (United States)

    Henning, Ryan K; Varghese, Joseph O; Das, Samir; Nag, Arundhati; Tang, Grace; Tang, Kevin; Sutherland, Alexander M; Heath, James R

    2016-04-01

    Abnormal signaling of the protein kinase Akt has been shown to contribute to human diseases such as diabetes and cancer, but Akt has proven to be a challenging target for drugging. Using iterative in situ click chemistry, we recently developed multiple protein-catalyzed capture (PCC) agents that allosterically modulate Akt enzymatic activity in a protein-based assay. Here, we utilize similar PCCs to exploit endogenous protein degradation pathways. We use the modularity of the anti-Akt PCCs to prepare proteolysis targeting chimeric molecules that are shown to promote the rapid degradation of Akt in live cancer cells. These novel proteolysis targeting chimeric molecules demonstrate that the epitope targeting selectivity of PCCs can be coupled with non-traditional drugging moieties to inhibit challenging targets. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

  12. Impact of protein uptake and degradation on recombinant protein secretion in yeast

    DEFF Research Database (Denmark)

    Tyo, Keith E. J.; Liu, Zihe; Magnusson, Ylva

    2014-01-01

    Protein titers, a key bioprocessing metric, depend both on the synthesis of protein and the degradation of protein. Secreted recombinant protein production in Saccharomyces cerevisiae is an attractive platform as minimal media can be used for cultivation, thus reducing fermentation costs...... and transcriptomics, we identify metabolic and regulatory markers that are consistent with uptake of whole proteins by endocytosis, followed by intracellular degradation and catabolism of substituent amino acids. Uptake and degradation of recombinant protein products may be common in S. cerevisiae protein secretion...... and simplifying downstream purification, compared to other systems that require complex media. As such, engineering S. cerevisiae to improve titers has been then the subject of significant attention, but the majority of previous efforts have been focused on improving protein synthesis. Here, we characterize...

  13. PROTEIN FRACTIONS AND IN VITRO FERMENTATION OF PROTEIN FEEDS FOR RUMINANTS

    Directory of Open Access Journals (Sweden)

    Angel L. Guevara-Mesa

    2011-05-01

    Full Text Available The objective of this study was to evaluate 20 protein feeds grouped in forages, vegetal by- products and animal by-products used for ruminant diets. Protein fractions (PF: A, non-protein nitrogen (NPN; B1, buffer-soluble protein; B2, buffer-insoluble, NDF-soluble protein; B3, NDF-insoluble, ADF-soluble protein; and C, ADF-insoluble protein, were determined for each ingredient.  Protein composition was correlated with total gas production in vitro (GP, gas production rate (S, lag time (L, DM disappearance (DMDIV and residual protein (RPIV. The completely randomised designed was analysed using mixed proc. and Tukey contrasts. Forages contained 18.29, 7.86, 66.00, 2.96, 4.89% of fractions A, B1, B2, B3 and C, respectively. Vegetable by-products contained 22.55, 4.55, 59.51, 8.84, 4.55% of each fraction, in the same order. Animal by-products contained 19.13, 4.52, 70.24, 3.74, 2.37% of each fraction, in the same order. Vetch, wheat bran and poultry litter had the greatest Vmax in each group. Vmax was correlated (P≤0.01 with total protein (r = -0.45, ADF (r = 0.27 and DMDIV (r = 0.61. In conclusion, there were differences in protein composition and kinetics of in vitro gas production among ingredients.

  14. In situ ruminal degradability of protein feeds with distinct physical ...

    African Journals Online (AJOL)

    Journal Home > Vol 47, No 1 (2017) > ... of protein feeds with different physical forms (meal versus grain) through a meta-analysis study. ... grains with the meal forms of soybean, peanut, sunflower, cottonseed, and corn gluten. The degradation parameters of dry matter did not differ significantly between meals and grains.

  15. Rate and extent of ruminal degradation of crude protein from ...

    African Journals Online (AJOL)

    Predicted crude protein degradation was calculated at rate constants for outflow of 0.04 and 0.06/h respect- ively. ... as buffers, an ionophore and an antibiotic according to general .... the non-bird resistant ('sweet') varieties. Ruminal .... have been affected by both the particle type and the math- ematical model we used.

  16. Endocytosis and intracellular protein degradation in cystic fibrosis fibroblasts

    International Nuclear Information System (INIS)

    Jessup, W.; Dean, R.T.

    1983-01-01

    Normal rates of pinocytosis of [ 3 H]sucrose were measured in cystic fibrosis fibroblasts, and were not affected by the addition of cystic fibrosis serum. Bulk protein degradation (a significant proportion of which occurs intralysosomally following autophagy) and its regulation by growth state were apparently identical in normal and cystic fibrosis cultures. (Auth.)

  17. Proteasomal and lysosomal protein degradation and heart disease.

    Science.gov (United States)

    Wang, Xuejun; Robbins, Jeffrey

    2014-06-01

    In the cell, the proteasome and lysosomes represent the most important proteolytic machineries, responsible for the protein degradation in the ubiquitin-proteasome system (UPS) and autophagy, respectively. Both the UPS and autophagy are essential to protein quality and quantity control. Alterations in cardiac proteasomal and lysosomal degradation are remarkably associated with most heart disease in humans and are implicated in the pathogenesis of congestive heart failure. Studies carried out in animal models and in cell culture have begun to establish both sufficiency and, in some cases, the necessity of proteasomal functional insufficiency or lysosomal insufficiency as a major pathogenic factor in the heart. This review article highlights some recent advances in the research into proteasome and lysosome protein degradation in relation to cardiac pathology and examines the emerging evidence for enhancing degradative capacities of the proteasome and/or lysosome as a new therapeutic strategy for heart disease. This article is part of a Special Issue entitled "Protein Quality Control, the Ubiquitin Proteasome System, and Autophagy". Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. Establishment of a ruminal protein degradation data base for dairy ...

    African Journals Online (AJOL)

    Establishment of a ruminal protein degradation data base for dairy cattle using the in situ polyester bag technique. 2. Energy sources. LJ Erasmus, J Prinsloo, PM Botha, HH Meissner. Abstract. No Abstract. Full Text: EMAIL FREE FULL TEXT EMAIL FREE FULL TEXT · DOWNLOAD FULL TEXT DOWNLOAD FULL TEXT.

  19. Establishment of a ruminal protein degradation data base for dairy ...

    African Journals Online (AJOL)

    Establishment of a ruminal protein degradation data base for dairy cattle using the in situ polyester bag technique. 3. Roughages. LJ Erasmus, J Prinsloo, PM Botha, HH Meissner. Abstract. No Abstract. Full Text: EMAIL FREE FULL TEXT EMAIL FREE FULL TEXT · DOWNLOAD FULL TEXT DOWNLOAD FULL TEXT.

  20. degradable protein sources on performance of high-producing dairy ...

    African Journals Online (AJOL)

    with high-quality, low·degradable protein sources prOViding47% UDP is advocated for ... saliva and through the rumen wall (Waldo, 1968). Based on this type of ... of the feed industry, but is based on very little solid evidence. (Huber, 1984). Chalupa ...... of rumen fermentation in relation to ammonia concentration. Br. J. Nutr.

  1. Degradation of Crude Protein in Groundnut Cake, Guinea Grass ...

    African Journals Online (AJOL)

    Three West African dwarf rams fitted with rumen cannula, were used in a completely randomized design for degradation of crude protein (CP) of groundnut cake (GNC), Panicum maximum, rumen epithelial scraping (RES), and diets containing increasing levels of RES. Concentrate diets were formulated such that 0% (A), ...

  2. Light-induced protein degradation in human-derived cells.

    Science.gov (United States)

    Sun, Wansheng; Zhang, Wenyao; Zhang, Chao; Mao, Miaowei; Zhao, Yuzheng; Chen, Xianjun; Yang, Yi

    2017-05-27

    Controlling protein degradation can be a valuable tool for posttranslational regulation of protein abundance to study complex biological systems. In the present study, we designed a light-switchable degron consisting of a light oxygen voltage (LOV) domain of Avena sativa phototropin 1 (AsLOV2) and a C-terminal degron. Our results showed that the light-switchable degron could be used for rapid and specific induction of protein degradation in HEK293 cells by light in a proteasome-dependent manner. Further studies showed that the light-switchable degron could also be utilized to mediate the degradation of secreted Gaussia princeps luciferase (GLuc), demonstrating the adaptability of the light-switchable degron in different types of protein. We suggest that the light-switchable degron offers a robust tool to control protein levels and may serves as a new and significant method for gene- and cell-based therapies. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Effect of Electron Beam Irradiation on Degradability Coefficients and Ruminalpostruminal Digestibility of Dry Matter and Crude Protein of some Plant Protein Sources

    Directory of Open Access Journals (Sweden)

    gasem tahan

    2016-06-01

    Full Text Available Effect of electron beam irradiation on degradability coefficients and ruminal- postruminal digestibility of dry matter and crude protein of soybean meal, canola meal and Lathyrus sativus seed, irradiated at doses of 50, 100 and 150 kGy was investigated. Ruminal degradability of dry matter and crude protein was determined by in situ method using two cannulated Holstein heifers. Ruminal- postruminal digestibility of dry matter and crude protein was determined by in situ (nylon bag-in vitro (daisy digestor techniques. Data analyzed using SAS software as randomized completely design and the treatment means were compared using Tukey test. The results indicated that irradiation had no effect on dry matter, ether extract and ash content of feeds. In soybean meal, washout fraction and potentially degradable fraction of dry matter and crude protein was higher and lower at dose of 150 kGy irradiation than other treatments, respectively, and degradation rate constant and ruminal effective degradability of dry matter and crude protein was lower at all doses of irradiation than untreated soybean meal. In canola meal, irradiation at doses of 50 and 100 kGy decreased washout fraction and increased potentially degradable fraction of crude protein compared with untreated canola meal. In Lathyrus sativus seed, only potentially degradable fraction of dry matter and crude protein was lower at dose of 150 kGy irradiation than untreated Lathyrus sativus seed. Ruminal digestibility of crude protein decreased in soybean meal at doses of 100 and 150 kGy irradiation and for canola meal at all doses of irradiation than untreated samples. Total tract digestibility of crude protein decreased in soybean meal at dose of 150 kGy irradiation and for canola meal at all doses of irradiation than untreated samples. In Lathyrus sativus seed, ruminal-postruminal digestibility and total tract digestibility of dry matter increased at doses of 100 and 150 kGy irradiation than untreated

  4. A critical role for protein degradation in the nucleus accumbens core in cocaine reward memory.

    Science.gov (United States)

    Ren, Zhen-Yu; Liu, Meng-Meng; Xue, Yan-Xue; Ding, Zeng-Bo; Xue, Li-Fen; Zhai, Suo-Di; Lu, Lin

    2013-04-01

    The intense associative memories that develop between cocaine-paired contexts and rewarding stimuli contribute to cocaine seeking and relapse. Previous studies have shown impairment in cocaine reward memories by manipulating a labile state induced by memory retrieval, but the mechanisms that underlie the destabilization of cocaine reward memory are unknown. In this study, using a Pavlovian cocaine-induced conditioned place preference (CPP) procedure in rats, we tested the contribution of ubiquitin-proteasome system-dependent protein degradation in destabilization of cocaine reward memory. First, we found that polyubiquitinated protein expression levels and polyubiquitinated N-ethylmaleimide-sensitive fusion (NSF) markedly increased 15 min after retrieval while NSF protein levels decreased 1 h after retrieval in the synaptosomal membrane fraction in the nucleus accumbens (NAc) core. We then found that infusion of the proteasome inhibitor lactacystin into the NAc core prevented the impairment of memory reconsolidation induced by the protein synthesis inhibitor anisomycin and reversed the effects of anisomycin on NSF and glutamate receptor 2 (GluR2) protein levels in the synaptosomal membrane fraction in the NAc core. We also found that lactacystin infusion into the NAc core but not into the shell immediately after extinction training sessions inhibited CPP extinction and reversed the extinction training-induced decrease in NSF and GluR2 in the synaptosomal membrane fraction in the NAc core. Finally, infusions of lactacystin by itself into the NAc core immediately after each training session or before the CPP retrieval test had no effect on the consolidation and retrieval of cocaine reward memory. These findings suggest that ubiquitin-proteasome system-dependent protein degradation is critical for retrieval-induced memory destabilization.

  5. In vitro estimation of rumen protein degradability using 35S to label the bacterial mass

    International Nuclear Information System (INIS)

    Khristov, A.; Aleksandrov, S.; Aleksiev, I.

    1994-01-01

    An experiment was carried out in order to simplify a previously developed 15 N-method for in vitro estimation of rumen protein degradability. Casein (Cas), whole soybeans (Sb) heated at 120 o C for 20 min (SbTherm) and sunflower (Sfl) were incubated at 39 o C for 4 hours in a water bathshaker with the following media: McDougall's buffer, strained and enriched with particle associated bacteria rumen fluid (2:1), rapidly (maltose, sucrose, glucose) and more slowly (pectin, soluble starch) degradable carbohydrates with final concentration of 815 mg/100 ml and 21.7 μCi/100 ml of 35 S (from Na 2 35 SO 4 ). After the incubation had been ceased, a bacterial fraction was isolated through differential centrifugation and specific activity of bacterial (Bac) and high speed total solids (TS) nitrogen was measured. The ratio was used to calculate bacterial mass in TS and through the Kjeldahl nitrogen concentration in TS - the net bacterial growth (against control vessels without protein). The level of ammonia-N in the supernate after blank correction was used to find the ammonia-N released from protein degradation. The data showed that the rate (and extend) of degradation for the Cas (as a standard protein) was lower compared to those obtained through the 15 N-method but it was higher than the rate derived through another in vitro method. The Cas equivalent of the Sb was higher than the figure we found in a previous experiment with solvent extracted soybean meal suggesting that the 35 S-method underestimated the degradability of the Cas. After being tested on a wider range of foodstuffs, the proposed 35 S-method might be considered as an alternative procedure which is less laborous than the 15 N-method. (author)

  6. Sulfur isotopic fractionation of carbonyl sulfide during degradation by soil bacteria and enzyme

    Science.gov (United States)

    Kamezaki, Kazuki; Hattori, Shohei; Ogawa, Takahiro; Toyoda, Sakae; Kato, Hiromi; Katayama, Yoko; Yoshida, Naohiro

    2017-04-01

    Carbonyl sulfide (COS) is an atmospheric trace gas that possess great potential for tracer of carbon cycle (Campbell et al., 2008). COS is taken up by vegetation during photosynthesis like absorption of carbon dioxide but COS can not emit by respiration of vegetation, suggesting possible tracer for gross primary production. However, some studies show the COS-derived GPP is larger than the estimates by using carbon dioxide flux because COS flux by photolysis and soil flux are not distinguished (e.g. Asaf et al., 2013). Isotope analysis is a useful tool to trace sources and transformations of trace gases. Recently our group developed a promising new analytical method for measuring the stable sulfur isotopic compositions of COS using nanomole level samples: the direct isotopic analytical technique of on-line gas chromatography-isotope ratio mass spectrometry (GC-IRMS) using fragmentation ions S+ enabling us to easily analyze sulfur isotopes in COS (Hattori et al., 2015). Soil is thought to be important as both a source and a sink of COS in the troposphere. In particular, soil has been reported as a large environmental sink for atmospheric COS. Bacteria isolated from various soils actively degrade COS, with various enzymes such as carbonic anhydrase and COSase (Ogawa et al., 2013) involved in COS degradation. However, the mechanism and the magnitude of bacterial contribution in terms of a sink for atmospheric COS is still uncertain. Therefore, it is important to quantitatively evaluate this contribution using COS sulfur isotope analysis. We present isotopic fractionation constants for COS by laboratory incubation experiments during degradation by soil bacteria and COSase. Incubation experiments were conducted using strains belonging to the genera Mycobacterium, Williamsia, Cupriavidus, and Thiobacillus, isolated from natural soil or activated sludge and enzyme purified from a bacteria. As a result, the isotopic compositions of OCS were increased during degradation of

  7. Protein aggregation and degradation during iodine labeling and its consequences for protein adsorption to biomaterials

    DEFF Research Database (Denmark)

    Holmberg, Maria; Jensen, Karin Bagger Stibius; Ndoni, Sokol

    2007-01-01

    Protein adsorption on modified and unmodified polymer surfaces investigated through radiolabeling experiments showed a tendency for higher than expected albumin and immunoglobulin G (IgG) adsorption. Possible enhanced protein aggregation and degradation caused by the iodine labeling method used w...

  8. Direct ubiquitin independent recognition and degradation of a folded protein by the eukaryotic proteasomes-origin of intrinsic degradation signals.

    Directory of Open Access Journals (Sweden)

    Amit Kumar Singh Gautam

    Full Text Available Eukaryotic 26S proteasomes are structurally organized to recognize, unfold and degrade globular proteins. However, all existing model substrates of the 26S proteasome in addition to ubiquitin or adaptor proteins require unstructured regions in the form of fusion tags for efficient degradation. We report for the first time that purified 26S proteasome can directly recognize and degrade apomyoglobin, a globular protein, in the absence of ubiquitin, extrinsic degradation tags or adaptor proteins. Despite a high affinity interaction, absence of a ligand and presence of only helices/loops that follow the degradation signal, apomyoglobin is degraded slowly by the proteasome. A short floppy F-helix exposed upon ligand removal and in conformational equilibrium with a disordered structure is mandatory for recognition and initiation of degradation. Holomyoglobin, in which the helix is buried, is neither recognized nor degraded. Exposure of the floppy F-helix seems to sensitize the proteasome and primes the substrate for degradation. Using peptide panning and competition experiments we speculate that initial encounters through the floppy helix and additional strong interactions with N-terminal helices anchors apomyoglobin to the proteasome. Stabilizing helical structure in the floppy F-helix slows down degradation. Destabilization of adjacent helices accelerates degradation. Unfolding seems to follow the mechanism of helix unraveling rather than global unfolding. Our findings while confirming the requirement for unstructured regions in degradation offers the following new insights: a origin and identification of an intrinsic degradation signal in the substrate, b identification of sequences in the native substrate that are likely to be responsible for direct interactions with the proteasome, and c identification of critical rate limiting steps like exposure of the intrinsic degron and destabilization of an unfolding intermediate that are presumably

  9. Model for Stress-induced Protein Degradation in Lemna minor1

    Science.gov (United States)

    Cooke, Robert J.; Roberts, Keith; Davies, David D.

    1980-01-01

    Transfer of Lemna minor fronds to adverse or stress conditions produces a large increase in the rate of protein degradation. Cycloheximide partially inhibits stress-induced protein degradation and also partially inhibits the protein degradation which occurs in the absence of stress. The increased protein degradation does not appear to be due to an increase in activity of soluble proteolytic enzymes. Biochemical evidence indicates that stress, perhaps acting via hormones, affects the permeability of certain membranes, particularly the tonoplast. A general model for stress-induced protein degradation is presented in which changes in membrane properties allow vacuolar proteolytic enzymes increased access to cytoplasmic proteins. PMID:16661588

  10. Sequential Isolation of Saturated, Aromatic, Resinic and Asphaltic Fractions Degrading Bacteria from Oil Contaminated Soil in South Sumatera

    Directory of Open Access Journals (Sweden)

    Pingkan Aditiawati

    2012-04-01

    Full Text Available Sequential isolation has been conducted to obtain isolates of saturated, aromatic, resin, and asphaltene fractions degrading bacteria from oil contaminated sites. Five soil samples were collected from South Sumatera. These were analyzed using soil extract medium enriched with oil recovery or Remaining-Oil recovery Degradated (ROD as sole carbon and energy sources according to the isolation stage. ROD at the end of every isolation stage analyzed oil fractions by use of the SARA analysis method. Six isolates of bacteria have been selected, one isolate was fraction saturates degrading bacteria that are Mycobacterium sp. T1H2D4-7 at degradation rate 0.0199 mgs/h with density 8.4x106 cfu/g from stage I. The isolate T2H1D2-4, identified as Pseudomonas sp. was fraction aromatics degrading bacteria at accelerate 0.0141 mgs/h with density 5.1x106 cfu/g are obtained at stage II. Two isolates namely Micrococcus sp. T3H2D4-2 and Pseudomonas sp. T1H1D5-5 were fraction resins degrading bacteria by accelerate 0.0088 mgs/h at density 5.6x106 cfu/g and 0.0089 mgs/h at density 5.7x106 cfu/g are obtained at stage III. Isolation of stage IV has been obtained two isolates Pseudomonas sp. T4H1D3-1and Pseudomonas sp. T4H3D5-4 were fraction asphaltenes degrading bacteria by accelerate 0.0057 mgs/h at density 5.6x106 cfu/g and accelerate 0.0058 mgs/h at density 5.7x106 cfu/g.

  11. Simulation of dual carbon-bromine stable isotope fractionation during 1,2-dibromoethane degradation.

    Science.gov (United States)

    Jin, Biao; Nijenhuis, Ivonne; Rolle, Massimo

    2018-06-01

    We performed a model-based investigation to simultaneously predict the evolution of concentration, as well as stable carbon and bromine isotope fractionation during 1,2-dibromoethane (EDB, ethylene dibromide) transformation in a closed system. The modelling approach considers bond-cleavage mechanisms during different reactions and allows evaluating dual carbon-bromine isotopic signals for chemical and biotic reactions, including aerobic and anaerobic biological transformation, dibromoelimination by Zn(0) and alkaline hydrolysis. The proposed model allowed us to accurately simulate the evolution of concentrations and isotope data observed in a previous laboratory study and to successfully identify different reaction pathways. Furthermore, we illustrated the model capabilities in degradation scenarios involving complex reaction systems. Specifically, we examined (i) the case of sequential multistep transformation of EDB and the isotopic evolution of the parent compound, the intermediate and the reaction product and (ii) the case of parallel competing abiotic pathways of EDB transformation in alkaline solution.

  12. Nutritional quality and fractionation of carbohydrates and protein in the forage components of an intensive silvopastoral system

    International Nuclear Information System (INIS)

    Gaviria, Xiomara; Rivera, J.E.; Barahona, R.

    2015-01-01

    The objective of this study was to evaluate the nutritional quality of the forage components of a SPSi based on Leucaena leucocephala associated to improved pastures, as well as its biomass production. The forage production was determined at several moments of the year and the nutritional quality was evaluated through the Cornell model. The soluble protein proportion (fraction A) was similar between the grasses and L. leucocephala, and represented as minimum 34 % of the total protein. The proportion of protein B2 (intermediate degradation) of the legume was higher than that of the grasses (53,7 vs. 30,2 %, respectively). Protein B3 of the diet (slow degradation) was around 22 % of the total protein, and more than 71 % of it can be considered degradable in rumen. L. leucocephala showed a higher concentration of soluble carbohydrates (16,7 %) and lower quantity of fraction B2 (14,94 %) than the grasses. Concerning the biomass availability, a production of 19,26 t DM/ha year-1 was reached. It is concluded that in SPSis a high quantity of quality forage is produced throughout the year, and that this offer is sufficient to cover the requirements of ruminants. (author)

  13. The relationship between protein synthesis and protein degradation in object recognition memory.

    Science.gov (United States)

    Furini, Cristiane R G; Myskiw, Jociane de C; Schmidt, Bianca E; Zinn, Carolina G; Peixoto, Patricia B; Pereira, Luiza D; Izquierdo, Ivan

    2015-11-01

    For decades there has been a consensus that de novo protein synthesis is necessary for long-term memory. A second round of protein synthesis has been described for both extinction and reconsolidation following an unreinforced test session. Recently, it was shown that consolidation and reconsolidation depend not only on protein synthesis but also on protein degradation by the ubiquitin-proteasome system (UPS), a major mechanism responsible for protein turnover. However, the involvement of UPS on consolidation and reconsolidation of object recognition memory remains unknown. Here we investigate in the CA1 region of the dorsal hippocampus the involvement of UPS-mediated protein degradation in consolidation and reconsolidation of object recognition memory. Animals with infusion cannulae stereotaxically implanted in the CA1 region of the dorsal hippocampus, were exposed to an object recognition task. The UPS inhibitor β-Lactacystin did not affect the consolidation and the reconsolidation of object recognition memory at doses known to affect other forms of memory (inhibitory avoidance, spatial learning in a water maze) while the protein synthesis inhibitor anisomycin impaired the consolidation and the reconsolidation of the object recognition memory. However, β-Lactacystin was able to reverse the impairment caused by anisomycin on the reconsolidation process in the CA1 region of the hippocampus. Therefore, it is possible to postulate a direct link between protein degradation and protein synthesis during the reconsolidation of the object recognition memory. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Antioxidant activity of cod (Gadus morhua) protein hydrolysates: Fractionation and characterisation of peptide fractions

    DEFF Research Database (Denmark)

    Farvin Habebullah, Sabeena; Andersen, Lisa Lystbæk; Otte, Jeanette

    2016-01-01

    This study aimed to characterise peptide fractions (>5 kDa, 3–5 kDa and fractions were dominated by Ala, Gly, Glu and Ser. The total amino acid composition had high proportions of Lys, Ala...... and Glu. The 3–5 kDa and fractions were further fractionated by size exclusion chromatography. All sub-fractions showed high Fe2+ chelating activity. The DPPH radical-scavenging activity of the 3–5 kDa fraction was exerted mainly by one sub-fraction dominated by peptides with masses below 600 Da....... The DPPH radical-scavenging activity of the fraction was exerted by sub-fractions with low molecular weight. The highest reducing power was found in a sub-fraction containing peptides rich in Arg, Tyr and Phe. Both free amino acids and low molecular weight peptides thus seemed to contribute...

  15. Accelerated degradation of the D2 protein of photosystem II under ultraviolet radiation

    International Nuclear Information System (INIS)

    Jansen, M.A.K.; Edelman, M.; Greenberg, B.M.; Gaba, V.

    1996-01-01

    The D2 protein of photosystem II is relatively stable in vivo under photosynthetic active radiation, but its degradation accelerates under UVB radiation. Little is known about accelerated D2 protein degradation. We characterized wavelength dependence and sensitivity toward photosystem II inhibitors. The in vivo D2 degradation spectrum resembles the pattern for the rapidly turning over D1 protein of photosystem II, with rates being maximal in the UVB region. We propose that D2 degradation, like D1 degradation, is activated by distinct photosensitizers in the UVB and visible regions of the spectrum. In both wavelength regions, photosystem II inhibitors that are known to be targeted to the D1 protein affect D2 degradation. This suggests that degradation of the two proteins is coupled, D2 degradation being influenced by events occurring at the Q B niche on the D1 protein. (Author)

  16. Secretion of intact proteins and peptide fragments by lysosomal pathways of protein degradation

    International Nuclear Information System (INIS)

    Isenman, L.D.; Dice, J.F.

    1989-01-01

    We report that degradation of proteins microinjected into human fibroblasts is accompanied by release into the culture medium of peptide fragments and intact proteins as well as single amino acids. For the nine proteins and polypeptides microinjected, acid-precipitable radioactivity, i.e. peptide fragments and/or intact proteins, ranged from 10 to 67% of the total released radioactivity. Peptide fragments and/or intact protein accounted for 60% of the radioactivity released into the medium by cells microinjected with ribonuclease A. Two major radiolabeled peptide fragments were found, and one was of an appropriate size to function as an antigen in antigen-presenting cells. The peptides released from microinjected ribonuclease A were derived from lysosomal pathways of proteolysis based on several lines of evidence. Previous studies have shown that microinjected ribonuclease A is degraded to single amino acids entirely within lysosomes. We show that release of free amino acids and peptide fragments and/or intact protein was equivalently stimulated by serum deprivation and equivalently inhibited by NH4Cl. We also show that lysosomal degradation of endocytosed [3H]ribonuclease A was accompanied by the release of two peptide fragments similar in size and charge to those from microinjected [ 3 H]ribonuclease A. These findings demonstrate that degradation within lysosomes occurs in a manner that spares specific peptides; they also suggest a previously unsuspected pathway by which cells can secrete cytosol-derived polypeptides

  17. Autoantibodies to myelin basic protein catalyze site-specific degradation of their antigen.

    Science.gov (United States)

    Ponomarenko, Natalia A; Durova, Oxana M; Vorobiev, Ivan I; Belogurov, Alexey A; Kurkova, Inna N; Petrenko, Alexander G; Telegin, Georgy B; Suchkov, Sergey V; Kiselev, Sergey L; Lagarkova, Maria A; Govorun, Vadim M; Serebryakova, Marina V; Avalle, Bérangère; Tornatore, Pete; Karavanov, Alexander; Morse, Herbert C; Thomas, Daniel; Friboulet, Alain; Gabibov, Alexander G

    2006-01-10

    Autoantibody-mediated tissue destruction is among the main features of organ-specific autoimmunity. This report describes "an antibody enzyme" (abzyme) contribution to the site-specific degradation of a neural antigen. We detected proteolytic activity toward myelin basic protein (MBP) in the fraction of antibodies purified from the sera of humans with multiple sclerosis (MS) and mice with induced experimental allergic encephalomyelitis. Chromatography and zymography data demonstrated that the proteolytic activity of this preparation was exclusively associated with the antibodies. No activity was found in the IgG fraction of healthy donors. The human and murine abzymes efficiently cleaved MBP but not other protein substrates tested. The sites of MBP cleavage determined by mass spectrometry were localized within immunodominant regions of MBP. The abzymes could also cleave recombinant substrates containing encephalytogenic MBP(85-101) peptide. An established MS therapeutic Copaxone appeared to be a specific abzyme inhibitor. Thus, the discovered epitope-specific antibody-mediated degradation of MBP suggests a mechanistic explanation of the slow development of neurodegeneration associated with MS.

  18. Estimation of the fraction of biologically active methyl tert-butyl ether degraders in a heterogeneous biomass sample

    DEFF Research Database (Denmark)

    Waul, Christopher Kevin; Arvin, Erik; Schmidt, Jens Ejbye

    2008-01-01

    The fraction of biologically active methyl tert-butyl ether degraders in reactors is just as important for prediction of removal rates as knowledge of the kinetic parameters. The fraction of biologically active methyl tert-butyl ether degraders in a heterogeneous biomass sample, taken from a packed...... bed reactor, was determined using a batch kinetic based approach. The procedure involved modeling of methyl tert-butyl ether removal rates from batch experiments followed by parameter estimations. It was estimated to be 5-14% (w/w) of the measured volatile suspended solids concentration in the reactor....

  19. Reticulophagy and Ribophagy: Regulated Degradation of Protein Production Factories

    Directory of Open Access Journals (Sweden)

    Eduardo Cebollero

    2012-01-01

    Full Text Available During autophagy, cytosol, protein aggregates, and organelles are sequestered into double-membrane vesicles called autophagosomes and delivered to the lysosome/vacuole for breakdown and recycling of their basic components. In all eukaryotes this pathway is important for adaptation to stress conditions such as nutrient deprivation, as well as to regulate intracellular homeostasis by adjusting organelle number and clearing damaged structures. For a long time, starvation-induced autophagy has been viewed as a nonselective transport pathway; however, recent studies have revealed that autophagy is able to selectively engulf specific structures, ranging from proteins to entire organelles. In this paper, we discuss recent findings on the mechanisms and physiological implications of two selective types of autophagy: ribophagy, the specific degradation of ribosomes, and reticulophagy, the selective elimination of portions of the ER.

  20. Protein Degradation in Normal and Beige (Chediak-Higashi) Mice

    Science.gov (United States)

    Lyons, Robert T.; Pitot, Henry C.

    1978-01-01

    The beige mouse, C57BL/6 (bg/bg), is an animal model for the Chediak-Higashi syndrome in man, a disease characterized morphologically by giant lysosomes in most cell types. Half-lives for the turnover of [14C]bicarbonate-labeled total soluble liver protein were determined in normal and beige mice. No significant differences were observed between the normal and mutant strain for both rapidly and slowly turning-over classes of proteins. Glucagon treatment during the time-course of protein degradation had similar effects on both normal and mutant strains and led to the conclusion that the rate of turnover of endogenous intracellular protein in the beige mouse liver does not differ from normal. The rates of uptake and degradation of an exogenous protein were determined in normal and beige mice by intravenously injecting 125I-bovine serum albumin and following, in peripheral blood, the loss with time of phosphotungstic acid-insoluble bovine serum albumin and the parallel appearance of phosphotungstic acid-soluble (degraded) material. No significant differences were observed between beige and normal mice in the uptake by liver lysosomes of 125I-bovine serum albumin (t½ = 3.9 and 2.8 h, respectively). However, it was found that lysosomes from livers of beige mice released phosphotungstic acid-soluble radioactivity at a rate significantly slower than normal (t½ = 6.8 and 3.1 h, respectively). This defect in beige mice could be corrected by chronic administration of carbamyl choline (t½ = 3.5 h), a cholinergic agonist which raises intracellular cyclic GMP levels. However, no significant differences between normal and beige mice were observed either in the ability of soluble extracts of liver and kidney to bind [3H]cyclic GMP in vitro or in the basal levels of cyclic AMP in both tissues. The relevance of these observations to the presumed biochemical defect underlying the Chediak-Higashi syndrome is discussed. PMID:202611

  1. Mass spectrometry analysis of proteome-wide proteolytic post-translational degradation of proteins

    OpenAIRE

    Shen, Yufeng; Hixson, Kim K.; Tolić, Nikola; Camp, David G.; Purvine, Samuel O.; Moore, Ronald J.; Smith, Richard D.

    2008-01-01

    Protein proteolytic degradation is an essential component to proper cell function and its life cycle. Here, we study the protein degradation in yeast Saccharomyces cerevisiae cells on a proteome-wide scale by detection of the intermediate peptides produced from the intracellular degradation of proteins using sequencing-based tandem mass spectrometry. By tracing the detected ~1,100 peptides and their ~200 protein substrate origins we obtain evidence for new insights into the proteome-wide prot...

  2. Protein degradation rate is the dominant mechanism accounting for the differences in protein abundance of basal p53 in a human breast and colorectal cancer cell line.

    Directory of Open Access Journals (Sweden)

    Eszter Lakatos

    Full Text Available We determine p53 protein abundances and cell to cell variation in two human cancer cell lines with single cell resolution, and show that the fractional width of the distributions is the same in both cases despite a large difference in average protein copy number. We developed a computational framework to identify dominant mechanisms controlling the variation of protein abundance in a simple model of gene expression from the summary statistics of single cell steady state protein expression distributions. Our results, based on single cell data analysed in a Bayesian framework, lends strong support to a model in which variation in the basal p53 protein abundance may be best explained by variations in the rate of p53 protein degradation. This is supported by measurements of the relative average levels of mRNA which are very similar despite large variation in the level of protein.

  3. Protein degradation rate is the dominant mechanism accounting for the differences in protein abundance of basal p53 in a human breast and colorectal cancer cell line.

    Science.gov (United States)

    Lakatos, Eszter; Salehi-Reyhani, Ali; Barclay, Michael; Stumpf, Michael P H; Klug, David R

    2017-01-01

    We determine p53 protein abundances and cell to cell variation in two human cancer cell lines with single cell resolution, and show that the fractional width of the distributions is the same in both cases despite a large difference in average protein copy number. We developed a computational framework to identify dominant mechanisms controlling the variation of protein abundance in a simple model of gene expression from the summary statistics of single cell steady state protein expression distributions. Our results, based on single cell data analysed in a Bayesian framework, lends strong support to a model in which variation in the basal p53 protein abundance may be best explained by variations in the rate of p53 protein degradation. This is supported by measurements of the relative average levels of mRNA which are very similar despite large variation in the level of protein.

  4. Exploring the mechanical behavior of degrading swine neural tissue at low strain rates via the fractional Zener constitutive model.

    Science.gov (United States)

    Bentil, Sarah A; Dupaix, Rebecca B

    2014-02-01

    The ability of the fractional Zener constitutive model to predict the behavior of postmortem swine brain tissue was examined in this work. Understanding tissue behavior attributed to degradation is invaluable in many fields such as the forensic sciences or cases where only cadaveric tissue is available. To understand how material properties change with postmortem age, the fractional Zener model was considered as it includes parameters to describe brain stiffness and also the parameter α, which quantifies the viscoelasticity of a material. The relationship between the viscoelasticity described by α and tissue degradation was examined by fitting the model to data collected in a previous study (Bentil, 2013). This previous study subjected swine neural tissue to in vitro unconfined compression tests using four postmortem age groups (week). All samples were compressed to a strain level of 10% using two compressive rates: 1mm/min and 5mm/min. Statistical analysis was used as a tool to study the influence of the fractional Zener constants on factors such as tissue degradation and compressive rate. Application of the fractional Zener constitutive model to the experimental data showed that swine neural tissue becomes less stiff with increased postmortem age. The fractional Zener model was also able to capture the nonlinear viscoelastic features of the brain tissue at low strain rates. The results showed that the parameter α was better correlated with compressive rate than with postmortem age. © 2013 Published by Elsevier Ltd.

  5. In vitro anthelmintic effects of Spigelia anthelmia protein fractions against Haemonchus contortus.

    Directory of Open Access Journals (Sweden)

    Sandra Alves Araújo

    Full Text Available Gastrointestinal nematodes are a significant concern for animal health and well-being, and anthelmintic treatment is mainly performed through the use of chemical products. However, bioactive compounds produced by plants have shown promise for development as novel anthelmintics. The aim of this study is to assess the anthelmintic activity of protein fractions from Spigelia anthelmia on the gastrointestinal nematode Haemonchus contortus. Plant parts were separated into leaves, stems and roots, washed with distilled water, freeze-dried and ground into a fine powder. Protein extraction was performed with sodium phosphate buffer (75 mM, pH 7.0. The extract was fractionated using ammonium sulfate (0-90% and extensively dialyzed. The resulting fractions were named LPF (leaf protein fraction, SPF (stem protein fraction and RPF (root protein fraction, and the protein contents and activities of the fractions were analyzed. H. contortus egg hatching (EHA, larval exsheathment inhibition (LEIA and larval migration inhibition (LMIA assays were performed. Proteomic analysis was conducted, and high-performance liquid chromatography (HPLC chromatographic profiles of the fractions were established to identify proteins and possible secondary metabolites. S. anthelmia fractions inhibited H. contortus egg hatching, with LPF having the most potent effects (EC50 0.17 mg mL-1. During LEIA, SPF presented greater efficiency than the other fractions (EC50 0.25 mg mL-1. According to LMIA, the fractions from roots, stems and leaves also reduced the number of larvae, with EC50 values of 0.11, 0.14 and 0.21 mg mL-1, respectively. Protein analysis indicated the presence of plant defense proteins in the S. anthelmia fractions, including protease, protease inhibitor, chitinase and others. Conversely, secondary metabolites were absent in the S. anthemia fractions. These results suggest that S. anthelmia proteins are promising for the control of the gastrointestinal nematode H

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

    International Nuclear Information System (INIS)

    Yu, P.

    2007-01-01

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

  7. Investigation of the impact of trace elements on anaerobic volatile fatty acid degradation using a fractional factorial experimental design.

    Science.gov (United States)

    Jiang, Ying; Zhang, Yue; Banks, Charles; Heaven, Sonia; Longhurst, Philip

    2017-11-15

    The requirement of trace elements (TE) in anaerobic digestion process is widely documented. However, little is understood regarding the specific requirement of elements and their critical concentrations under different operating conditions such as substrate characterisation and temperature. In this study, a flask batch trial using fractional factorial design is conducted to investigate volatile fatty acids (VFA) anaerobic degradation rate under the influence of the individual and combined effect of six TEs (Co, Ni, Mo, Se, Fe and W). The experiment inoculated with food waste digestate, spiked with sodium acetate and sodium propionate both to 10 g/l. This is followed by the addition of a selection of the six elements in accordance with a 2 6-2 fractional factorial principle. The experiment is conducted in duplicate and the degradation of VFA is regularly monitored. Factorial effect analysis on the experimental results reveals that within these experimental conditions, Se has a key role in promoting the degradation rates of both acetic and propionic acids; Mo and Co are found to have a modest effect on increasing propionic acid degradation rate. It is also revealed that Ni shows some inhibitory effects on VFA degradation, possibly due to its toxicity. Additionally, regression coefficients for the main and second order effects are calculated to establish regression models for VFA degradation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Induced oligomerization targets Golgi proteins for degradation in lysosomes.

    Science.gov (United States)

    Tewari, Ritika; Bachert, Collin; Linstedt, Adam D

    2015-12-01

    Manganese protects cells against forms of Shiga toxin by down-regulating the cycling Golgi protein GPP130. Down-regulation occurs when Mn binding causes GPP130 to oligomerize and traffic to lysosomes. To determine how GPP130 is redirected to lysosomes, we tested the role of GGA1 and clathrin, which mediate sorting in the canonical Golgi-to-lysosome pathway. GPP130 oligomerization was induced using either Mn or a self-interacting version of the FKBP domain. Inhibition of GGA1 or clathrin specifically blocked GPP130 redistribution, suggesting recognition of the aggregated GPP130 by the GGA1/clathrin-sorting complex. Unexpectedly, however, GPP130's cytoplasmic domain was not required, and redistribution also occurred after removal of GPP130 sequences needed for its normal cycling. Therefore, to test whether aggregate recognition might be a general phenomenon rather than one involving a specific GPP130 determinant, we induced homo-oligomerization of two unrelated Golgi-targeted constructs using the FKBP strategy. These were targeted to the cis- and trans-Golgi, respectively, using domains from mannosidase-1 and galactosyltransferase. Significantly, upon oligomerization, each redistributed to peripheral punctae and was degraded. This occurred in the absence of detectable UPR activation. These findings suggest the unexpected presence of quality control in the Golgi that recognizes aggregated Golgi proteins and targets them for degradation in lysosomes. © 2015 Tewari et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  9. Rapid degradation of abnormal proteins in vacuoles from Acer pseudoplatanus L. cells

    International Nuclear Information System (INIS)

    Canut, H.; Alibert, G.; Carrasco, A.; Boudet, A.M.

    1986-01-01

    In Acer pseudoplatanus cells, the proteins synthesized in the presence of an amino acid analog ([ 14 C]p-fluorophenylalanine), were degraded more rapidly than normal ones ([ 14 C]phenylalanine as precursor). The degradation of an important part of these abnormal proteins occurred inside the vacuoles. The degradation process was not apparently associated to a specific proteolytic system but was related to a preferential transfer of these aberrant proteins from the cytoplasm to the vacuole

  10. Nanobody-Directed Specific Degradation of Proteins by the 26S-Proteasome in Plants

    OpenAIRE

    Baudisch, Bianca; Pfort, Ingrid; Sorge, Eberhard; Conrad, Udo

    2018-01-01

    Here, we present data showing the directed degradation of target proteins recognized by a specific nanobody in transgenic plants. Green fluorescent protein was depleted by a chimeric nanobody fused to a distinct F-box domain, which enables protein degradation via the ubiquitin proteasome pathway. This technique could thus be used to knock out other proteins of interest in planta using specific, high-affinity binding proteins.

  11. Nanobody-Directed Specific Degradation of Proteins by the 26S-Proteasome in Plants

    Directory of Open Access Journals (Sweden)

    Bianca Baudisch

    2018-02-01

    Full Text Available Here, we present data showing the directed degradation of target proteins recognized by a specific nanobody in transgenic plants. Green fluorescent protein was depleted by a chimeric nanobody fused to a distinct F-box domain, which enables protein degradation via the ubiquitin proteasome pathway. This technique could thus be used to knock out other proteins of interest in planta using specific, high-affinity binding proteins.

  12. Nanobody-Directed Specific Degradation of Proteins by the 26S-Proteasome in Plants.

    Science.gov (United States)

    Baudisch, Bianca; Pfort, Ingrid; Sorge, Eberhard; Conrad, Udo

    2018-01-01

    Here, we present data showing the directed degradation of target proteins recognized by a specific nanobody in transgenic plants. Green fluorescent protein was depleted by a chimeric nanobody fused to a distinct F-box domain, which enables protein degradation via the ubiquitin proteasome pathway. This technique could thus be used to knock out other proteins of interest in planta using specific, high-affinity binding proteins.

  13. GCK-MODY diabetes associated with protein misfolding, cellular self-association and degradation.

    Science.gov (United States)

    Negahdar, Maria; Aukrust, Ingvild; Johansson, Bente B; Molnes, Janne; Molven, Anders; Matschinsky, Franz M; Søvik, Oddmund; Kulkarni, Rohit N; Flatmark, Torgeir; Njølstad, Pål Rasmus; Bjørkhaug, Lise

    2012-11-01

    GCK-MODY, dominantly inherited mild fasting hyperglycemia, has been associated with >600 different mutations in the glucokinase (GK)-encoding gene (GCK). When expressed as recombinant pancreatic proteins, some mutations result in enzymes with normal/near-normal catalytic properties. The molecular mechanism(s) of GCK-MODY due to these mutations has remained elusive. Here, we aimed to explore the molecular mechanisms for two such catalytically 'normal' GCK mutations (S263P and G264S) in the F260-L270 loop of GK. When stably overexpressed in HEK293 cells and MIN6 β-cells, the S263P- and G264S-encoded mutations generated misfolded proteins with an increased rate of degradation (S263P>G264S) by the protein quality control machinery, and a propensity to self-associate (G264S>S263P) and form dimers (SDS resistant) and aggregates (partly Triton X-100 insoluble), as determined by pulse-chase experiments and subcellular fractionation. Thus, the GCK-MODY mutations S263P and G264S lead to protein misfolding causing destabilization, cellular dimerization/aggregation and enhanced rate of degradation. In silico predicted conformational changes of the F260-L270 loop structure are considered to mediate the dimerization of both mutant proteins by a domain swapping mechanism. Thus, similar properties may represent the molecular mechanisms for additional unexplained GCK-MODY mutations, and may also contribute to the disease mechanism in other previously characterized GCK-MODY inactivating mutations. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. Total proteins and protein fractions levels in pregnant rats subjected to whole-body gamma irradiation

    International Nuclear Information System (INIS)

    Mansour, M.A.; Roushdy, H.M.; Mazhar, F.M.; Abu-Gabal, H.A.

    1986-01-01

    A total number of 180 mature rats (120 females and 60 males) weighing from 120-140 g were used to study the effect of two doses (2 and 4 Gy) whole-body gamma irradiation on the level of total protein and protein fractions in serum of pregnant rats during the period of organogenesis. It was found that the levels of total protein, albumin and gamma globulins significantly decreased according to the doses of exposure. The levels of alpha and beta globulins significantly increased more in the serum of rats exposed to 2 Gy than in rats exposed to 4 Gy. The level of A/G ratio significantly decreased more in the serum of rats exposed to 2Gy than in those exposed to 4 Gy

  15. Characterization of immunogenic Clonorchis sinensis protein fractions by gel fitration chromatography

    Directory of Open Access Journals (Sweden)

    Duan Pham Ngoc

    2015-04-01

    Full Text Available Objective: To characterize immunogenic protein fraction of Clonorchis sinensis (C. sinensis by partial purification. Methods: A total of 30 hamsters were infected with 50 C. sinensis metacercariae, and then C. sinensis protein was purified by gel filtration chromatography. Indirect ELISA and immunoblot were used to detect the antibody in sera of hamsters infected with C. sinensis. Results: The gel filtration showed 2 peaks at high (fraction No. 10 to 14 and low (fraction No. 21 to 26 molecular weight proteins. Indirect ELISA showed that both antibodies of clonorchiasis and opisthorchiasis reacted strongly with early fractions (6 to 14 and the reaction was gradually reduced at middle and late fractions (15 to 50. Both antibodies showed different individual fraction of C. sinensis by immunoblot. It showed several protein bands that the 34 and 37 kDa were major proteins. The 53 kDa protein which was only found in the clonorchiasis reacted with fraction 20. Conclusions: The purified antigen of C. sinensis reacted similarly with both antibodies of clonorchiasis and opisthorchiasis where strong reaction was seen with early fractions. The C. sinensis protein fraction No. 20 may be useful for immunodiagnosis of clonorchiasis.

  16. DEGRADABILIDADE IN SITU DA MATÉRIA SECA, DA PROTEÍNA BRUTA E DA FRAÇÃO FIBROSA DE CONCENTRADOS E SUBPRODUTOS AGROINDUSTRIAIS IN SITU DEGRADABILITY OF DRY MATTER, CRUDE PROTEIN AND FIBROUS FRACTION OF CONCENTRATE AND AGROINDUSTRIAL BY-PRODUCTS

    Directory of Open Access Journals (Sweden)

    Gleidson Giordano Pinto de Carvalho Carvalho

    2009-09-01

    Full Text Available Objetivou-se avaliar a degradabilidade ruminal da matéria seca (MS, da proteína bruta (PB, da fibra em detergente neutro (FDN e da fibra em detergente ácido (FDA do milho (Zea mays, do farelo de soja (Glicyne max L., da torta de dendê (Elaeis guineensis Jacq. e do farelo de cacau (Theobroma cacao L.. Incubaram-se amostras de cada alimento no rúmen de três novilhos por períodos de 0; 3; 6; 12; 24 e 48 horas. As degradabilidades efetivas da MS, PB, FDN e FDA, para a taxa de passagem de 5%/hora, foram relativamente baixas (abaixo de 60%, exceto para a PB do farelo de soja (acima de 65%. O farelo de soja apresentou os maiores coeficientes de degradação, tanto para MS e PB como também para os constituintes da parede celular, seguido do milho, torta de dendê e farelo de cacau. O farelo de cacau apresentou as menores taxas de degradação ruminal.

    PALAVRAS-CHAVES: Farelo de cacau, incubação ruminal, torta de dendê. The objective of the experiment was to evaluate the dry matter (DM, crude protein (CP, neutral detergent fiber (NDF and acid detergent fiber (ADF ruminal degradability of corn (Zea mays, soybean meal (Glicyne max L., palm kernel cake (Elaeis guineensis Jacq. and cocoa meal (Theobroma cacao L.. Samples of each feed were incubated in rumens of three steers for periods of 0; 3; 6; 12; 24 and 48 hours. The DM, CP, NDF and ADF effective degradabilities, for a passage rate of 5%/hour, were relatively low (lower than 60%, except for soybean meal CP (higher than 65%. Soybean meal showed the greatest degradation coefficients for DM and CP as so as for cellular wall constituents, followed by corn, palm kernel cake and cocoa meal. Cocoa meal showed the lowest ruminal degradation rates.

    KEY WORDS: Cocoa meal, incubation ruminal, palm kernel cake.

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

    Science.gov (United States)

    An economical and environmentally friendly whey protein fractionation process was developed using supercritical carbon dioxide (sCO2) as an acid to produce enriched fractions of alpha-lactalbumin (alpha-La) and beta-lactoglobulin (beta-Lg) from a commercial whey protein isolate (WPI) containing 55% ...

  18. Fractional rate of degradation (kd) of starch in the rumen and its relation to in vivo rumen and total digestibility

    DEFF Research Database (Denmark)

    Hvelplund, Torben; Larsen, Mogens; Lund, Peter

    2009-01-01

    in different ways both chemically and physically. The starch sources were fed in mixed diets together with grass silage and soya bean meal and allocated ad libitum to fistulated dairy cows. The starch content varied between 13 and 35% in ration dry matter for the different starch sources. The design...... was a series of cross-over experiments with two cows and two periods. Ruminal starch pool was estimated from rumen evacuation and starch flow was estimated by duodenal and faeces sampling. Fractional rate of rumen degradation was estimated from the equation [kd = rumen degraded/rumen pool] and rumen and total...

  19. Assessment of Bacterial Degradation of Aromatic Hydrocarbons in the Environment by Analysis of Stable Carbon Isotope Fractionation

    Energy Technology Data Exchange (ETDEWEB)

    Meckenstock, Rainer U. [Eberhard-Karls University of Tuebingen, Center for Applied Geoscience (Germany)], E-mail: rainer.meckenstock@uni-tuebingen.de; Morasch, Barbara [University of Konstanz, Faculty of Biology (Germany); Kaestner, Matthias; Vieth, Andrea; Richnow, Hans Hermann [Center for Environmental Research, Department of Remediation Research (Germany)

    2002-05-15

    {sup 13}C/{sup 12}C stable carbon isotope fractionation was used to assess biodegradation in contaminated aquifers with toluene as a model compound. Different strains of anaerobic bacteria (Thauera aromatica, Geobacter metallireducens, and the sulfate-reducing strain TRM1) showed consistent {sup 13}C/{sup 12}C carbon isotope fractionation with fractionation factors between {alpha}C = 1.0017 and 1.0018. In contrast, three cultures of aerobic organisms, using different mono- and dioxygenase enzyme systems to initiate toluene degradation, showed variable isotope fractionation factors of {alpha}C = 1.0027 (Pseudomonasputida strain mt-2), {alpha}C = 1.0011 (Ralstonia picketii), and{alpha}C = 1.0004 (Pseudomonas putida strain F1). The great variability of isotope fractionation between different aerobic bacterial strains suggests that interpretation of isotope data in oxic habitats can only be qualitative. A soil column was run as a model system for contaminated aquifers with toluene as the carbon source and sulfate as the electron acceptor and samples were taken at different ports along the column. Microbial toluene degradation was calculated based on the {sup 13}C/{sup 12}C isotope fractionation factors of the batch culture experiments together with the observed {sup 13}C/{sup 12}C isotope shifts of the residual toluene fractions. The calculated percentage of biodegradation, B, correlated well with the decreasing toluene concentrations at the sampling ports and indicated the increasing extent of biodegradation along the column. The theoretical toluene concentrations as calculated based on the isotope values matched the measured concentrations at the different sampling ports indicating that the Rayleigh equation can be used to calculate biodegradation in quasi closed systems based on measured isotope shifts. A similar attempt was performed to assess toluene degradation in a contaminated, anoxic aquifer. A transect of groundwater wells was monitored along the main

  20. Assessment of Bacterial Degradation of Aromatic Hydrocarbons in the Environment by Analysis of Stable Carbon Isotope Fractionation

    International Nuclear Information System (INIS)

    Meckenstock, Rainer U.; Morasch, Barbara; Kaestner, Matthias; Vieth, Andrea; Richnow, Hans Hermann

    2002-01-01

    13 C/ 12 C stable carbon isotope fractionation was used to assess biodegradation in contaminated aquifers with toluene as a model compound. Different strains of anaerobic bacteria (Thauera aromatica, Geobacter metallireducens, and the sulfate-reducing strain TRM1) showed consistent 13 C/ 12 C carbon isotope fractionation with fractionation factors between αC = 1.0017 and 1.0018. In contrast, three cultures of aerobic organisms, using different mono- and dioxygenase enzyme systems to initiate toluene degradation, showed variable isotope fractionation factors of αC = 1.0027 (Pseudomonasputida strain mt-2), αC = 1.0011 (Ralstonia picketii), andαC = 1.0004 (Pseudomonas putida strain F1). The great variability of isotope fractionation between different aerobic bacterial strains suggests that interpretation of isotope data in oxic habitats can only be qualitative. A soil column was run as a model system for contaminated aquifers with toluene as the carbon source and sulfate as the electron acceptor and samples were taken at different ports along the column. Microbial toluene degradation was calculated based on the 13 C/ 12 C isotope fractionation factors of the batch culture experiments together with the observed 13 C/ 12 C isotope shifts of the residual toluene fractions. The calculated percentage of biodegradation, B, correlated well with the decreasing toluene concentrations at the sampling ports and indicated the increasing extent of biodegradation along the column. The theoretical toluene concentrations as calculated based on the isotope values matched the measured concentrations at the different sampling ports indicating that the Rayleigh equation can be used to calculate biodegradation in quasi closed systems based on measured isotope shifts. A similar attempt was performed to assess toluene degradation in a contaminated, anoxic aquifer. A transect of groundwater wells was monitored along the main direction of the groundwater flow and revealed decreasing

  1. Influence of anabolic agents on protein synthesis and degradation in muscle cells grown in culture

    Energy Technology Data Exchange (ETDEWEB)

    Roeder, R.A.; Thorpe, S.D.; Byers, F.M.; Schelling, G.T.; Gunn, J.M.

    Muscle cell culture (L/sub 6/) studies were conducted to determine whether anabolic agents have a direct effect on the muscle cell. The effect of zeranol, testosterone propionate, estradiol benzoate, progesterone, dexamethasone and anabolic agent-dexamethasone combinations on protein synthesis and degradation were measured. Myoblast and myotube cultures were pretreated with 1 ..mu..M compounds for 12, 24 and 48 h before a 6-h synthesis or degradation measuring period. Protein synthesis was determined as cpm of (/sup 3/H) leucine incorporated per mg cell protein. Protein degradation was measured by a pulse-chase procedure using (/sup 3/H) leucine and expressed as the percentage labeled protein degraded in 6 h. Progesterone slightly increased protein synthesis in myoblast cultures. Testosterone propionate had no effect on synthesis. Protein synthesis was decreased by estradiol benzoate in myotube cultures. Protein degradation was not altered appreciably by anabolic agents. Protein synthesis was initially inhibited in myotubes by dexamethasone, but increased in myoblasts and myotubes in the extended incubation time. Dexamethasone also consistently increased protein degradation, but this required several hours to be expressed. Anabolic agents did not interfere with dexamethasone-induced increases in protein synthesis and degradation. The magnitude of response and sensitivity were similar for both the myoblast and the more fully differentiated myotube for all compounds tested. These results indicate that anabolic agents at the 1 ..mu..M level do not have a direct anabolic effect on muscle or alter glucocorticoid-induced catabolic response in muscle.

  2. Influence of anabolic agents on protein synthesis and degradation in muscle cells grown in culture

    International Nuclear Information System (INIS)

    Roeder, R.A.; Thorpe, S.D.; Byers, F.M.; Schelling, G.T.; Gunn, J.M.

    1986-01-01

    Muscle cell culture (L 6 ) studies were conducted to determine whether anabolic agents have a direct effect on the muscle cell. The effect of zeranol, testosterone propionate, estradiol benzoate, progesterone, dexamethasone and anabolic agent-dexamethasone combinations on protein synthesis and degradation were measured. Myoblast and myotube cultures were pretreated with 1 μM compounds for 12, 24 and 48 h before a 6-h synthesis or degradation measuring period. Protein synthesis was determined as cpm of [ 3 H] leucine incorporated per mg cell protein. Protein degradation was measured by a pulse-chase procedure using [ 3 H] leucine and expressed as the percentage labeled protein degraded in 6 h. Progesterone slightly increased protein synthesis in myoblast cultures. Testosterone propionate had no effect on synthesis. Protein synthesis was decreased by estradiol benzoate in myotube cultures. Protein degradation was not altered appreciably by anabolic agents. Protein synthesis was initially inhibited in myotubes by dexamethasone, but increased in myoblasts and myotubes in the extended incubation time. Dexamethasone also consistently increased protein degradation, but this required several hours to be expressed. Anabolic agents did not interfere with dexamethasone-induced increases in protein synthesis and degradation. The magnitude of response and sensitivity were similar for both the myoblast and the more fully differentiated myotube for all compounds tested. These results indicate that anabolic agents at the 1 μM level do not have a direct anabolic effect on muscle or alter glucocorticoid-induced catabolic response in muscle

  3. Biochemistry of cellulose degradation and cellulose utilization for feeds and for protein

    Energy Technology Data Exchange (ETDEWEB)

    Sadara, J C; Lachke, A H; Shewale, J G

    1979-01-01

    A review discussing production of single-cell protein, fuel, and glucose from cellulose decomposition; surface or solid fermentations of single-cell protein; production of cellulases; and the biochemistry of cellulose degradation was presented.

  4. The different roles of selective autophagic protein degradation in mammalian cells.

    Science.gov (United States)

    Wang, Da-wei; Peng, Zhen-ju; Ren, Guang-fang; Wang, Guang-xin

    2015-11-10

    Autophagy is an intracellular pathway for bulk protein degradation and the removal of damaged organelles by lysosomes. Autophagy was previously thought to be unselective; however, studies have increasingly confirmed that autophagy-mediated protein degradation is highly regulated. Abnormal autophagic protein degradation has been associated with multiple human diseases such as cancer, neurological disability and cardiovascular disease; therefore, further elucidation of protein degradation by autophagy may be beneficial for protein-based clinical therapies. Macroautophagy and chaperone-mediated autophagy (CMA) can both participate in selective protein degradation in mammalian cells, but the process is quite different in each case. Here, we summarize the various types of macroautophagy and CMA involved in determining protein degradation. For this summary, we divide the autophagic protein degradation pathways into four categories: the post-translational modification dependent and independent CMA pathways and the ubiquitin dependent and independent macroautophagy pathways, and describe how some non-canonical pathways and modifications such as phosphorylation, acetylation and arginylation can influence protein degradation by the autophagy lysosome system (ALS). Finally, we comment on why autophagy can serve as either diagnostics or therapeutic targets in different human diseases.

  5. Activation of chromatin degradation by a protein factor of thymocyte cytoplasm of irradiated mice

    International Nuclear Information System (INIS)

    Soldatenkov, V.A.; Filippovich, I.V.

    1986-01-01

    A cytoplasmic thymocyte fraction isolated 1 h after irradiation of mice accelerates chromatin degradation in isolated nuclei. Treatment of the cytoplasmic fraction by heat and injection of cycloheximide to mice prevent the acceleration of DNA degradation. The analysis of the chromatin degradation products and the kinetics of this process at acid and alkaline pH shows that activation of DNA degradation in thymocytes by a factor obtained from the irradiated cell cytoplasm is specific for a Ca 2+ , Mg 2+ -dependent enzyme. The time- and dose-dependent parameters of the appearance in the thymocyte cytoplasm of the factor influencing degradation of chromatin are in a good agreement with both the time of the onset of its postirradiation degradation and the dose dependence of this process

  6. Transient degradation of NF-κB proteins in macrophages after interaction with mast cell granules

    Directory of Open Access Journals (Sweden)

    Noriko Ito

    1998-01-01

    Full Text Available The exposure of the macrophage cell line, J774 to mast cell granules (MCG led to the form ation of altered nuclear transcription factor proteins (NFκBx, which had faster electrophoretic mobility than the p50 homodimer of NF-κB, but retained comparable DNA binding capacity. Antibodies to N-terminal peptides of p50, p52, p65 or c-Rel supershifted only a fraction of NF-κBx. Western blot analyses revealed that nuclear p65 and c-Rel were progressively degraded after exposure to MCG, whereas nuclear p50 appeared to be unaffected. In contrast, cytoplasmic p50, p65, c-Rel as well as IkBα remained intact after MCG treatment, although p52 was clearly degraded. In comparison to J774 cells, incubation of m ouse peritoneal macrophages with MCG resulted in more extensive alterations to NF-κB proteins. The alterations in NF-κB proteins did not affect the expression of inducible nitric oxide synthase (iNOS or TNF-α mRNA in J774 cells. These data indicate that exposure of J774 cells to MCG leads to generation of altered nuclear p52, p65 and c-Rel, which retain intact N-terminal peptides, specific oligonucleotide binding and transactivating activity. On the other hand, in peritoneal macrophages, MCG induce more extensive modifications to NF-κB proteins with associated inhibition of iNOS or TNF-α mRNA expression.

  7. Serum protein fractionation using supported molecular matrix electrophoresis.

    Science.gov (United States)

    Dong, Weijie; Matsuno, Yu-ki; Kameyama, Akihiko

    2013-08-01

    Supported molecular matrix electrophoresis (SMME), in which a hydrophilic polymer such as PVA serves as a support within a porous PVDF membrane, was recently developed. This method is similar to cellulose acetate membrane electrophoresis but differs in the compatibility to glycan analysis of the separated bands. In this report, we describe the first instance of the application of SMME to human serum fractionation, and demonstrate the differences with serum fractionation by cellulose acetate membrane electrophoresis. The SMME membrane exhibited almost no EOF during electrophoresis, unlike the cellulose acetate membrane, but afforded comparative results for serum fractionation. The visualization of each fraction was achieved by conventional staining with dye such as Direct Blue-71, and objective quantification was obtained by densitometry after inducing membrane transparency with 1-nonene. Immunostaining was also achieved. Moreover, mass spectrometric analysis of both N-linked and O-linked glycans from the separated bands was demonstrated. Serum fractionation and glycan profiling of each fraction using SMME will enable novel insights into the relationships between various glycosylation profiles and disease states. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. 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.

  9. 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.

  10. Degradation of misfolded proteins in neurodegenerative diseases: therapeutic targets and strategies.

    Science.gov (United States)

    Ciechanover, Aaron; Kwon, Yong Tae

    2015-03-13

    Mammalian cells remove misfolded proteins using various proteolytic systems, including the ubiquitin (Ub)-proteasome system (UPS), chaperone mediated autophagy (CMA) and macroautophagy. The majority of misfolded proteins are degraded by the UPS, in which Ub-conjugated substrates are deubiquitinated, unfolded and cleaved into small peptides when passing through the narrow chamber of the proteasome. The substrates that expose a specific degradation signal, the KFERQ sequence motif, can be delivered to and degraded in lysosomes via the CMA. Aggregation-prone substrates resistant to both the UPS and the CMA can be degraded by macroautophagy, in which cargoes are segregated into autophagosomes before degradation by lysosomal hydrolases. Although most misfolded and aggregated proteins in the human proteome can be degraded by cellular protein quality control, some native and mutant proteins prone to aggregation into β-sheet-enriched oligomers are resistant to all known proteolytic pathways and can thus grow into inclusion bodies or extracellular plaques. The accumulation of protease-resistant misfolded and aggregated proteins is a common mechanism underlying protein misfolding disorders, including neurodegenerative diseases such as Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), prion diseases and Amyotrophic Lateral Sclerosis (ALS). In this review, we provide an overview of the proteolytic pathways in neurons, with an emphasis on the UPS, CMA and macroautophagy, and discuss the role of protein quality control in the degradation of pathogenic proteins in neurodegenerative diseases. Additionally, we examine existing putative therapeutic strategies to efficiently remove cytotoxic proteins from degenerating neurons.

  11. A genetic bistable switch utilizing nonlinear protein degradation.

    Science.gov (United States)

    Huang, Daniel; Holtz, William J; Maharbiz, Michel M

    2012-07-09

    Bistability is a fundamental property in engineered and natural systems, conferring the ability to switch and retain states. Synthetic bistable switches in prokaryotes have mainly utilized transcriptional components in their construction. Using both transcriptional and enzymatic components, creating a hybrid system, allows for wider bistable parameter ranges in a circuit. In this paper, we demonstrate a tunable family of hybrid bistable switches in E. coli using both transcriptional components and an enzymatic component. The design contains two linked positive feedback loops. The first loop utilizes the lambda repressor, CI, and the second positive feedback loop incorporates the Lon protease found in Mesoplasma florum (mf-Lon). We experimentally tested for bistable behavior in exponential growth phase, and found that our hybrid bistable switch was able to retain its state in the absence of an input signal throughout 40 cycles of cell division. We also tested the transient behavior of our switch and found that switching speeds can be tuned by changing the expression rate of mf-Lon. To our knowledge, this work demonstrates the first use of dynamic expression of an orthogonal and heterologous protease to tune a nonlinear protein degradation circuit. The hybrid switch is potentially a more robust and tunable topology for use in prokaryotic systems.

  12. Effects of gamma irradiation on chemical composition and ruminal protein degradation of canola meal

    Science.gov (United States)

    Shawrang, P.; Nikkhah, A.; Zare-Shahneh, A.; Sadeghi, A. A.; Raisali, G.; Moradi-Shahrebabak, M.

    2008-07-01

    Gamma irradiation of canola meal (at doses of 25, 50 and 75 kGy) could alter its ruminal protein degradation characteristics by cross-linking of the polypeptide chains. This processing resulted in decrease (linear effect, Pruminal protein degradation and increase (linear effect, Pruminant nutrition.

  13. Binding of inorganic mercury by subcellular fractions and proteins of rat kidneys

    Energy Technology Data Exchange (ETDEWEB)

    Komsta-Szumska, E; Chmielnicka, J; Piotrowski, J K

    1976-01-01

    Inorganic mercury, administered to rats in a single dose of 0.5 mg Hg/kg is accumulated in the kidneys mainly in the soluble (54 percent) and nuclear (30 percent) fractions, showing decreasing tendency with time. Mitochondrial and microsomal fractions, initially accumulating approximately 11 and 6 percent of total Hg, show a tendency to increase the absolute level of Hg for the first week after administration. In the soluble fraction low-molecular weight, metallothioneinlike proteins are mainly responsible for the accumulation of mercury; in other fractions proteins of higher molecular weight prevail.

  14. Joint interpretation of enantiomer and stable isotope fractionation for chiral pesticides degradation

    DEFF Research Database (Denmark)

    Jin, Biao; Rolle, Massimo

    2016-01-01

    introduce a modeling approach with the aim of unifying and integrating the interpretation of isotopic and enantiomeric fractionation. The model is based on the definition of enantiomer-specific isotopologues and jointly predicts the evolution of concentration, enantiomer fractionation, as well as changes...

  15. Identification of proteins in the postsynaptic density fraction by mass spectrometry

    DEFF Research Database (Denmark)

    Walikonis, R S; Jensen, Ole Nørregaard; Mann, M

    2000-01-01

    Our understanding of the organization of postsynaptic signaling systems at excitatory synapses has been aided by the identification of proteins in the postsynaptic density (PSD) fraction, a subcellular fraction enriched in structures with the morphology of PSDs. In this study, we have completed...... not previously known to be constituents of the PSD fraction and 24 that had previously been associated with the PSD by other methods. The newly identified proteins include the heavy chain of myosin-Va (dilute myosin), a motor protein thought to be involved in vesicle trafficking, and the mammalian homolog...

  16. 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

  17. Dry fractionation for sustainable production of plant protein concentrates

    NARCIS (Netherlands)

    Pelgrom, P.J.M.

    2015-01-01

    The global demand for protein-rich foods is expected to double in the coming decades due to the increasing prosperity and world population. To keep up with the demand, the transition from an animal to a plant-based protein supply is desirable from long-term economic and environmental

  18. Enantiomeric fractioning, degradation and metabolite formation of Mecoprop in subsoils with a phenoxy acid contamination history

    DEFF Research Database (Denmark)

    Frkova, Zuzana; Johansen, Anders; Karlson, Ulrich G.

    2015-01-01

    for their ability to degrade mecoprop under natural and amended conditions. Degradation of mecoprop was studied at elevated and environmentally relevant mecoprop concentrations as affected by nitrate and glucose at nitrate-reducing conditions and at a presence of oxygen (mimicking purging the soil with air. Results......As persistence and toxicity of the enantiomers of chiral pesticides are different a more comprehensive understanding of the fate of enantiomers of agrochemicals in the environment is necessary. Subsoils sampled vertically (2.5-6 m) at a site with a history of phenoxy acid contamination were used...... and enantioselectivity. Glucose hinders mecoprop degradation and changes the EF. Changing EF confirmed enzymatic dgradation of mecoprop in soils, which was well interpreted using the Michaelis-Menten kinetic model. The highest mecoprop degradation rate was measured in soils incubated at nitrate-reducing conditions...

  19. Modeling position-specific isotope fractionation of organic micropollutants degradation via different reaction pathways

    DEFF Research Database (Denmark)

    Jin, Biao; Rolle, Massimo

    : dichlorobenzamide (BAM), isoproturon (IPU) and diclofenac (DCF). The model successfully reproduces the multi-element isotope data, and precisely captures the dual element isotope trends, characterizing the different degradation pathways. Besides illustrating the model capability of mechanistic evaluation...

  20. Prefoldin Promotes Proteasomal Degradation of Cytosolic Proteins with Missense Mutations by Maintaining Substrate Solubility.

    Directory of Open Access Journals (Sweden)

    Sophie A Comyn

    2016-07-01

    Full Text Available Misfolded proteins challenge the ability of cells to maintain protein homeostasis and can accumulate into toxic protein aggregates. As a consequence, cells have adopted a number of protein quality control pathways to prevent protein aggregation, promote protein folding, and target terminally misfolded proteins for degradation. In this study, we employed a thermosensitive allele of the yeast Guk1 guanylate kinase as a model misfolded protein to investigate degradative protein quality control pathways. We performed a flow cytometry based screen to identify factors that promote proteasomal degradation of proteins misfolded as the result of missense mutations. In addition to the E3 ubiquitin ligase Ubr1, we identified the prefoldin chaperone subunit Gim3 as an important quality control factor. Whereas the absence of GIM3 did not impair proteasomal function or the ubiquitination of the model substrate, it led to the accumulation of the poorly soluble model substrate in cellular inclusions that was accompanied by delayed degradation. We found that Gim3 interacted with the Guk1 mutant allele and propose that prefoldin promotes the degradation of the unstable model substrate by maintaining the solubility of the misfolded protein. We also demonstrated that in addition to the Guk1 mutant, prefoldin can stabilize other misfolded cytosolic proteins containing missense mutations.

  1. Polysorbates 20 and 80 used in the formulation of protein biotherapeutics: structure and degradation pathways.

    Science.gov (United States)

    Kerwin, Bruce A

    2008-08-01

    Polysorbates 20 and 80 (Tween 20 and Tween 80) are used in the formulation of biotherapeutic products for both preventing surface adsorption and as stabilizers against protein aggregation. The polysorbates are amphipathic, nonionic surfactants composed of fatty acid esters of polyoxyethylene sorbitan being polyoxyethylene sorbitan monolaurate for polysorbate 20 and polyoxyethylene sorbitan monooleate for polysorbate 80. The polysorbates used in the formulation of biopharmaceuticals are mixtures of different fatty acid esters with the monolaurate fraction of polysorbate 20 making up only 40-60% of the mixture and the monooleate fraction of polysorbate 80 making up >58% of the mixture. The polysorbates undergo autooxidation, cleavage at the ethylene oxide subunits and hydrolysis of the fatty acid ester bond. Autooxidation results in hydroperoxide formation, side-chain cleavage and eventually formation of short chain acids such as formic acid all of which could influence the stability of a biopharmaceutical product. Oxidation of the fatty acid moiety while well described in the literature has not been specifically investigated for polysorbate. This review focuses on the chemical structure of the polysorbates, factors influencing micelle formation and factors and excipients influencing stability and degradation of the polyoxyethylene and fatty acid ester linkages.

  2. Heating and reduction affect the reaction with tannins of wine protein fractions differing in hydrophobicity.

    Science.gov (United States)

    Marangon, Matteo; Vincenzi, Simone; Lucchetta, Marco; Curioni, Andrea

    2010-02-15

    During the storage, bottled white wines can manifest haziness due to the insolubilisation of the grape proteins that may 'survive' in the fermentation process. Although the exact mechanism of this occurrence is not fully understood, proteins and tannins are considered two of the key factors involved in wine hazing, since their aggregation leads to the formation of insoluble particles. To better understand this complex interaction, proteins and tannins from the same unfined Pinot grigio wine were separated. Wine proteins were then fractionated by hydrophobic interaction chromatography (HIC). A significant correlation between hydrophobicity of the wine protein fractions and the haze formed after reacting with wine tannins was found, with the most reactive fractions revealing (by SDS-PAGE and RP-HPLC analyses) the predominant presence of thaumatin-like proteins. Moreover, the effects of both protein heating and disulfide bonds reduction (with dithiotreithol) on haze formation in the presence of tannins were assessed. These treatments generally resulted in an improved reactivity with tannins, and this phenomenon was related to both the surface hydrophobicity and composition of the protein fractions. Therefore, haze formation in wines seems to be related to hydrophobic interactions occurring among proteins and tannins. These interactions should occur on hydrophobic tannin-binding sites, whose exposition on the proteins can depend on both protein heating and reduction. Copyright 2009 Elsevier B.V. All rights reserved.

  3. Evaluation of certain crop residues for carbohydrate and protein fractions by cornell net carbohydrate and protein system

    Directory of Open Access Journals (Sweden)

    Venkateswarulu Swarna

    2015-06-01

    Full Text Available Four locally available crop residues viz., jowar stover (JS, maize stover (MS, red gram straw (RGS and black gram straw (BGS were evaluated for carbohydrate and protein fractions using Cornell Net Carbohydrate and Protein (CNCP system. Lignin (% NDF was higher in legume straws as compared to cereal stovers while Non-structural carbohydrates (NSC (% DM followed the reverse trend. The carbohydrate fractions A and B1 were higher in BGS while B2 was higher in MS as compared to other crop residues. The unavailable cell wall fraction (C was higher in legume straws when compared to cereal stovers. Among protein fractions, B1 was higher in legume straws when compared to cereal stovers while B2 was higher in cereal stovers as compared to legume straws. Fraction B3 largely, bypass protein was highest in MS as compared to other crop residues. Acid detergent insoluble crude protein (ADICP (% CP or unavailable protein fraction C was lowest in MS and highest in BGS. It is concluded that MS is superior in nutritional value for feeding ruminants as compared to other crop residues.

  4. In vitro degradation of the 32kDa PS II reaction centre protein

    International Nuclear Information System (INIS)

    Eckenswiller, L.C.; Greenberg, B.M.

    1989-01-01

    The 32kDa thylakoid membrane protein is an integral component of the PS II reaction centre. The protein, although stable in the dark, undergoes light dependent turnover. Light from the UV, visible and far-red spectral regions induce 32kDa protein degradation. To better understand 32kDa protein metabolism, an in vitro degradation system is being developed. It consists of isolated thylakoid membranes than contain radiolabelled protein. The 32kDa protein is actively and specifically degraded when the thylakoid preparation is exposed to UV or visible radiation. The protein is stable in the dark. The herbicides (atrazine and DCMU) inhibit degradation in the in vitro system as they do in vivo. Additionally, several methods of isolating thylakoids are being compared to optimize the 32kDa protein degradation reaction. The preparations will be evaluated based on their ability to permit light dependent degradation of the 32kDa protein without affecting the other membrane components

  5. Polypeptide composition of fraction 1 protein of the somatic hybrid between Petunia parodii and Petunia parviflora.

    Science.gov (United States)

    Kumar, A; Wilson, D; Cocking, E C

    1981-04-01

    The analysis of the subunit polypeptide composition of Fraction 1 protein provides information on the expression of both chloroplast and nuclear genomes. Fraction 1 protein, isolated from leaves of the somatic hybrid plants derived form the fusion of protoplasts of Petunia parodii and P. parviflora, was analyzed for its subunit polypeptide composition by isoelectric focusing in 8 M urea. The fraction 1 protein enzyme oligomer in the somatic hybrid plants contained small subunits resulting from the expression of both parental nuclear genomes, but probably only one of the parental large subunits, namely that of P. parodii. The relevance of such somatic hybrid material for the study of nucleocytoplasmic interrelationship is discussed, as well as the use of these fraction 1 protein isoelectric focusing patterns for the analysis of taxonomic relationships in Petunia.

  6. Effect of disintegration wave grinding on fractional protein and amino acid composition of chickpea

    Directory of Open Access Journals (Sweden)

    G. O. Magomedov

    2013-01-01

    Full Text Available The study of fractional changes and amino acid composition of proteins in the application of chickpea disintegration wave grinding. Comparative analysis of six varieties of chickpea before and after grinding.

  7. Effect of disintegration wave grinding on fractional protein and amino acid composition of chickpea

    OpenAIRE

    G. O. Magomedov; M. K. Sadigova; S. I. Lukina; V. Y. Kustov

    2013-01-01

    The study of fractional changes and amino acid composition of proteins in the application of chickpea disintegration wave grinding. Comparative analysis of six varieties of chickpea before and after grinding.

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

    NARCIS (Netherlands)

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

    2006-01-01

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

  9. In-depth analysis of low abundant proteins in bovine colostrum using different fractionation techniques

    DEFF Research Database (Denmark)

    Nissen, Asger; Bendixen, Emøke; Ingvartsen, Klaus Lønne

    2012-01-01

    Bovine colostrum is well known for its large content of bioactive components and its importance for neonatal survival. Unfortunately, the colostrum proteome is complicated by a wide dynamic range, because of a few dominating proteins that hamper sensitivity and proteome coverage achieved on low...... abundant proteins. Moreover, the composition of colostrum is complex and the proteins are located within different physical fractions that make up the colostrum. To gain a more exhaustive picture of the bovine colostrum proteome and gather information on protein location, we performed an extensive pre......-analysis fractionation of colostrum prior to 2D-LC-MS/MS analysis. Physical and chemical properties of the proteins and colostrum were used alone or in combination for the separation of proteins. ELISA was used to quantify and verify the presence of proteins in colostrum. In total, 403 proteins were identified...

  10. Sequential fractionation and isolation of subcellular proteins from tissue or cultured cells

    OpenAIRE

    Sabina Baghirova; Bryan G. Hughes; Michael J. Hendzel; Richard Schulz

    2015-01-01

    Many types of studies require the localization of a protein to, or isolation of enriched protein from a specific cellular compartment. Many protocols in the literature and from commercially available kits claim to yield pure cellular fractions. However, in our hands, the former often do not work effectively and the latter may be prohibitively expensive if a large number of fractionations are required. Furthermore, the largely proprietary composition of reagents in commercial kits means that t...

  11. Sequence and 3D structure based analysis of TNT degrading proteins in Arabidopsis thaliana.

    Science.gov (United States)

    Bhattacherjee, Amrita; Mandal, Rahul Shubhra; Das, Santasabuj; Kundu, Sudip

    2014-03-01

    TNT, accidentally released at several manufacturing sites, contaminates ground water and soil. It has a toxic effect to algae and invertebrate, and chronic exposure to TNT also causes harmful effects to human. On the other hand, many plants including Arabidopsis thaliana have the ability to metabolize TNT either completely or at least to a reduced less toxic form. In A. thaliana, the enzyme UDP glucosyltransferase (UDPGT) can further conjugate the reduced forms 2-HADNT and 4-HADNT (2-hydroxylamino-4, 6- dinitrotoluene and 4-hydroxylamino-2, 6- dinitrotoluene) of TNT. Based on the experimental analysis, existing literature and phylogenetic analysis, it is evident that among 107 UDPGT proteins only six are involved in the TNT degrading process. A total of 13 UDPGT proteins including five of these TNT degrading proteins fall within the same group of phylogeny. Thus, these 13 UDPGT proteins have been classified into two groups, TNT-degrading and TNT-non-degrading proteins. To understand the differences in TNT-degrading capacities; using homology modeling we first predicted two structures, taking one representative sequence from both the groups. Next, we performed molecular docking of the modeled structure and TNT reduced form 2-hydroxylamino-4, 6- dinitrotoluene (2-HADNT). We observed that while the Trp residue located within the active site region of the TNT- degrading protein showed π-Cation interaction; such type of interaction was absent in TNT-non-degrading protein, as the respective Trp residue lay outside of the pocket in this case. We observed the conservation of this π-Cation interaction during MD simulation of TNT-degrading protein. Thus, the position and the orientation of the active site residue Trp could explain the presence and absence of TNT-degrading capacity of the UDPGT proteins.

  12. Amino Acid Composition, Molecular Weight Distribution and Gel Electrophoresis of Walnut (Juglans regia L. Proteins and Protein Fractionations

    Directory of Open Access Journals (Sweden)

    Xiaoying Mao

    2014-01-01

    Full Text Available As a by-product of oil production, walnut proteins are considered as an additional source of plant protein for human food. To make full use of the protein resource, a comprehensive understanding of composition and characteristics of walnut proteins are required. Walnut proteins have been fractionated and characterized in this study. Amino acid composition, molecular weight distribution and gel electrophoresis of walnut proteins and protein fractionations were analyzed. The proteins were sequentially separated into four fractions according to their solubility. Glutelin was the main component of the protein extract. The content of glutelin, albumin, globulin and prolamin was about 72.06%, 7.54%, 15.67% and 4.73% respectively. Glutelin, albumin and globulin have a balanced content of essential amino acids, except for methionine, with respect to the FAO pattern recommended for adults. SDS-PAGE patterns of albumin, globulin and glutelin showed several polypeptides with molecular weights 14.4 to 66.2 kDa. The pattern of walnut proteins in two-dimension electrophoresis (2-DE showed that the isoelectric point was mainly in the range of 4.8–6.8. The results of size exclusion chromatogram indicated molecular weight of the major components of walnut proteins were between 3.54 and 81.76 kDa.

  13. Monitoring the Erosion of Hydrolytically-Degradable Nanogels via Multiangle Light Scattering Coupled to Asymmetrical Flow Field-Flow Fractionation

    Science.gov (United States)

    Smith, Michael H.; South, Antoinette B.; Gaulding, Jeffrey C.; Lyon, L. Andrew

    2009-01-01

    We describe the synthesis and characterization of degradable nanogels that display bulk erosion under physiologic conditions (pH = 7.4, 37 °C). Erodible poly(N-isopropylmethacrylamide) nanogels were synthesized by copolymerization with N,O-(dimethacryloyl)hydroxylamine, a cross-linker previously used in the preparation of non-toxic and biodegradable bulk hydrogels. To monitor particle degradation, we employed multiangle light scattering and differential refractometry detection following asymmetrical flow field-flow fractionation. This approach allowed the detection of changes in nanogel molar mass and topology as a function of both temperature and pH. Particle erosion was evident from both an increase in nanogel swelling and a decrease in scattering intensity as a function of time. Following these analyses, the samples were recovered for subsequent characterization by direct particle tracking, which yields hydrodynamic size measurements and enables number density determination. Additionally, we confirmed the conservation of nanogel stimuli-responsivity through turbidity measurements. Thus, we have demonstrated the synthesis of degradable nanogels that erode under conditions and on timescales that are relevant for many drug delivery applications. The combined separation and light scattering detection method is demonstrated to be a versatile means to monitor erosion and should also find applicability in the characterization of other degradable particle constructs. PMID:20000662

  14. A Critical Appraisal of Quantitative Studies of Protein Degradation in the Framework of Cellular Proteostasis

    Directory of Open Access Journals (Sweden)

    Beatriz Alvarez-Castelao

    2012-01-01

    Full Text Available Protein homeostasis, proteostasis, is essential to understand cell function. Protein degradation is a crucial component of the proteostatic mechanisms of the cell. Experiments on protein degradation are nowadays present in many investigations in the field of molecular and cell biology. In the present paper, we focus on the different experimental approaches to study protein degradation and present a critical appraisal of the results derived from steady-state and kinetic experiments using detection of unlabelled and labelled protein methodologies with a proteostatic perspective. This perspective allows pinpointing the limitations in interpretation of results and the need of further experiments and/or controls to establish “definitive evidence” for the role of protein degradation in the proteostasis of a given protein or the entire proteome. We also provide a spreadsheet for simple calculations of mRNA and protein decays for mimicking different experimental conditions and a checklist for the analysis of experiments dealing with protein degradation studies that may be useful for researchers interested in the area of protein turnover.

  15. Degradation of protein translation machinery by amino acid starvation-induced macroautophagy

    DEFF Research Database (Denmark)

    Gretzmeier, Christine; Eiselein, Sven; Johnson, Gregory R.

    2017-01-01

    , unbiased approaches relying on quantitative mass spectrometry-based proteomics. Macroautophagy is induced by rapamycin treatment, and by amino acid and glucose starvation in differentially, metabolically labeled cells. Protein dynamics are linked to image-based models of autophagosome turnover. Depending...... on the inducing stimulus, protein as well as organelle turnover differ. Amino acid starvation-induced macroautophagy leads to selective degradation of proteins important for protein translation. Thus, protein dynamics reflect cellular conditions in the respective treatment indicating stimulus-specific pathways...

  16. Variability in the fractionation of stable isotopes during degradation of two intertidal red algae

    Science.gov (United States)

    Hill, Jaclyn M.; McQuaid, Christopher D.

    2009-04-01

    Macroalgae contribute to intertidal food webs primarily as detritus, with unclear implications for food web studies using stable isotope analysis. We examined differences in the thallus parts of two South African rhodophytes ( Gelidium pristoides and Hypnea spicifera) and changes in overall δ13C, δ15N signatures and C:N ratios during degradation in both the field and laboratory. We hypothesized that both degrading macroalgal tissue and macroalgal-derived suspended particulate material (SPM) would show negligible changes in δ13C, but enriched δ15N signatures and lower C:N ratios relative to healthy plants. Only C:N laboratory ratios conformed to predictions, with both species of macroalgae showing decomposition related changes in δ13C and significant depletions in δ15N in both the field and laboratory. In the laboratory, algal tissue and SPM from each species behaved similarly (though some effects were non-significant) but with differing strengths. Gelidium pristoides δ13C increased and C:N ratios decreased over time in tissue and SPM; δ15N became depleted only in SPM. Hypnea spicifera, δ13C, δ15N and C:N ratios all decreased during degradation in both SPM and algae. Over 60 days in the field, δ13C was depleted in both species (1-2‰) and in naturally senescent Gelidium pristoides fronds. δ15N was depleted in Hypnea spicifera (approx. 1‰), while C:N ratios of both species were unaffected. The two species differed in δ13C, δ15N and C:N after degradation, but only in C:N beforehand. We suggest isotope changes in the laboratory mainly reflect microbial effects, while in the field these are combined with leaching due to constant water replenishment and agitation. Differences between these two species in the isotope responses to degradation highlight the difficulty of linking the signature of SPM to its multiple sources.

  17. Toscana virus NSs protein promotes degradation of double-stranded RNA-dependent protein kinase.

    Science.gov (United States)

    Kalveram, Birte; Ikegami, Tetsuro

    2013-04-01

    Toscana virus (TOSV), which is transmitted by Phlebotomus spp. sandflies, is a major etiologic agent of aseptic meningitis and encephalitis in the Mediterranean. Like other members of the genus Phlebovirus of the family Bunyaviridae, TOSV encodes a nonstructural protein (NSs) in its small RNA segment. Although the NSs of Rift Valley fever virus (RVFV) has been identified as an important virulence factor, which suppresses host general transcription, inhibits transcription from the beta interferon promoter, and promotes the proteasomal degradation of double-stranded RNA-dependent protein kinase (PKR), little is known about the functions of NSs proteins encoded by less-pathogenic members of this genus. In this study we report that TOSV is able to downregulate PKR with similar efficiency as RVFV, while infection with the other phleboviruses-i.e., Punta Toro virus, sandfly fever Sicilian virus, or Frijoles virus-has no effect on cellular PKR levels. In contrast to RVFV, however, cellular transcription remains unaffected during TOSV infection. TOSV NSs protein promotes the proteasome-dependent downregulation of PKR and is able to interact with kinase-inactive PKR in infected cells.

  18. Desempenho e características de carcaça de bezerros confinados recebendo dietas com diferentes degradabilidades da fração protéica Performance and carcass traits of feedlot calves receiving diets with different degradabilities of the protein fraction

    Directory of Open Access Journals (Sweden)

    Rafael da Costa Cervieri

    2001-10-01

    Full Text Available O objetivo deste trabalho foi avaliar as respostas do fornecimento de dietas com diferentes níveis de proteína degradável no rúmen (PDR, durante a fase inicial de confinamento, no desempenho, nas características de crescimento monitoradas in vivo por ultra-sonografia e nos parâmetros de carcaça e qualidade de carne de bovinos jovens. Foram utilizados 60 bezerros Brangus não castrados, com 8 meses e peso vivo médio de 243,2 kg, submetidos a três tratamentos nas fases de crescimento (Fase 1 e terminação (Fase 2: T1 - 69% PDR nas Fases 1 e 2; T2 - 77% PDR na Fase 1 e 69% PDR na Fase 2; T3 - 61% PDR na Fase 1 e 69% PDR na Fase 2. Na Fase 1, os animais do tratamento T2 apresentaram menor ganho de peso médio diário que os animais do tratamento T1 e pior conversão alimentar que T1 e T3. O peso de carcaça quente, seu rendimento e os pesos de dianteiro, ponta de agulha e traseiro, bem como a área de olho de lombo, gordura subcutânea, força de cisalhamento e composição química da carne, não apresentaram diferença estatística entre tratamentos. A utilização de dietas com menor teor de PDR melhorou a conversão alimentar e o ganho de peso médio diário em até 11,5 e 16,9%, respectivamente, na fase de crescimento, indicando a utilização de menores níveis de PDR neste período.The objective of this study was to evaluate the effects of different levels of degradable intake protein (DIP diets during the feedlot-growing phase, on performance, in vivo growth parameters measured by ultrasound and on carcass traits and meat quality of young bulls. Sixty Brangus calves, 8 months old, averaging 243,2 kg, were submitted to three treatments in growing (Phase 1 and finishing (Phase 2 phases: T1 - 69% DIP in Phases 1 and 2; T2 - 77% DIP in Phase 1 and 69% DIP in Phase 2; T3 - 61% DIP in Phase 1 and 69% DIP in Phase 2. In Phase 1, animals on treatments T2 showed a lower average daily gain than those on treatments T1 and worst feed

  19. The Arabidopsis CROWDED NUCLEI genes regulate seed germination by modulating degradation of ABI5 protein.

    Science.gov (United States)

    Zhao, Wenming; Guan, Chunmei; Feng, Jian; Liang, Yan; Zhan, Ni; Zuo, Jianru; Ren, Bo

    2016-07-01

    In Arabidopsis, the phytohormone abscisic acid (ABA) plays a vital role in inhibiting seed germination and in post-germination seedling establishment. In the ABA signaling pathway, ABI5, a basic Leu zipper transcription factor, has important functions in the regulation of seed germination. ABI5 protein localizes in nuclear bodies, along with AFP, COP1, and SIZ1, and was degraded through the 26S proteasome pathway. However, the mechanisms of ABI5 nuclear body formation and ABI5 protein degradation remain obscure. In this study, we found that the Arabidopsis CROWDED NUCLEI (CRWN) proteins, predicted nuclear matrix proteins essential for maintenance of nuclear morphology, also participate in ABA-controlled seed germination by regulating the degradation of ABI5 protein. During seed germination, the crwn mutants are hypersensitive to ABA and have higher levels of ABI5 protein compared to wild type. Genetic analysis suggested that CRWNs act upstream of ABI5. The observation that CRWN3 colocalizes with ABI5 in nuclear bodies indicates that CRWNs might participate in ABI5 protein degradation in nuclear bodies. Moreover, we revealed that the extreme C-terminal of CRWN3 protein is necessary for its function in the response to ABA in germination. Our results suggested important roles of CRWNs in ABI5 nuclear body organization and ABI5 protein degradation during seed germination. © 2015 Institute of Botany, Chinese Academy of Sciences.

  20. Muscle protein degradation and amino acid metabolism during prolonged knee-extensor exercise in humans

    DEFF Research Database (Denmark)

    Van Hall, Gerrit; Saltin, B; Wagenmakers, A J

    1999-01-01

    to a substantial increase in net muscle protein degradation, and that a lowering of the starting muscle glycogen content leads to a further increase. The carbon atoms of the branched-chain amino acids (BCAA), glutamate, aspartate and asparagine, liberated by protein degradation, and the BCAA and glutamate......The aim of this study was to investigate whether prolonged one-leg knee-extensor exercise enhances net protein degradation in muscle with a normal or low glycogen content. Net amino acid production, as a measure of net protein degradation, was estimated from leg exchange and from changes...... in the concentrations of amino acids that are not metabolized in skeletal muscle. Experiments were performed at rest and during one-leg knee-extensor exercise in six subjects having one leg with a normal glycogen content and the other with a low glycogen content. Exercise was performed for 90 min at a workload of 60...

  1. Degradation of extracytoplasmic catalysts for protein folding in Bacillus subtilis

    NARCIS (Netherlands)

    Krishnappa, Laxmi; Monteferrante, Carmine G; Neef, Jolanda; Dreisbach, Annette; van Dijl, Jan Maarten

    The general protein secretion pathway of Bacillus subtilis has a high capacity for protein export from the cytoplasm, which is exploited in the biotechnological production of a wide range of enzymes. These exported proteins pass the membrane in an unfolded state, and accordingly, they have to fold

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

    Science.gov (United States)

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

    2015-11-01

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

  3. White clover fractions as protein source for monogastrics - Dry matter digestibility and Protein Digestibility-Corrected Amino Acid Scores

    DEFF Research Database (Denmark)

    Stødkilde, Lene; Damborg, Vinni K; Jørgensen, Henry

    2018-01-01

    BACKGROUND: The aim was to evaluate white clover as an alternative protein source for monogastrics. White clover plant and leaves were processed using a screw-press resulting in a solid pulp and a juice from which protein was acid-precipitated. The chemical composition of all fractions...... was determined and digestibility of dry matter (DM) and protein was assessed in an experiment with growing rats. RESULTS: Protein concentrates were produced with crude protein (CP) content of 451 g/kg DM and 530 g/kg DM for white clover plant and leaves, respectively and a pulp with CP content of 313 and 374 g...

  4. Antioxidant activities and functional properties of protein and peptide fractions isolated from salted herring brine

    DEFF Research Database (Denmark)

    Taheri, Ali; Farvin, Sabeena; Jacobsen, Charlotte

    2014-01-01

    In the present study proteins isolated from herring brine, which is a by-product of marinated herring production were evaluated for their functional properties and antioxidant activity. Herring brine was collected from the local herring industry and proteins were precipitated by adjusting the p...... to delay iron catalyzed lipid oxidation in 5% fish oil in water emulsions and the 10–50kDa fraction was the best. These results show the potential of proteins and peptide fractions recovered from waste water from the herring industry as source of natural antioxidants for use in food products....

  5. Inhibition of platelet [3H]- imipramine binding by human plasma protein fractions

    International Nuclear Information System (INIS)

    Strijewski, A.; Chudzik, J.; Tang, S.W.

    1988-01-01

    Inhibition of high-affinity [ 3 H]-imipramine binding to platelet membranes by human plasma fractions and isolated plasma proteins was investigated. Several plasma proteins were found to contribute to the observed apparent inhibition and this contribution was assessed in terms of inhibitor units. Alpha 1 acid glycoprotein, high density and low density lipoprotein, IgG and α 1 -antitrypsin were identified as effective non-specific inhibitors. Alpha-1-acid glycoprotein was confirmed to be the most potent plasma protein inhibitor. Cohn fractions were evaluated for the presence of the postulated endocoid of [ 3 H]-imipramine binding site

  6. Lead Intoxication On Protein Fractions, Testicular Tissues And Ameliorative Effect Of ANTOX On Male Albino Rats

    International Nuclear Information System (INIS)

    HASSANIN, M.M.; EL-MAHDY, A.A.; ZAKI, Z.T.; EMARAH, E.A.M.; HUSSEIN, A.M.M.

    2010-01-01

    Lead (heavy metal) was given as lead acetate for two groups of adult male albino rats (Rattus rattus) in drinking water at dose level of 100 mg/litre for 3 and 6 weeks to evaluate its toxic effects on protein and its fractions by using electrophoresis technique, serum testosterone using radioimmunoassay and histological investigation of the testis of male rats.Administration of 100 mg/L lead acetate in drinking water for 3 and 6 weeks induced fluctuated changes in serum total proteins, protein fractions and significant decrease in serum testosterone hormone.Histopathological examination showed cellular changes and degeneration of the seminiferous tubules after 3 and 6 weeks of administration of lead acetate.The treatment of rats with antox (10 mg/kg) during the experimental period caused improvement in protein fraction and testosterone level, and the testes sections appeared more or less normal.

  7. Protein fraction heterogeneity in donkey’s milk analysed by proteomic methods

    Directory of Open Access Journals (Sweden)

    G. D'Urso

    2010-04-01

    Full Text Available Donkey’s milk is often well tolerate by patients affected by cow’s milk protein allergy, probably thanks to its protein composition. This empiric evidence, confirmed by some clinical trials, needs to be better investigated. A preliminary survey on the protein fraction of donkey’s milk was carried out: fifty-six individual milk samples have been collected and analysed by IEF and SDS-PAGE. Five different IEF patterns have been identified, showing a marked heterogeneity both in casein and whey protein fractions. A single IEF pattern showed an apparent reduced amount of casein fraction highlighted by SDS. Three of the five IEF patterns have been further investigated by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS.

  8. ORGANIC MATTER AND CRUDE PROTEIN DEGRADATION SYNCHRONY IN DIETS SELECTED BY RANGE GOATS.

    Directory of Open Access Journals (Sweden)

    Rafael Ramírez Orduña

    2010-09-01

    Full Text Available The study was carried out with the aim to asses the synchrony of organic matter and crude protein degradation in the rumen of diets selected by range goats through two years. Five esophageal cannulated adult male goats were used to collect extrusa samples during summer (August 9–13 and autumn (November 29 –December 3 of 2006, winter (February 20 – 24, spring (April 29 –May 5, summer (September 10–15 and autumn (December 4–8 of 2007 and winter (February 20 – 25 and spring (May 9 –13 of 2008. Extrusa samples were subjected to chemical analysis to determine organic matter (OM, crude protein (CP in situ and in vitro true digestibility of dry matter. OM and CP intake were estimated by total fecal collection. Effective extent of degradation of the OM and CP was calculated hourly and total 24 hours. From the hourly quantity of OM and CP degraded, a synchrony index of CP to OM was calculated, and from the total 24 hours degradation, degraded organic matter intake and crude protein intake were also estimated. Sampling date was the main effect that determined the variation of diet OM and CP degradation parameters. Degraded crude protein intake as a proportion of degraded OM was affected by sampling date and was correlated to rainfall. During winter of the first year degraded crude protein intake was below the requirements for maintenance or to promote growth for range goats weighing 40 kg. Even though, synchrony index between OM and CP degradation was affected by sampling date goats maintained a high synchrony index throughout the years.

  9. Effects of gamma irradiation on chemical composition and ruminal protein degradation of canola meal

    Energy Technology Data Exchange (ETDEWEB)

    Shawrang, P. [Agriculture, Medical and Industrial Research School, Nuclear Science and Technology Research Institute, Atomic Energy Organization of Iran, P.O. Box 31485-498, Karaj (Iran, Islamic Republic of); Department of Animal Science, Faculty of Agriculture, Tehran University P.O. Box 4111, Karaj (Iran, Islamic Republic of)], E-mail: parvinshawrang@yahoo.co.uk; Nikkhah, A.; Zare-Shahneh, A. [Department of Animal Science, Faculty of Agriculture, Tehran University P.O. Box 4111, Karaj (Iran, Islamic Republic of); Sadeghi, A.A. [Department of Animal Science, Faculty of Agriculture, Science and Research Branch, Islamic Azad University, P.O. Box 14515-4933, Tehran (Iran, Islamic Republic of); Raisali, G. [Radiation Applications Research School, Nuclear Science and Technology Research Institute, Atomic Energy Organization of Iran, P.O. Box 11365-3486, Tehran (Iran, Islamic Republic of); Moradi-Shahrebabak, M. [Department of Animal Science, Faculty of Agriculture, Tehran University P.O. Box 4111, Karaj (Iran, Islamic Republic of)

    2008-07-15

    Gamma irradiation of canola meal (at doses of 25, 50 and 75 kGy) could alter its ruminal protein degradation characteristics by cross-linking of the polypeptide chains. This processing resulted in decrease (linear effect, P<0.001) of ruminal protein degradation and increase (linear effect, P<0.001) of intestinal protein digestibility. The results showed that gamma irradiation at doses higher than 25 kGy can be used as a cross-linking agent to improve protein properties of supplements in ruminant nutrition.

  10. Effects of gamma irradiation on chemical composition and ruminal protein degradation of canola meal

    International Nuclear Information System (INIS)

    Shawrang, P.; Nikkhah, A.; Zare-Shahneh, A.; Sadeghi, A.A.; Raisali, G.; Moradi-Shahrebabak, M.

    2008-01-01

    Gamma irradiation of canola meal (at doses of 25, 50 and 75 kGy) could alter its ruminal protein degradation characteristics by cross-linking of the polypeptide chains. This processing resulted in decrease (linear effect, P<0.001) of ruminal protein degradation and increase (linear effect, P<0.001) of intestinal protein digestibility. The results showed that gamma irradiation at doses higher than 25 kGy can be used as a cross-linking agent to improve protein properties of supplements in ruminant nutrition

  11. Colorimetric determination of in vitro feed protein degradation

    African Journals Online (AJOL)

    bag technique. Diazotized f ish meal, maize gluten meal and soya-bean were incubated with strained rumen fluid and a suitable growth medium. Only 49o/o and 18% of the colour bound to soya-bean was bound to fish meal and corn gluten meal, respectively. Degradation values expressed as a percen- tage of the total ...

  12. Critical lysine residues of Klf4 required for protein stabilization and degradation

    Energy Technology Data Exchange (ETDEWEB)

    Lim, Key-Hwan; Kim, So-Ra; Ramakrishna, Suresh; Baek, Kwang-Hyun, E-mail: baek@cha.ac.kr

    2014-01-24

    Highlights: • Klf4 undergoes the 26S proteasomal degradation by ubiquitination on its multiple lysine residues. • Essential Klf4 ubiquitination sites are accumulated between 190–263 amino acids. • A mutation of lysine at 232 on Klf4 elongates protein turnover. • Klf4 mutants dramatically suppress p53 expression both under normal and UV irradiated conditions. - Abstract: The transcription factor, Krüppel-like factor 4 (Klf4) plays a crucial role in generating induced pluripotent stem cells (iPSCs). As the ubiquitination and degradation of the Klf4 protein have been suggested to play an important role in its function, the identification of specific lysine sites that are responsible for protein degradation is of prime interest to improve protein stability and function. However, the molecular mechanism regulating proteasomal degradation of the Klf4 is poorly understood. In this study, both the analysis of Klf4 ubiquitination sites using several Klf4 deletion fragments and bioinformatics predictions showed that the lysine sites which are signaling for Klf4 protein degradation lie in its N-terminal domain (aa 1–296). The results also showed that Lys32, 52, 232, and 252 of Klf4 are responsible for the proteolysis of the Klf4 protein. These results suggest that Klf4 undergoes proteasomal degradation and that these lysine residues are critical for Klf4 ubiquitination.

  13. Degradation of structurally characterized proteins injected into HeLa cells. Basic measurements

    International Nuclear Information System (INIS)

    Rogers, S.W.; Rechsteiner, M.

    1988-01-01

    Thirty-five proteins of known x-ray structure were labeled by chloramine-T radioiodination or by reaction with 125I-Bolton-Hunter reagent and introduced into HeLa cells using red cell-mediated microinjection. Degradation rates of the injected proteins were then determined over the next 50 h by measuring the release of soluble isotope to the culture medium. Control experiments demonstrated that the measured rates were not compromised by proteolysis within RBCs, the presence of unfused RBCs, or degradation of protein released from RBCs to the medium. Degradation of some injected proteins was faster during the first 12 h after fusion than at later times, apparently a response of HeLa cells to trypsinization. However, all proteins exhibited first-order degradation rates between 24 and 48 h post injection. Except for seven proteins, stabilities measured during this interval were unaffected by the labeling procedure. Reductive methylation was used to choose among the seven discordant values, and half-lives for the 35 proteins ranged from 16 h for lysozyme to 214 h for yeast alcohol dehydrogenase. Since half-lives for six of the injected proteins closely match values obtained by in vivo measurements, we consider our estimates of the metabolic stabilities of the injected proteins to be generally accurate. Therefore, the half-lives obtained by microinjection should prove useful in the search for relationships between protein structure and intracellular stability

  14. Polyubiquitin chain assembly and organisation determine the dynamics of protein activation and degradation

    Directory of Open Access Journals (Sweden)

    Lan K. Nguyen

    2014-01-01

    Full Text Available Protein degradation via ubiquitination is a major proteolytic mechanism in cells. Once a protein is destined for degradation, it is tagged by multiple ubiquitin molecules. The synthesised polyubiquitin chains can be recognised by the 26S proteosome where proteins are degraded. These chains form through multiple ubiquitination cycles that are similar to multi-site phosphorylation cycles. As kinases and phosphatases, two opposing enzymes (E3 ligases and deubiquitinases DUBs catalyse (deubiquitination cycles. Although multi-ubiquitination cycles are fundamental mechanisms of controlling protein concentrations within a cell, their dynamics have never been explored. Here, we fill this knowledge gap. We show that under permissive physiological conditions, the formation of polyubiquitin chain of length greater than two and subsequent degradation of the ubiquitinated protein, which is balanced by protein synthesis, can display bistable, switch-like responses. Interestingly, the occurrence of bistability becomes pronounced, as the chain grows, giving rise to all-or-none regulation at the protein levels. We give predictions of protein distributions under bistable regime awaiting experimental verification. Importantly, we show for the first time that sustained oscillations can robustly arise in the process of formation of ubiquitin chain, largely due to the degradation of the target protein. This new feature is opposite to the properties of multi-site phosphorylation cycles, which are incapable of generating oscillation if the total abundance of interconverted protein forms is conserved. We derive structural and kinetic constraints for the emergence of oscillations, indicating that a competition between different substrate forms and the E3 and DUB is critical for oscillation. Our work provides the first detailed elucidation of the dynamical features brought about by different molecular setups of the polyubiquitin chain assembly process responsible for

  15. Common Prairie feeds with different soluble and insoluble fractions used for CPM diet formulation in dairy cattle: Impact of carbohydrate-protein matrix structure on protein and other primary nutrient digestion

    Science.gov (United States)

    Peng, Quanhui; Wang, Zhisheng; Zhang, Xuewei; Yu, Peiqiang

    2014-03-01

    An experiment was conducted to investigate the relationship of carbohydrates molecular spectral characteristics to rumen degradability of primary nutrients in Prairie feeds in dairy cattle. In total, 12 different types of feeds were selected, each type of feed was from three different source with total 37 samples. Six types of them were energy-sourced feeds and the others were protein-sourced feeds. The carbohydrates molecular spectral intensity of various functional groups were collected using Fourier transform infrared attenuated total reflectance (ATR-FT/IR) spectroscopy. In the in situ study, the results showed that the rumen digestibility and digestible fractions of primary nutrients (DM, OM, NCP, and CP) were significantly different (P digestibility and digestible fractions of DM, OM and NCP. Spectral intensities of H_1150, H_1015, A_1, and A_3 were weakly negatively associated with in situ rumen degradation of CP. Spectral intensities of A_1240 and H_1240, mainly associated with cellulosic compounds, were correlated with rumen CP degradation. The multiple regression analysis demonstrated that the spectral intensities of A_3 and H_1415 played the most important role and could be used as a potential tool to predict rumen protein degradation of feeds in dairy cattle. In conclusion, this study showed that the carbohydrates as a whole have an effect on protein rumen degradation, rather than cellulose alone, indicating carbohydrate-protein matrix structure impact protein utilization in dairy cattle. The non-invasive molecular spectral technique (ATR-FT/IR) could be used as a rapid potential tool to predict rumen protein degradation of feedstuffs by using molecular spectral bands intensities in carbohydrate fingerprint region.

  16. Common Prairie feeds with different soluble and insoluble fractions used for CPM diet formulation in dairy cattle: impact of carbohydrate-protein matrix structure on protein and other primary nutrient digestion.

    Science.gov (United States)

    Peng, Quanhui; Wang, Zhisheng; Zhang, Xuewei; Yu, Peiqiang

    2014-01-01

    An experiment was conducted to investigate the relationship of carbohydrates molecular spectral characteristics to rumen degradability of primary nutrients in Prairie feeds in dairy cattle. In total, 12 different types of feeds were selected, each type of feed was from three different source with total 37 samples. Six types of them were energy-sourced feeds and the others were protein-sourced feeds. The carbohydrates molecular spectral intensity of various functional groups were collected using Fourier transform infrared attenuated total reflectance (ATR-FT/IR) spectroscopy. In the in situ study, the results showed that the rumen digestibility and digestible fractions of primary nutrients (DM, OM, NCP, and CP) were significantly different (P<0.05) among the feeds. The spectral bands features were significantly different (P<0.05) among the feeds. Spectral intensities of A_Cell, H_1415 and H_1370 were weakly positively correlated with in situ rumen digestibility and digestible fractions of DM, OM and NCP. Spectral intensities of H_1150, H_1015, A_1, and A_3 were weakly negatively associated with in situ rumen degradation of CP. Spectral intensities of A_1240 and H_1240, mainly associated with cellulosic compounds, were correlated with rumen CP degradation. The multiple regression analysis demonstrated that the spectral intensities of A_3 and H_1415 played the most important role and could be used as a potential tool to predict rumen protein degradation of feeds in dairy cattle. In conclusion, this study showed that the carbohydrates as a whole have an effect on protein rumen degradation, rather than cellulose alone, indicating carbohydrate-protein matrix structure impact protein utilization in dairy cattle. The non-invasive molecular spectral technique (ATR-FT/IR) could be used as a rapid potential tool to predict rumen protein degradation of feedstuffs by using molecular spectral bands intensities in carbohydrate fingerprint region. Copyright © 2013 Elsevier B

  17. Antioxidant activities of bambara groundnut (Vigna subterranea) protein hydrolysates and their membrane ultrafiltration fractions.

    Science.gov (United States)

    Arise, Abimbola K; Alashi, Adeola M; Nwachukwu, Ifeanyi D; Ijabadeniyi, Oluwatosin A; Aluko, Rotimi E; Amonsou, Eric O

    2016-05-18

    In this study, the bambara protein isolate (BPI) was digested with three proteases (alcalase, trypsin and pepsin), to produce bambara protein hydrolysates (BPHs). These hydrolysates were passed through ultrafiltration membranes to obtain peptide fractions of different sizes (fractions were investigated for antioxidant activities. The membrane fractions showed that peptides with sizes 3 kDa. This is in agreement with the result obtained for the ferric reducing power, metal chelating and hydroxyl radical scavenging activities where higher molecular weight peptides exhibited better activity (p fractions. However, for all the hydrolysates, the low molecular weight peptides were more effective diphenyl-1-picrylhydrazyl (DPPH) radical scavengers but not superoxide radicals when compared to the bigger peptides. In comparison with glutathione (GSH), BPHs and their membrane fractions had better (p fractions that did not show any metal chelating activity. However, the 5-10 kDa pepsin hydrolysate peptide fractions had greater (88%) hydroxyl scavenging activity than GSH, alcalase and trypsin hydrolysates (82%). These findings show the potential use of BPHs and their peptide fraction as antioxidants in reducing food spoilage or management of oxidative stress-related metabolic disorders.

  18. Soil Organic Carbon Fractions and Stocks Respond to Restoration Measures in Degraded Lands by Water Erosion.

    Science.gov (United States)

    Nie, Xiaodong; Li, Zhongwu; Huang, Jinquan; Huang, Bin; Xiao, Haibing; Zeng, Guangming

    2017-05-01

    Assessing the degree to which degraded soils can be recovered is essential for evaluating the effects of adopted restoration measures. The objective of this study was to determine the restoration of soil organic carbon under the impact of terracing and reforestation. A small watershed with four typical restored plots (terracing and reforestation (four different local plants)) and two reference plots (slope land with natural forest (carbon-depleted) and abandoned depositional land (carbon-enriched)) in subtropical China was studied. The results showed that soil organic carbon, dissolved organic carbon and microbial biomass carbon concentrations in the surface soil (10 cm) of restored lands were close to that in abandoned depositional land and higher than that in natural forest land. There was no significant difference in soil organic carbon content among different topographic positions of the restored lands. Furthermore, the soil organic carbon stocks in the upper 60 cm soils of restored lands, which were varied between 50.08 and 62.21 Mg C ha -1 , were higher than 45.90 Mg C ha -1 in natural forest land. Our results indicated that the terracing and reforestation could greatly increase carbon sequestration and accumulation and decrease carbon loss induced by water erosion. And the combination measures can accelerate the restoration of degraded soils when compared to natural forest only. Forest species almost have no impact on the total amount of soil organic carbon during restoration processes, but can significantly influence the activity and stability of soil organic carbon. Combination measures which can provide suitable topography and continuous soil organic carbon supply could be considered in treating degraded soils caused by water erosion.

  19. Soil Organic Carbon Fractions and Stocks Respond to Restoration Measures in Degraded Lands by Water Erosion

    Science.gov (United States)

    Nie, Xiaodong; Li, Zhongwu; Huang, Jinquan; Huang, Bin; Xiao, Haibing; Zeng, Guangming

    2017-05-01

    Assessing the degree to which degraded soils can be recovered is essential for evaluating the effects of adopted restoration measures. The objective of this study was to determine the restoration of soil organic carbon under the impact of terracing and reforestation. A small watershed with four typical restored plots (terracing and reforestation (four different local plants)) and two reference plots (slope land with natural forest (carbon-depleted) and abandoned depositional land (carbon-enriched)) in subtropical China was studied. The results showed that soil organic carbon, dissolved organic carbon and microbial biomass carbon concentrations in the surface soil (10 cm) of restored lands were close to that in abandoned depositional land and higher than that in natural forest land. There was no significant difference in soil organic carbon content among different topographic positions of the restored lands. Furthermore, the soil organic carbon stocks in the upper 60 cm soils of restored lands, which were varied between 50.08 and 62.21 Mg C ha-1, were higher than 45.90 Mg C ha-1 in natural forest land. Our results indicated that the terracing and reforestation could greatly increase carbon sequestration and accumulation and decrease carbon loss induced by water erosion. And the combination measures can accelerate the restoration of degraded soils when compared to natural forest only. Forest species almost have no impact on the total amount of soil organic carbon during restoration processes, but can significantly influence the activity and stability of soil organic carbon. Combination measures which can provide suitable topography and continuous soil organic carbon supply could be considered in treating degraded soils caused by water erosion.

  20. Stage of lactation and corresponding diets affect in situ protein degradation by dairy cows.

    Science.gov (United States)

    Schadt, I; Mertens, D R; Van Soest, P J; Azzaro, G; Licitra, G

    2014-12-01

    The influence of stage of lactation and corresponding diets on rates of protein degradation (kd) is largely unstudied. Study objectives were to measure and compare in situ ruminal kd of crude protein (CP) and estimate rumen CP escape (rumen-undegradable protein; RUP) of selected feeds by cows at 3 stages of lactation fed corresponding diets, and to determine the incubation times needed in an enzymatic in vitro procedure, using 0.2 units of Streptomyces griseus protease per percent of true CP, that predicted in situ RUP. Residue CP was measured after in situ fermentation for 4, 8, 12, 24, 36, 48, and 72 h of 5 protein sources and 3 total mixed rations, which were fed to the in situ cows. Two nonlactating (dry) cows and 2 cows each at 190 (mid) and 90 (peak) days of lactation were used. Each pair of cows was offered free-choice diets that differed in composition to meet their corresponding nutrient requirements. Diets had decreasing proportions of forages and contained (dry matter basis) 11.9, 15.1 and 16.4% CP and 54.3, 40.3 and 35.3% neutral detergent fiber, for dry, mid, and peak TMR (TMR1, TMR2, and TMR3), respectively. Intakes were 10.3, 21.4, and 23.8kg of dry matter/d, respectively. Kinetic CP fractions (extractable, potentially degradable, undegradable, or slowly degradable) were unaffected by treatment. Lag time and kd varied among feeds. The kd was faster for all feeds (0.136/h) when incubated in dry-TMR1 cows compared with mid-TMR2 (0.097/h) or peak-TMR3 (0.098/h) cows, and no differences in lag time were detected. Calculated RUP, using estimated passage rates for each cow based on intake, differed between dry-TMR1 (0.382) and mid-TMR2 (0.559) or peak-TMR3 (0.626) cows, with a tendency for mid-TMR2 to be different from peak-TMR3. Using the average kd and lag time obtained from dry-TMR1 to calculate RUP for mid-TMR2 and peak-TMR3 cows using their passage rates reduced RUP values by 6.3 and 9.5 percentage units, respectively. Except for that of herring meal

  1. Effects of Synchronicity of Carbohydrate and Protein Degradation on Rumen Fermentation Characteristics and Microbial Protein Synthesis

    Directory of Open Access Journals (Sweden)

    J. K. Seo

    2013-03-01

    Full Text Available A series of in vitro studies were carried out to determine i the effects of enzyme and formaldehyde treatment on the degradation characteristics of carbohydrate and protein sources and on the synchronicity of these processes, and ii the effects of synchronizing carbohydrate and protein supply on rumen fermentation and microbial protein synthesis (MPS in in vitro experiments. Untreated corn (C and enzyme-treated corn (EC were combined with soy bean meal with (ES and without (S enzyme treatment or formaldehyde treatment (FS. Six experimental feeds (CS, CES, CFS, ECS, ECES and ECFS with different synchrony indices were prepared. Highly synchronous diets had the greatest dry matter (DM digestibility when untreated corn was used. However, the degree of synchronicity did not influence DM digestibility when EC was mixed with various soybean meals. At time points of 12 h and 24 h of incubation, EC-containing diets showed lower ammonia-N concentrations than those of C-containing diets, irrespective of the degree of synchronicity, indicating that more efficient utilization of ammonia-N for MPS was achieved by ruminal microorganisms when EC was offered as a carbohydrate source. Within C-containing treatments, the purine base concentration increased as the diets were more synchronized. This effect was not observed when EC was offered. There were significant effects on VFA concentration of both C and S treatments and their interactions. Similar to purine concentrations, total VFA production and individual VFA concentration in the groups containing EC as an energy source was higher than those of other groups (CS, CES and CFS. The results of the present study suggested that the availability of energy or the protein source are the most limiting factors for rumen fermentation and MPS, rather than the degree of synchronicity.

  2. Effect of pH 5 enzyme from liver on the protein synthesis by mammary gland subcellular fractions in vitro

    International Nuclear Information System (INIS)

    Singh, Jaspal; Singh, Ajit; Ganguli, N.C.

    1976-01-01

    The effect of pH 5 enzyme fraction of liver on the protein synthesizing activity of the subcellular fractions of the mammary gland has been investigated. Results indicate that (1) lactating liver pH 5 enzyme stimulates protein synthesis which is enhanced by the addition of ATP-generating system and (2) the enzyme fractions from the non-lactating liver inhibits the protein synthesis by mammary fractions, but in some cases like mitochondrial and supernatant fractions of mammary it elevates the synthesis when supplemented with ATP-generating system. Chlorella protein hydrolysate- 14 C was used as a tracer and rabits were used as experimental animals. (M.G.B.)

  3. Fractionation, amino acid profiles, antimicrobial and free radical scavenging activities of Citrullus lanatus seed protein.

    Science.gov (United States)

    Dash, Priyanka; Ghosh, Goutam

    2017-12-01

    In the present study, a modified Osborne fractionation method was followed to isolate albumin (C alb ), globulin (C glo ), prolamin (C pro ) and glutelin (C glu ) successively from seeds of Citrullus lanatus (watermelon). This research work was undertaken to investigate the antimicrobial and antioxidant activities of isolated protein fractions of C. lanatus seed. Amino acid composition and molecular weight distribution were determined to establish their relationship with antimicrobial and antioxidant activity. Among all the fractions, C pro was found to be most effective against A. baumannii followed by C alb and C glo . The results showed that growth of inhibition of these protein fractions differ significantly from each other (p ≤ 0.05). In view of antioxidant potential, C glo exhibited strongest antioxidant capacity while C glu showed weakest antioxidant potential.

  4. Glucose Deprivation Triggers Protein Kinase C-dependent β-Catenin Proteasomal Degradation*

    Science.gov (United States)

    Choi, Seung-Won; Song, Jun-Kyu; Yim, Ye-Seal; Yun, Ho-Geun; Chun, Kyung-Hee

    2015-01-01

    Autophagy is a conserved process that contributes to cell homeostasis. It is well known that induction mainly occurs in response to nutrient starvation, such as starvation of amino acids and insulin, and its mechanisms have been extensively characterized. However, the mechanisms behind cellular glucose deprivation-induced autophagy are as of now poorly understood. In the present study, we determined a mechanism by which glucose deprivation induced the PKC-dependent proteasomal degradation of β-catenin, leading to autophagy. Glucose deprivation was shown to cause a sub-G1 transition and enhancement of the LC3-II protein levels, whereas β-catenin protein underwent degradation in a proteasome-dependent manner. Moreover, the inhibition of GSK3β was unable to abolish the glucose deprivation-mediated β-catenin degradation or up-regulation of LC3-II protein levels, which suggested GSK3β-independent protein degradation. Intriguingly, the inhibition of PKCα using a pharmacological inhibitor and transfection of siRNA for PKCα was observed to effectively block glucose deprivation-induced β-catenin degradation as well as the increase in LC3-II levels and the accumulation of a sub-G1 population. Together, our results demonstrated a molecular mechanism by which glucose deprivation can induce the GSK3β-independent protein degradation of β-catenin, leading to autophagy. PMID:25691573

  5. Light-induced protein nitration and degradation with HONO emission

    Science.gov (United States)

    Meusel, Hannah; Elshorbany, Yasin; Kuhn, Uwe; Bartels-Rausch, Thorsten; Reinmuth-Selzle, Kathrin; Kampf, Christopher J.; Li, Guo; Wang, Xiaoxiang; Lelieveld, Jos; Pöschl, Ulrich; Hoffmann, Thorsten; Su, Hang; Ammann, Markus; Cheng, Yafang

    2017-10-01

    Proteins can be nitrated by air pollutants (NO2), enhancing their allergenic potential. This work provides insight into protein nitration and subsequent decomposition in the presence of solar radiation. We also investigated light-induced formation of nitrous acid (HONO) from protein surfaces that were nitrated either online with instantaneous gas-phase exposure to NO2 or offline by an efficient nitration agent (tetranitromethane, TNM). Bovine serum albumin (BSA) and ovalbumin (OVA) were used as model substances for proteins. Nitration degrees of about 1 % were derived applying NO2 concentrations of 100 ppb under VIS/UV illuminated conditions, while simultaneous decomposition of (nitrated) proteins was also found during long-term (20 h) irradiation exposure. Measurements of gas exchange on TNM-nitrated proteins revealed that HONO can be formed and released even without contribution of instantaneous heterogeneous NO2 conversion. NO2 exposure was found to increase HONO emissions substantially. In particular, a strong dependence of HONO emissions on light intensity, relative humidity, NO2 concentrations and the applied coating thickness was found. The 20 h long-term studies revealed sustained HONO formation, even when concentrations of the intact (nitrated) proteins were too low to be detected after the gas exchange measurements. A reaction mechanism for the NO2 conversion based on the Langmuir-Hinshelwood kinetics is proposed.

  6. Ruminal degradation kinetics of protein foods by in vitro gas production technique

    Directory of Open Access Journals (Sweden)

    Ivone Yurika Mizubuti

    2014-02-01

    Full Text Available Chemical analysis of carbohydrates and nitrogen fractions, as well as, determination their carbohydrates digestion rates in soyben meal (SM, crambe meal (CM, radish meal (RM, wet brewery residue (WBR and dehydrated silkworm chrysalis (SCD were accomplished. The kinetics parameters of non-fibrous carbohydrates (NFC and B2 fraction were estimated using cumulative gas production technique. Among the foods studied there was considerable variation in chemical composition. The crambe meal was the only food that did not present synchronism between carbohydrate and nitrogen fractions. In this food there was predominance of A+B1 carbohydrates fractions and B1+B2 nitrogen compounds fraction, and for the other predominated B2 carbohydrate fraction and B1+ B2 nitrogen compounds fraction. There were differences among the digestive kinetic parameters for all foods. The greater participation in gas production due to non-fibrous carbohydrates was found in the crambe meal and oilseed radish meal. The fermentation of fibrous carbohydrates provided higher gas volume in the wet brewery residue and in the soybean meal, however, the soybean meal was food with higher total gas volume. Non fibrous carbohydrates degradation rates of wet brewery residue and dehydrated silkworm chrysalis were far below the limits of degradation of this fraction. Due to the parameters obtained by the cumulative gas production, the soybean meal was the best food, however, all others have potential for use in animal nutrition. The cumulative gas production technique allows the estimative of degradation rates and provides further information about the ruminal fermentation kinetics of foods.

  7. Context Memory Formation Requires Activity-Dependent Protein Degradation in the Hippocampus

    Science.gov (United States)

    Cullen, Patrick K.; Ferrara, Nicole C.; Pullins, Shane E.; Helmstetter, Fred J.

    2017-01-01

    Numerous studies have indicated that the consolidation of contextual fear memories supported by an aversive outcome like footshock requires de novo protein synthesis as well as protein degradation mediated by the ubiquitin-proteasome system (UPS). Context memory formed in the absence of an aversive stimulus by simple exposure to a novel…

  8. Role of Proteasome-Dependent Protein Degradation in Long-Term Operant Memory in "Aplysia"

    Science.gov (United States)

    Lyons, Lisa C.; Gardner, Jacob S.; Gandour, Catherine E.; Krishnan, Harini C.

    2017-01-01

    We investigated the in vivo role of protein degradation during intermediate (ITM) and long-term memory (LTM) in "Aplysia" using an operant learning paradigm. The proteasome inhibitor MG-132 inhibited the induction and molecular consolidation of LTM with no effect on ITM. Remarkably, maintenance of steady-state protein levels through…

  9. 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.

  10. Optically degradable dendrons for temporary adhesion of proteins to DNA.

    Science.gov (United States)

    Kostiainen, Mauri A; Kotimaa, Juha; Laukkanen, Marja-Leena; Pavan, Giovanni M

    2010-06-18

    Experimental studies and molecular dynamics modeling demonstrate that multivalent dendrons can be used to temporarily glue proteins and DNA together with high affinity. We describe N-maleimide-cored polyamine dendrons that can be conjugated with free cysteine residues on protein surfaces through 1,4-conjugate addition to give one-to-one protein-polymer conjugates. We used a genetically engineered cysteine mutant of class II hydrophobin (HFBI) and a single-chain Fragment variable (scFv) antibody as model proteins for the conjugation reactions. The binding affinity of the protein-dendron conjugates towards DNA was experimentally assessed by using the ethidium bromide displacement assay. The binding was found to depend on the generation of the dendron, with the second generation having a stronger affinity than the first generation. Thermodynamic parameters of the binding were obtained from molecular dynamics modeling, which showed that the high binding affinity for each system is almost completely driven by a strong favorable binding enthalpy that is opposed by unfavorable binding entropy. A short exposure to UV (lambda approximately 350 nm) can cleave the photolabile o-nitrobenzyl-linked binding ligands from the surface of the dendron, which results in loss of the multivalent binding interactions and triggers the release of the DNA and protein. The timescale of the release is very rapid and the binding partners can be efficiently released after 3 min of UV exposure.

  11. Proteins in Soy Might Have a Higher Role in Cancer Prevention than Previously Expected: Soybean Protein Fractions Are More Effective MMP-9 Inhibitors Than Non-Protein Fractions, Even in Cooked Seeds

    Directory of Open Access Journals (Sweden)

    Ana Lima

    2017-02-01

    Full Text Available The search for anticancer MMP-9 inhibitors (MMPIs in food products has become a major goal for research. MMPIs in soy have been related only to saponins and isoflavones, but recently, low specific protein fractions in soybeans were shown to reduce MMP-9 activity as well. The present work aimed at comparing the MMPI potential of protein fractions (P and non-protein fractions (NP isolated from soybean seeds, before and after soaking and cooking, mimicking dietary exposures. Reverse and substrate zymography, as well as a fluoregenic DQ gelatin assay were used to evaluate MMP-9 activities. Colon cancer cell migration and proliferation was also tested in HT29 cells. Regarding MMP-9 inhibition, proteins in soy presented IC50 values 100 times lower than non-protein extracts, and remained active after cooking, suggesting that proteins may be more effective MMP-9 inhibitors than non-protein compounds. Using the determined IC50 concentrations, NP fractions were able to induce higher inhibitions of HT29 cell migration and proliferation, but not through MMP-9 inhibition, whilst protein fractions were shown to specifically inhibit MMP-9 activity. Overall, our results show that protein fractions in soybeans might have a higher role in soy-related cancer prevention as MMPIs than previously expected. Being nontoxic and active at lower concentrations, the discovery of these heat-resistant specific MMPI proteins in soy can be of significant importance for cancer preventive diets, particularly considering the increasing use of soy proteins in food products and the controversy around isoflavones amongst consumers.

  12. Does altered protein metabolism interfere with postmortem degradation analysis for PMI estimation?

    Science.gov (United States)

    Zissler, A; Ehrenfellner, B; Foditsch, E E; Monticelli, F C; Pittner, S

    2018-03-02

    An accurate estimation of the postmortem interval (PMI) is a central aspect in forensic routine. Recently, a novel approach based on the analysis of postmortem muscle protein degradation has been proposed. However, a number of questions remain to be answered until sensible application of this method to a broad variety of forensic cases is possible. To evaluate whether altered in vivo protein metabolism interferes with postmortem degradation patterns, we conducted a comparative study. We developed a standardized animal degradation model in rats, and collected additional muscle samples from animals recovering from muscle injury and from rats with developed disuse muscle atrophy after induced spinal cord injury. All samples were analyzed by SDS-PAGE and Western blot, labeling well-characterized muscle proteins. Tropomyosin was found to be stable throughout the investigated PMI and no alterations were detected in regenerating and atrophic muscles. In contrast, significant predictable postmortem changes occurred in desmin and vinculin protein band patterns. While no significant deviations from native patterns were detected in at-death samples of disuse muscle atrophy, interestingly, samples of rats recovering from muscle injury revealed additional desmin and vinculin degradation bands that did not occur in this form in any of the examined postmortem samples regardless of PMI. It remains to be investigated whether in vivo-altered metabolism influences postmortem degradation kinetics or if such muscle samples undergo postmortem degradation in a regular fashion.

  13. The mitochondrial translocator protein, TSPO, inhibits HIV-1 envelope glycoprotein biosynthesis via the endoplasmic reticulum-associated protein degradation pathway.

    Science.gov (United States)

    Zhou, Tao; Dang, Ying; Zheng, Yong-Hui

    2014-03-01

    The HIV-1 Env glycoprotein is folded in the endoplasmic reticulum (ER), which is necessary for viral entry and replication. Currently, it is still unclear how this process is regulated. The glycoprotein folding in the ER is controlled by the ER-associated protein degradation (ERAD) pathway, which specifically targets misfolded proteins for degradation. Previously, we reported that HIV-1 replication is restricted in the human CD4(+) T cell line CEM.NKR (NKR). To understand this mechanism, we first analyzed cellular protein expression in NKR cells and discovered that levels of the mitochondrial translocator protein TSPO were upregulated by ∼64-fold. Notably, when NKR cells were treated with TSPO antagonist PK-11195, Ro5-4864, or diazepam, HIV restriction was completely disrupted, and TSPO knockdown by short hairpin RNAs (shRNAs) achieved a similar effect. We next analyzed viral protein expression, and, interestingly, we discovered that Env expression was specifically inhibited. Both TSPO knockdown and treatment with TSPO antagonist could restore Env expression in NKR cells. We further discovered that Env proteins were rapidly degraded and that kifunensine, an ERAD pathway inhibitor, could restore Env expression and viral replication, indicating that Env proteins were misfolded and degraded through the ERAD pathway in NKR cells. We also knocked out the TSPO gene in 293T cells using CRISPR/Cas9 (clustered, regularly interspaced, short palindromic repeat [CRISPR]/CRISPR-associated-9) technology and found that TSPO could similarly inhibit Env expression in these cells. Taken together, these results demonstrate that TSPO inhibits Env protein expression through the ERAD pathway and suggest that mitochondria play an important role in regulating the Env folding process. The HIV-1 Env glycoprotein is absolutely required for viral infection, and an understanding of its expression pathway in infected cells will identify new targets for antiretroviral therapies. Env proteins

  14. [L-arginine metabolism enzyme activities in rat liver subcellular fractions under condition of protein deprivation].

    Science.gov (United States)

    Kopyl'chuk, G P; Buchkovskaia, I M

    2014-01-01

    The features of arginase and NO-synthase pathways of arginine's metabolism have been studied in rat liver subcellular fractions under condition of protein deprivation. During the experimental period (28 days) albino male rats were kept on semi synthetic casein diet AIN-93. The protein deprivation conditions were designed as total absence of protein in the diet and consumption of the diet partially deprived with 1/2 of the casein amount compared to in the regular diet. Daily diet consumption was regulated according to the pair feeding approach. It has been shown that the changes of enzyme activities, involved in L-arginine metabolism, were characterized by 1.4-1.7 fold decrease in arginase activity, accompanied with unchanged NO-synthase activity in cytosol. In mitochondrial fraction the unchanged arginase activity was accompanied by 3-5 fold increase of NO-synthase activity. At the terminal stages of the experiment the monodirectional dynamics in the studied activities have been observed in the mitochondrial and cytosolfractions in both experimental groups. In the studied subcellular fractions arginase activity decreased (2.4-2.7 fold with no protein in the diet and 1.5 fold with partly supplied protein) and was accompanied by NO-synthase activity increase by 3.8 fold in cytosole fraction, by 7.2 fold in mitochondrial fraction in the group with no protein in the diet and by 2.2 and 3.5 fold in the group partialy supplied with protein respectively. The observed tendency is presumably caused by the switch of L-arginine metabolism from arginase into oxidizing NO-synthase parthway.

  15. Role of foam drainage in producing protein aggregates in foam fractionation.

    Science.gov (United States)

    Li, Rui; Zhang, Yuran; Chang, Yunkang; Wu, Zhaoliang; Wang, Yanji; Chen, Xiang'e; Wang, Tao

    2017-10-01

    It is essential to obtain a clear understanding of the foam-induced protein aggregation to reduce the loss of protein functionality in foam fractionation. The major effort of this work is to explore the roles of foam drainage in protein aggregation in the entire process of foam fractionation with bovine serum albumin (BSA) as a model protein. The results show that enhancing foam drainage increased the desorption of BSA molecules from the gas-liquid interface and the local concentration of desorbed molecules in foam. Therefore, it intensified the aggregation of BSA in foam fractionation. Simultaneously, it also accelerated the flow of BSA aggregates from rising foam into the residual solution along with the drained liquid. Because enhancing foam drainage increased the relative content of BSA molecules adsorbed at the gas-liquid interface, it also intensified the aggregation of BSA during both the defoaming process and the storage of the foamate. Furthermore, enhancing foam drainage more readily resulted in the formation of insoluble BSA aggregates. The results are highly important for a better understanding of foam-induced protein aggregation in foam fractionation. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Recent Advance in Division of Carbohydrate and Protein Fractions of Ruminant Feed and Their Metabolism in Digestive Tract

    Institute of Scientific and Technical Information of China (English)

    Xiaohua; PAN; Liang; YANG; Hairui; XIN; Benhai; XIONG

    2016-01-01

    Accurate assessment of feed’s Carbohydrate( CHO) and protein nutritional values and rumen metabolism are significant for dairy production. Cornell Net Carbohydrate and Protein System( CNCPS) as an important method to evaluate feedstuff nutritional values,hasn’t been widely used in China. In order to illustrate updates of CNCPS systems deeply,the following sections were reviewed:( i) CHO and protein fractions were updated,CA was subdivided into CA1,CA2,CA3 and CA4 in CNCPS v6. 1,protein was reclassified into PA1,PA2,PB1,PB2 and PC after CNCPS v6. 1. Content of CHO and protein fractions vary in different feedstuff and affected by feed processing;( ii) Degradation rates( Kd) values for the new CA expanded scheme were updated to 0,7,5,40- 60 % h-1respectively,Kd for PA and PB1 decreased to 200 % h-1and 10- 40 % h-1;( iii) Equations for passage rate( Kp) initially includes Kpf( Kp of forages) and Kpc( Kp of concentrates),and adjusted by effective NDF( e NDF),while in CNCPS v5. 0,Kpl( Kp of liquids) equation was added and e NDF was replaced by physically effective NDF( pe NDF). In CNCPS v6. 1,Fp BW and Cp BW were integrated into Kp equations and pe NDF was abandoned.( iv)The relationship and difference among Weende system of proximate analysis,Van Soest fiber analysis[35],NRC( 2001)[28]and CNCPS were analyzed. The first two systems laid the foundation for NRC( 2001) and CNCPS system. The latter two systems are different in CHO and protein division,also NRC( 2001) developed separate Kp equations for wet and dry forages but no equation for Kpl. CNCPS developed a Kp equation that work for wet and dry forages,and Kpl equation was established. In conclusion,the division and development of CHO and protein fractions,the update of Kd and Kp equation were reviewed systematically.

  17. Proteomes and Ubiquitylomes Analysis Reveals the Involvement of Ubiquitination in Protein Degradation in Petunias1

    Science.gov (United States)

    Liu, Juanxu; Wei, Qian; Wang, Rongmin; Yang, Weiyuan; Ma, Yueyue; Chen, Guoju

    2017-01-01

    Petal senescence is a complex programmed process. It has been demonstrated previously that treatment with ethylene, a plant hormone involved in senescence, can extensively alter transcriptome and proteome profiles in plants. However, little is known regarding the impact of ethylene on posttranslational modification (PTM) or the association between PTM and the proteome. Protein degradation is one of the hallmarks of senescence, and ubiquitination, a major PTM in eukaryotes, plays important roles in protein degradation. In this study, we first obtained reference petunia (Petunia hybrida) transcriptome data via RNA sequencing. Next, we quantitatively investigated the petunia proteome and ubiquitylome and the association between them in petunia corollas following ethylene treatment. In total, 51,799 unigenes, 3,606 proteins, and 2,270 ubiquitination sites were quantified 16 h after ethylene treatment. Treatment with ethylene resulted in 14,448 down-regulated and 6,303 up-regulated unigenes (absolute log2 fold change > 1 and false discovery rate petunia. Several putative ubiquitin ligases were up-regulated at the protein and transcription levels. Our results showed that the global proteome and ubiquitylome were negatively correlated and that ubiquitination could be involved in the degradation of proteins during ethylene-mediated corolla senescence in petunia. Ethylene regulates hormone signaling transduction pathways at both the protein and ubiquitination levels in petunia corollas. In addition, our results revealed that ethylene increases the ubiquitination levels of proteins involved in endoplasmic reticulum-associated degradation. PMID:27810942

  18. The comparison of naturally weathered oil and artificially photo-degraded oil at the molecular level by a combination of SARA fractionation and FT-ICR MS

    International Nuclear Information System (INIS)

    Islam, Ananna; Cho, Yunju; Yim, Un Hyuk; Shim, Won Joon; Kim, Young Hwan; Kim, Sunghwan

    2013-01-01

    Highlights: • Weathered oils from the Hebei Spirit oil spill and photo degraded oils are compared. • We investigate changes of polar species at the molecular level by 15T FT-ICR MS. • Significant reduction of sulfur class compounds in saturates fraction is observed. • The relative abundance of protonated compounds (presumably basic nitrogen compounds) increase after degradation. • Changes of polar compounds occurred by natural and photo degradation are similar. -- Abstract: Two sets of oil samples, one obtained from different weathering stages of the M/V Hebei Spirit oil spill site and the other prepared by an in vitro photo-degradation experiment, were analyzed and compared at the molecular level by atmospheric pressure photo-ionization coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). For a more detailed comparison at the molecular level, the oil samples were separated into saturate, aromatic, resin, and asphaltene (SARA) fractions before MS analysis. Gravimetric analysis of the SARA fractions revealed a decreased weight percentage of the aromatic fraction and an increased resin fraction in both sets of samples. Molecular-level investigations of the SARA fractions showed a significant reduction in the S 1 class in the saturate fraction and increase of S 1 O 1 class compounds with high DBE values in resin fraction. Levels of N 1 and N 1 O 1 class compounds resulting in protonated ions (presumably basic nitrogen compounds) increased after degradation compared to compounds generating molecular ions (presumably non-basic nitrogen compounds). This study revealed changes occurring in heteroatom polar species of crude oils such as sulfur and nitrogen containing compounds that have not been easily detected with conventional GC based techniques

  19. Long-term aversive taste memory requires insular and amygdala protein degradation.

    Science.gov (United States)

    Rodriguez-Ortiz, Carlos J; Balderas, Israela; Saucedo-Alquicira, Fernando; Cruz-Castañeda, Paulina; Bermudez-Rattoni, Federico

    2011-03-01

    Some reports have shown that the ubiquitin-proteasome system (UPS) is necessary to degrade repressor factors to produce new proteins essential to memory consolidation. Furthermore, recent evidence suggests that memory updating also relies on protein degradation through the UPS. To evaluate whether degradation of proteins is part of the cellular events needed for long-term storage of taste aversion, we injected lactacystin--an UPS inhibitor--into the amygdala and/or insular cortex 30 min before the first or second training trials. The results revealed that degradation of proteins in either the amygdala or insular cortex suffices for long-term stabilization of first-time encounter taste aversion. On the other hand, lactacystin applied in the insula, but not in the amygdala, before the second training prevented long-term storage of updated information. Our results support that degradation of proteins by means of the UPS is required every time taste aversion is to be stored in long-term memory. Copyright © 2010 Elsevier Inc. All rights reserved.

  20. Monoubiquitination of Tob/BTG family proteins competes with degradation-targeting polyubiquitination

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Toru, E-mail: toru@ims.u-tokyo.ac.jp [Division of Oncology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Kim, Minsoo [Division of Bacterial Infection, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Kozuka-Hata, Hiroko [Medical Proteomics Laboratory, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Watanabe, Masato [Department of Medical Genome Science, School of Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa 277-8562 (Japan); Oyama, Masaaki [Medical Proteomics Laboratory, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Tsumoto, Kouhei [Medical Proteomics Laboratory, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Department of Medical Genome Science, School of Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa 277-8562 (Japan); Yamamoto, Tadashi, E-mail: tyamamot@ims.u-tokyo.ac.jp [Division of Oncology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Cell Signal Unit, Okinawa Institute of Science and Technology, 1919-1 Onna-son, Kunigami, Okinawa 904-0412 (Japan)

    2011-05-27

    Highlights: {yields} Tob/BTG family proteins are monoubiquitinated in the absence of E3s in vitro. {yields} Monoubiquitination sites of Tob are identified by mass spectrometry. {yields} The monoubiquitination event correlates with lower levels of polyubiquitination. -- Abstract: Tob belongs to the anti-proliferative Tob/BTG protein family. The expression level of Tob family proteins is strictly regulated both transcriptionally and through post-translational modification. Ubiquitin (Ub)/proteosome-dependent degradation of Tob family proteins is critical in controlling cell cycle progression and DNA damage responses. Various Ub ligases (E3s) are responsible for degradation of Tob protein. Here, we show that Tob family proteins undergo monoubiquitination even in the absence of E3s in vitro. Determination of the ubiquitination site(s) in Tob by mass spectrometric analysis revealed that two lysine residues (Lys48 and Lys63) located in Tob/BTG homology domain are ubiquitinated. A mutant Tob, in which both Lys48 and Lys63 are substituted with alanine, is more strongly polyubiquitinated than wild-type Tob in vivo. These data suggest that monoubiquitination of Tob family proteins confers resistance against polyubiquitination, which targets proteins for degradation. The strategy for regulating the stability of Tob family proteins suggests a novel role for monoubiquitination.

  1. Monoubiquitination of Tob/BTG family proteins competes with degradation-targeting polyubiquitination

    International Nuclear Information System (INIS)

    Suzuki, Toru; Kim, Minsoo; Kozuka-Hata, Hiroko; Watanabe, Masato; Oyama, Masaaki; Tsumoto, Kouhei; Yamamoto, Tadashi

    2011-01-01

    Highlights: → Tob/BTG family proteins are monoubiquitinated in the absence of E3s in vitro. → Monoubiquitination sites of Tob are identified by mass spectrometry. → The monoubiquitination event correlates with lower levels of polyubiquitination. -- Abstract: Tob belongs to the anti-proliferative Tob/BTG protein family. The expression level of Tob family proteins is strictly regulated both transcriptionally and through post-translational modification. Ubiquitin (Ub)/proteosome-dependent degradation of Tob family proteins is critical in controlling cell cycle progression and DNA damage responses. Various Ub ligases (E3s) are responsible for degradation of Tob protein. Here, we show that Tob family proteins undergo monoubiquitination even in the absence of E3s in vitro. Determination of the ubiquitination site(s) in Tob by mass spectrometric analysis revealed that two lysine residues (Lys48 and Lys63) located in Tob/BTG homology domain are ubiquitinated. A mutant Tob, in which both Lys48 and Lys63 are substituted with alanine, is more strongly polyubiquitinated than wild-type Tob in vivo. These data suggest that monoubiquitination of Tob family proteins confers resistance against polyubiquitination, which targets proteins for degradation. The strategy for regulating the stability of Tob family proteins suggests a novel role for monoubiquitination.

  2. The F-box protein FBXO44 mediates BRCA1 ubiquitination and degradation.

    Science.gov (United States)

    Lu, Yunzhe; Li, Jiezhi; Cheng, Dongmei; Parameswaran, Balaji; Zhang, Shaohua; Jiang, Zefei; Yew, P Renee; Peng, Junmin; Ye, Qinong; Hu, Yanfen

    2012-11-30

    BRCA1 mutations account for a significant proportion of familial breast and ovarian cancers. In addition, reduced BRCA1 protein is associated with sporadic cancer cases in these tissues. At the cellular level, BRCA1 plays a critical role in multiple cellular functions such as DNA repair and cell cycle checkpoint control. Its protein level is regulated in a cell cycle-dependent manner. However, regulation of BRCA1 protein stability is not fully understood. Our earlier study showed that the amino terminus of BRCA1 harbors a degron sequence that is sufficient and necessary for conferring BRCA1 degradation. In the current study, we used mass spectrometry to identify Skp1 that regulates BRCA1 protein stability. Small interfering RNA screening that targets all human F-box proteins uncovered FBXO44 as an important protein that influences BRCA1 protein level. The Skp1-Cul1-F-box-protein44 (SCF(FBXO44)) complex ubiquitinates full-length BRCA1 in vitro. Furthermore, the N terminus of BRCA1 mediates the interaction between BRCA1 and FBXO44. Overexpression of SCF(FBXO44) reduces BRCA1 protein level. Taken together, our work strongly suggests that SCF(FBXO44) is an E3 ubiquitin ligase responsible for BRCA1 degradation. In addition, FBXO44 expression pattern in breast carcinomas suggests that SCF(FBXO44)-mediated BRCA1 degradation might contribute to sporadic breast tumor development.

  3. The F-box Protein FBXO44 Mediates BRCA1 Ubiquitination and Degradation*

    Science.gov (United States)

    Lu, Yunzhe; Li, Jiezhi; Cheng, Dongmei; Parameswaran, Balaji; Zhang, Shaohua; Jiang, Zefei; Yew, P. Renee; Peng, Junmin; Ye, Qinong; Hu, Yanfen

    2012-01-01

    BRCA1 mutations account for a significant proportion of familial breast and ovarian cancers. In addition, reduced BRCA1 protein is associated with sporadic cancer cases in these tissues. At the cellular level, BRCA1 plays a critical role in multiple cellular functions such as DNA repair and cell cycle checkpoint control. Its protein level is regulated in a cell cycle-dependent manner. However, regulation of BRCA1 protein stability is not fully understood. Our earlier study showed that the amino terminus of BRCA1 harbors a degron sequence that is sufficient and necessary for conferring BRCA1 degradation. In the current study, we used mass spectrometry to identify Skp1 that regulates BRCA1 protein stability. Small interfering RNA screening that targets all human F-box proteins uncovered FBXO44 as an important protein that influences BRCA1 protein level. The Skp1-Cul1-F-box-protein44 (SCFFBXO44) complex ubiquitinates full-length BRCA1 in vitro. Furthermore, the N terminus of BRCA1 mediates the interaction between BRCA1 and FBXO44. Overexpression of SCFFBXO44 reduces BRCA1 protein level. Taken together, our work strongly suggests that SCFFBXO44 is an E3 ubiquitin ligase responsible for BRCA1 degradation. In addition, FBXO44 expression pattern in breast carcinomas suggests that SCFFBXO44-mediated BRCA1 degradation might contribute to sporadic breast tumor development. PMID:23086937

  4. Crude protein changes on grassland along a degradation gradient ...

    African Journals Online (AJOL)

    The aim with this investigation was to quantify the impact of different veld condition classes, viz. poor, moderate and good on soil-water utilization (SWU: crude protein produced per unit of evapotranspiration), during four growing seasons (2000/01 – 2003/2004). Evapotranspiration was determined by quantifying the ...

  5. Crude protein changes on grassland along a degradation gradient ...

    African Journals Online (AJOL)

    Evapotranspiration was determined by quantifying the soil-water balance equation with the aid of runoff plots and soil-water content measurements. Crude protein ... The study shows that it is important to keep grassland in optimal condition to utilize limited soil water for sustainable plant and therefore animal production.

  6. Short communication In situ ruminal degradability of protein feeds ...

    African Journals Online (AJOL)

    2016-12-28

    Dec 28, 2016 ... ISSN 0375-1589 (print), ISSN 2221-4062 (online) ... protein at a passage rate of 2% per hour was higher in meals than in grains. .... beef cattle reduced the consumption rate, but improved the feed conversion rate, increasing.

  7. Determination of ABA-binding proteins contents in subcellular fractions isolated from cotton seedlings using radioimmunoanalysis

    International Nuclear Information System (INIS)

    Tursunkhodjayeva, F.M.

    2004-01-01

    Full text: Knowledge of plants' hormone receptor sites is essential to understanding of the principles of phytohormone action in cells and tissues. The hormone abscisic acid (ABA) takes part in many important physiological processes of plants, including water balance and resistance to salt stress. The detection of salt tolerance in the early stages of ontogenesis is desirable for effective cultivation of cotton. Usually such characteristics are determined visually after genetic analysis of hybrids over several generations. This classic method of genetics requires a long time to grow several generations of cotton plants. In this connection we study ABA-binding protein contents in subcellular fractions isolated from seedlings of several kinds of cotton with different tolerance to salt stress. The contents of ABA-binding protein in nuclei and chloroplasts fractions isolated from cotton seedlings were determined using radioimmunoanalysis. The subcellular fractions were prepared by ultracentrifugation in 0,25 - 2,2 M sucrose gradient. ABA-binding protein was isolated from cotton seedlings by affinity chromatography. The antibodies against ABA-binding protein of cotton were developed in rabbits according standard protocols. Than the antibodies were labelled by radioisotope J 125 according Greenwood et al. It was shown, that the nuclei and chloroplasts fractions isolated from cotton with high tolerance to salt stress contain ABA-binding protein up to 1,5-1,8 times more, than the same fractions from cotton with low tolerance to salt stress. So, the ABA-binding protein contents in cotton seedlings may be considered as a marker for screening of cotton kinds, which may potentially have high tolerance to salt stress

  8. Caracterização, fracionamento protéico, degradabilidade ruminal e digestibilidade in vitro da matéria seca e proteína bruta do resíduo de cervejaria úmido e fermentado - DOI: 10.4025/actascianimsci.v29i3.558 Characterization, protein fractioning, dry matter and crude protein rumen degradability and in vitro digestibility of wet and fermented brewer’s grain - DOI: 10.4025/actascianimsci.v29i3.558

    Directory of Open Access Journals (Sweden)

    Jacco Arnoud Erke

    2007-12-01

    Full Text Available Foram avaliadas as frações da proteína e dos carboidratos, a degradabilidade ruminal efetiva (DE da matéria seca (MS e proteína bruta (PB, a digestibilidade ruminal in vitro (DRIV da MS e PB, a digestibilidade intestinal in vitro (DIIV da proteína nãodegradada no rúmen (PNDR e os perfis de aminoácidos (AA e de ácidos graxos (AG do resíduo de cervejaria úmido (RCU e fermentado (RCF. O RCF foi obtido pelo processo de fermentação microbiana do RCU. Para determinar a DE da MS e PB do RCU e RCF, foram utilizados três novilhos da raça Holandesa, portadores de cânula ruminal. A DIIV da PNDR foi obtida pelo método de três estágios. Os dados obtidos para DE da MS e PB foram submetidos à análise de variância, em delineamento inteiramente casualizado. A fração A da PB do RCU foi de 7,9% e do RCF de 13,1% da PB. A DE da PB a 5% h-1 não diferiu (p IV da PB do RCF foi de 8,7% e as DIIV da PNDR do RCU e do RCF foram de 70,5 e 72,5%, respectivamente. Os perfis de AA e AG do RCU e RCF foram similares. O processo de fermentação anaeróbico não alterou as características nutricionais do RCU.The study evaluated the protein and carbohydrate fraction, dry matter (DM and crude protein (CP effective rumen degradability (ED, DM and CP in vitro ruminal digestibility (RDIV, rumen-undegradable protein (RUDP in vitro intestinal digestion (IDIV and amino-acid (AA and fatty acid (FA profile of the wet brewer’s grain (WBG, and fermented brewer’s grain (FBG. FBG was obtained from WBG fermentation. The DM and CP ED of WBG and FBG were determined in three Holstein steers with ruminal cannula. The IDIV of RUDP was obtained by the three-stage method. The values obtained for DM and CP ED were submitted to variance analysis, in a randomized design. The A fraction of WBG CP was 7.9%, and for FBG 13.1% of CP. The CP RD in a rate of 5% h-1 did not differ (p > 0.05 between WBG and FBG. The crude protein RDIV of FBG was 8.7% and IDIV of RUDP of WBG and

  9. Hydrolysis of Whey Protein Isolate with Bacillus licheniformis Protease: Fractionation and Identification of Aggregating Peptides

    NARCIS (Netherlands)

    Creusot, N.P.; Gruppen, H.

    2007-01-01

    The objective of this work was to identify the dominant aggregating peptides from a whey protein hydrolysate (degree of hydrolysis of 6.8%) obtained with Bacillus licheniformis protease. The aggregating peptides were fractionated with preparative reversed-phase chromatography and identified with

  10. Biosynthetically directed fractional 13C labeling facilitates identification of Phe and Tyr aromatic signals in proteins

    International Nuclear Information System (INIS)

    Jacob, Jaison; Louis, John M.; Nesheiwat, Issa; Torchia, Dennis A.

    2002-01-01

    Analysis of 2D [ 13 C, 1 H]-HSQC spectra of biosynthetic fractionally 13 C labeled proteins is a reliable, straightforward means to obtain stereospecific assignments of Val and Leu methyl sites in proteins. Herein we show that the same fractionally labeled protein sample facilitates observation and identification of Phe and Tyr aromatic signals. This is the case, in part, because the fractional 13 C labeling yields aromatic rings in which some of the 13 C- 13 C J-couplings, present in uniformly labeled samples, are absent. Also, the number of homonuclear J-coupling partners differs for the δ-, ε- and ζ-carbons. This enabled us to vary their signal intensities in distinctly different ways by appropriately setting the 13 C constant-time period in 2D [ 13 C, 1 H]-HSQC spectra. We illustrate the application of this approach to an 18 kDa protein, c-VIAF, a modulator of apoptosis. In addition, we show that cancellation of the aromatic 13 C CSA and 13 C- 1 H dipolar interactions can be fruitfully utilized in the case of the fractionally labeled sample to obtain high resolution 13 C constant-time spectra with good sensitivity

  11. Concentration of Proteins and Protein Fractions in Blood Plasma of Chickens Hatched from Eggs Irradiated with Low Level Gamma Rays

    International Nuclear Information System (INIS)

    Kraljevic, P.; Vilic, M.; Simpraga, M.; Matisic, D.; Miljanic, S.

    2011-01-01

    In literature there are many results which have shown that low dose radiation can stimulate many physiological processes of living organism. In our earlier paper it was shown that low dose of gamma radiation has a stimulative effect upon metabolic process in chickens hatched from eggs irradiated before incubation. This was proved by increase of body weight gain and body weight, as well as by increase of two enzymes activities in blood plasma (aspartate aminotransferase and alanine aminotransferase) which play an important role in protein metabolism. Therefore, an attempt was made to determine the effect of eggs irradiation by low dose gamma rays upon concentration of total proteins and protein fractions in the blood plasma of chickens hatched from irradiated eggs. The eggs of heavy breed chickens were irradiated with a dose of 0.15 Gy gamma radiation (60Co) before incubation. Along with the chickens which were hatched from irradiated eggs, there was a control group of chickens hatched from nonirradiated eggs. All other conditions were the same for both groups of chickens. Blood samples were taken from the right jugular vein on the 1 s t and 3 r d day, or from the wing vein on days 5 and 7 after hatching. The total proteins concentration in the blood plasma was determined by the biuret method using Boehringer Mannheim GmbH optimized kits. The protein fractions (albumin, α 1 -globulin, α 2 -globulin, β- and γ-globulins) were estimated electrophoretically on Cellogel strips. The total proteins concentration was significantly decreased in blood plasma of chickens hatched from irradiated eggs on days 3 (P t h day (P 2 -globulin was decreased on days 1 (P t h day of life. Obtained results indicate that low dose of gamma radiation has mostly inhibitory effect upon concentration of total proteins and protein fractions in the blood plasma of chickens hatched from irradiated eggs before incubation. (author)

  12. White clover fractions as protein source for monogastrics: dry matter digestibility and protein digestibility-corrected amino acid scores.

    Science.gov (United States)

    Stødkilde, Lene; Damborg, Vinni K; Jørgensen, Henry; Laerke, Helle N; Jensen, Søren K

    2018-05-01

    The present study aimed to evaluate the use of white clover as an alternative protein source for monogastrics. White clover plant and leaves were processed using a screw-press resulting in a solid pulp and a juice from which protein was acid-precipitated. The chemical composition of all fractions was determined and digestibility of dry matter (DM) and protein was assessed in an experiment with growing rats. Protein concentrates were produced with crude protein (CP) contents of 451 g kg -1 and 530 g kg -1 DM for white clover plant and leaves, respectively, and a pulp with CP contents of 313 and 374 g kg -1 DM from plant and leaves, respectively. The amino acid composition ranged from 4.72 to 6.49 g per 16 g of nitrogen (N) for lysine, 1.82-2.6 g per 16 g N for methionine and cysteine, and 3.66-5.24 g per 16 g N for threonine. True faecal digestibility of protein varied from 0.81 to 0.88, whereas DM digestibility was in the range 0.72-0.80. Methionine and cysteine were found to be limiting in all fractions, regardless of the reference group used. A high digestibility of white clover protein was found irrespective of the physical fractionation. Together with a well-balanced amino acid composition, this makes white clover a promising protein source for monogastrics. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  13. The use of a tannin crude extract from Cistus ladanifer L. to protect soya-bean protein from degradation in the rumen.

    Science.gov (United States)

    Dentinho, M T P; Moreira, O C; Pereira, M S; Bessa, R J B

    2007-06-01

    Cistus ladanifer L. (CL) is a perennial shrub abundant in dry woods and dry land of Mediterranean zone, with high level of tannins. Tannins bind to protein, preventing its degradation in the digestive compartments. This tannin/protein complex may be advantageous when partially protecting good-quality feed protein from excessive rumen protein degradation. The objective of this trial was to use a CL phenol crude extract to prevent excessive rumen degradation of soya-bean meal protein. The phenolic compounds were extracted using an acetone/water solution (70:30, v/v). Soya-bean meal was then treated with this crude CL extract, containing 640 g of total phenols (TP) per kg of dry matter (DM), in order to obtain mixtures with 0, 12.5, 25, 50, 100 and 150 g of TP per kg DM. Three rumen-cannulated rams were used to assess in sacco rumen degradability of DM and nitrogen (N). The three-step in vitro procedure was used to determine intestinal digestibility. Increasing extract concentrations quadratically decreased the N-soluble fraction a (R2 = 0.96, P = 0.0001) and increased the non-soluble degradable fraction b (R2 = 0.92, P = 0.005). The rate of degradation c linearly decreased with CL extract doses (R2 = 0.44, P = 0.0065). For the effective rumen degradability of N, a linear reduction (R2 = 0.94, P < 0.0001) was observed. The in vitro intestinal digestibility of protein (ivID) quadratically decreased (R2 = 0.99, P < 0.0001) with TP inclusion and the rumen undegradable protein (RUP) showed a quadratic increase (R2 = 0.94, P = 0.0417). Total intestinal protein availability, computed from the RUP and ivID, linearly decreased with TP inclusion level (R2 = 0.45, P = 0.0033).

  14. Identifiability study of the proteins degradation model, based on ADM1, using simultaneous batch experiments

    DEFF Research Database (Denmark)

    Flotats, X.; Palatsi, J.; Ahring, Birgitte Kiær

    2006-01-01

    are not inhibiting the hydrolysis process. The ADM1 model adequately expressed the consecutive steps of hydrolysis and acidogenesis, with estimated kinetic values corresponding to a fast acidogenesis and slower hydrolysis. The hydrolysis was found to be the rate limiting step of anaerobic degradation. Estimation...... of yield coefficients based on the relative initial slopes of VFA profiles obtained in a simple batch experiment produced satisfactory results. From the identification study, it was concluded that it is possible to determine univocally the related kinetic parameter values for protein degradation...... if the evolution of amino acids is measured in simultaneous batch experiments, with different initial protein and amino acids concentrations....

  15. Elemental analysis of human serum and serum protein fractions by thermal neutron activation

    International Nuclear Information System (INIS)

    Woittiez, J.R.W.

    1984-01-01

    Some applications of thermal neutron activation for the determination of elemental contents in human serum and human serum protein fractions are presented. Firstly total serum is dealt with, secondly serum protein fractions obtained by gel filtration are described. A brief review on the role of (trace) elements in human health and disease and a compilation of literature data for elemental contents in human serum, as obtained by neutron activation techniques, are given. The most important sources of statistical and systematic errors are evaluated. Results for the contents of sodium, potassium, magnesium, bromine, iron, copper, zinc, selenium, rubidium, cesium and antimony in serum are given, with emphasis on control of accuracy and precision. The possible relation between selenium in blood and cancer occurrence in humans is discussed. The results of elemental analyses from cancer patients and from a patient receiving a cytostatic treatment are presented. A survey of literature results for the determination of protein-bound elemental contents in serum is presented. Subsequently, results from a study on the behaviour of elements during gel filtration are discussed. Gel-element and protein-element interactions are studied. Finally the protein-bound occurrence of trace elements in human serum is determined by gel filtration and neutron activation analysis. Results for both desalting and fractionation are given, for the elements bromine, copper, manganese, vanadium, selenium, zinc, rubidium, iron and iodine. (Auth.)

  16. Quantity and functionality of protein fractions in chicken breast fillets affected by white striping.

    Science.gov (United States)

    Mudalal, S; Babini, E; Cavani, C; Petracci, M

    2014-08-01

    Recently, white striations parallel to muscle fibers direction have been observed on the surface of chicken breast, which could be ascribed to intensive growth selection. The aim of this study was to evaluate the effect of white striping on chemical composition with special emphasis on myofibrillar and sarcoplasmic protein fractions that are relevant to the processing features of chicken breast meat. During this study, a total of 12 pectoralis major muscles from both normal and white striped fillets were used to evaluate chemical composition, protein solubility (sarcoplasmic, myofibrillar, and total protein solubility), protein quantity (sarcoplasmic, myofibrillar, and stromal proteins), water holding capacity, and protein profile by SDS-PAGE analysis. White-striped fillets exhibited a higher percentage of moisture (75.4 vs. 73.8%; P cooking loss (33.7 vs. 27.4%; P chicken breast meat with white striping defect had different chemical composition (more fat and less protein) and protein quality and quantity (low content of myofibrillar proteins and high content of stromal proteins) with respect to normal meat. Furthermore, white striped fillets had lower protein functionality (higher cooking loss). All the former changes indicate that white striping has great impact on quality characteristics of chicken breast meat. © Poultry Science Association Inc.

  17. Integrin-like proteins are localized to plasma membrane fractions, not plastids, in Arabidopsis

    Science.gov (United States)

    Swatzell, L. J.; Edelmann, R. E.; Makaroff, C. A.; Kiss, J. Z.

    1999-01-01

    Integrins are a large family of integral membrane proteins that function in signal transduction in animal systems. These proteins are conserved in vertebrates, invertebrates, and fungi. Evidence from previous research suggests that integrin-like proteins may be present in plants as well, and that these proteins may function in signal transduction during gravitropism. In past studies, researchers have used monoclonal and polyclonal antibodies to localize beta 1 integrin-like proteins in plants. However, there is a disparity between data collected from these studies, especially since molecular weights obtained from these investigations range from 55-120 kDa for integrin-like proteins. To date, a complete investigation which employs all three basic immunolabeling procedures, immunoblotting, immunofluorescence microscopy, and immunogold labeling, in addition to extensive fractionation and exhaustive controls, has been lacking. In this paper, we demonstrate that use of a polyclonal antibody against the cytoplasmic domain of avian beta 1-integrin can produce potential artifacts in immunolocalization studies. However, these problems can be eliminated through use of starchless mutants or proper specimen preparation prior to electrophoresis. We also show that this antibody, when applied within the described parameters and with careful controls, identifies a large (100 kDa) integrin-like protein that is localized to plasma membrane fractions in Arabidopsis.

  18. Mare’s milk: composition and protein fraction in comparison with different milk species

    Directory of Open Access Journals (Sweden)

    Krešimir Kuterovac

    2011-06-01

    Full Text Available The usage of the mare’s milk as functional food especial for children intolerant to cow’s milk, with neurodermitis, allergies and similar disorders desiring to improve the quality of life is fiercely debated for last decades but there were no scientific studies to suggest such use of mare’s milk based on scientific research. The objectives of this study were to determine similarities of mare’s milk in comparison with milk of ruminants (cattle, sheep and goat and human milk in terms of milk composition and protein fraction as whey proteins, caseins and micelles size. All differences were discussed regarding usage of mare’s milk in human diet and compared to milk which is usually used in human nutrition. Regarding composition, the mare’s milk is similar to human milk in of crude protein, salt and lactose content, but it has significantly lower content of fat. Fractions of main proteins are similar between human and mare’s milk, except nitrogen casein (casein N which has twice lower content in human than in mare’s milk. Content of casein N from all ruminants’ milk differ much more. Just for true whey N and non-protein nitrogen (NPN similar content as human and mare’s milk has also goat milk. The casein content is the lowest in human milk; this content is three times greater in mare’s milk and six to seven times greater in goat’s and cow’s milk, while in sheep’s milk it is more than 10 times grater. In many components and fractions mare’s milk is more similar to human milk than milk of ruminants. A detail comparison of protein fraction shows quite large differences between milk of different species. More study and clinical research are needed that can recommend usage of mare’s milk in human diet as functional food on scientific bases.

  19. Ultrastructure and electrophoretic protein pattern of a nuclear fraction enriched in interchromatin granule conglomerations

    Energy Technology Data Exchange (ETDEWEB)

    Krzyzowska-Gruca, S.; Zborek, A.; Gruca, S.

    1986-01-01

    Rats were injected with a cytostatic 1-nitro-9/3'-dimethylpropyloamine/acridine.2HCl to induce aggregation of interchromatin granules (IG). The conglomerations of IG were well preserved in isolated liver nuclei and in nuclear structures deprived of chromatin. This feature enabled obtaining a nuclear fraction enriched in IG. The method consisted in extraction of isolated nuclei with a non-ionic detergent and digestion with DNase I in a high ionic strength. Each step of isolation was ultrastructurally monitored using both the routine electron microscopy as well as a preferential staining of IG with bismuth. Presence of spots of tightly packed granules within IG conglomerations in the final fraction like in the nuclei in situ was a good ultrastructural marker of IG. The resulting fraction consisted predominantly of IG conglomerations. Their preferential staining with bismuth was well preserved. Minute amounts of fibrillar material originating from nuclear matrix and residual nuclei could be observed. Protein composition of the fraction enriched in IG was studied by SDS-polyacrylamide gel electrophoresis. After electrotransfer, nitrocellulose filters were fixed with glutaraldehyde and stained with bismuth method in order to identify IG proteins. The results of ultrastructural and cytochemical studies in comparison to electrophoretic protein pattern are discussed.

  20. 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...

  1. Fiber digestion kinetics and protein degradability characteristics of stockpiled Tifton 85 bermudagrass.

    Science.gov (United States)

    Sechler, S R; Mullenix, M K; Holland, C M; Muntifering, R B

    2017-09-01

    A 2-yr study was conducted to determine effects of N fertilization level on fiber digestion kinetics and protein degradability characteristics of stockpiled Tifton 85 bermudagrass (T85). Six 0.76-ha pastures of stockpiled T85 were cut to a 10-cm stubble height on August 1 of each yr and fertilized with 56 (56N), 112 (112N), or 168 (168N) kg N/ha (2 pastures/treatment). Fiber digestion kinetics included the 72-hr potential extent of NDF digestion (PED), rate of NDF digestion, and lag time. In yr 1 and 2, PED decreased over the stockpile season. Rates of NDF digestion did not differ ( > 0.05) among N fertilization treatments in either yr. In yr 1, rate of NDF digestion was greatest ( digestion decreased ( digestion rates were similar for November and January 21 sampling dates. Lag time was greater ( digestion ( = -0.60 and -0.25 in yr 1 and 2, respectively) was observed. There was a trend ( = 0.06) for lignin concentration to be positively correlated with lag time ( = 0.39) in yr 1, and a strong relationship was observed in yr 2 ( = 0.91; digestion in stockpiled T85 were influenced more by temporal changes over the stockpile season than by N fertilization level. Supplement formulations based on kinetic parameters of fiber digestion may require periodic adjustment to insure that energy-yielding components of NDF are sufficient to meet animal requirements throughout the stockpile season. The CP fraction in stockpiled T85 contains sufficient RDP to support fibrolytic activity and growth of ruminal microorganisms throughout the stockpile season. Toward the latter end of the season, supplementation with sources of digestible fiber and RDP could be expected to increase MP supply to the host animal.

  2. Variability in carbon isotope fractionation of trichloroethene during degradation by persulfate activated with zero-valent iron: Effects of inorganic anions

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Yunde [State Key Laboratory of Biogeology and Environmental Geology, China University of Geosciences, Wuhan 430074 (China); School of Environmental Studies, China University of Geosciences, Wuhan 430074 (China); Zhou, Aiguo, E-mail: aiguozhou@cug.edu.cn [State Key Laboratory of Biogeology and Environmental Geology, China University of Geosciences, Wuhan 430074 (China); School of Environmental Studies, China University of Geosciences, Wuhan 430074 (China); Gan, Yiqun; Li, Xiaoqian [State Key Laboratory of Biogeology and Environmental Geology, China University of Geosciences, Wuhan 430074 (China); School of Environmental Studies, China University of Geosciences, Wuhan 430074 (China)

    2016-04-01

    Stable carbon isotope analysis has the potential to be used for assessing the performance of in situ remediation of organic contaminants. Successful application of this isotope technique requires understanding the magnitude and variability in carbon isotope fractionation associated with the reactions under consideration. This study investigated the influence of inorganic anions (sulfate, bicarbonate, and chloride) on carbon isotope fractionation of trichloroethene (TCE) during its degradation by persulfate activated with zero-valent iron. The results demonstrated that the significant carbon isotope fractionation (enrichment factors ε ranging from − 3.4 ± 0.3 to − 4.3 ± 0.3 ‰) was independent on the zero-iron dosage, sulfate concentration, and bicarbonate concentration. However, the ε values (ranging from − 7.0 ± 0.4 to − 13.6 ± 1.2 ‰) were dependent on the chloride concentration, indicating that chloride could significantly affect carbon isotope fractionation during TCE degradation by persulfate activated with zero-valent iron. The dependence of ε values on chloride concentration, indicated that TCE degradation mechanisms may be different from the degradation mechanism caused by sulfate radical (SO{sub 4}·{sup −}). Ignoring the effect of chloride on ε value may cause numerous uncertainties in quantitative assessment of the performance of the in situ chemical oxidation (ISCO). - Highlights: • Significant C isotope fractionation for TCE degradation by Fe{sup 0} activated persulfate. • The enrichment factors was independent of Fe{sup 0}, SO{sub 4}{sup 2−}, or HCO{sub 3}{sup −} concentration. • Cl{sup −} significantly influenced the carbon isotope fractionation.

  3. Variability in carbon isotope fractionation of trichloroethene during degradation by persulfate activated with zero-valent iron: Effects of inorganic anions

    International Nuclear Information System (INIS)

    Liu, Yunde; Zhou, Aiguo; Gan, Yiqun; Li, Xiaoqian

    2016-01-01

    Stable carbon isotope analysis has the potential to be used for assessing the performance of in situ remediation of organic contaminants. Successful application of this isotope technique requires understanding the magnitude and variability in carbon isotope fractionation associated with the reactions under consideration. This study investigated the influence of inorganic anions (sulfate, bicarbonate, and chloride) on carbon isotope fractionation of trichloroethene (TCE) during its degradation by persulfate activated with zero-valent iron. The results demonstrated that the significant carbon isotope fractionation (enrichment factors ε ranging from − 3.4 ± 0.3 to − 4.3 ± 0.3 ‰) was independent on the zero-iron dosage, sulfate concentration, and bicarbonate concentration. However, the ε values (ranging from − 7.0 ± 0.4 to − 13.6 ± 1.2 ‰) were dependent on the chloride concentration, indicating that chloride could significantly affect carbon isotope fractionation during TCE degradation by persulfate activated with zero-valent iron. The dependence of ε values on chloride concentration, indicated that TCE degradation mechanisms may be different from the degradation mechanism caused by sulfate radical (SO_4·"−). Ignoring the effect of chloride on ε value may cause numerous uncertainties in quantitative assessment of the performance of the in situ chemical oxidation (ISCO). - Highlights: • Significant C isotope fractionation for TCE degradation by Fe"0 activated persulfate. • The enrichment factors was independent of Fe"0, SO_4"2"−, or HCO_3"− concentration. • Cl"− significantly influenced the carbon isotope fractionation.

  4. ESCRT-dependent degradation of ubiquitylated plasma membrane proteins in plants.

    Science.gov (United States)

    Isono, Erika; Kalinowska, Kamila

    2017-12-01

    To control the abundance of plasma membrane receptors and transporters is crucial for proper perception and response to extracellular signals from surrounding cells and the environment. Posttranslational modification of plasma membrane proteins, especially ubiquitin conjugation or ubiquitylation, is key for the determination of stability for many transmembrane proteins localized on the cell surface. The targeted degradation is ensured by a complex network of proteins among which the endosomal sorting complex required for transport (ESCRT) plays a central role. This review focuses on progresses made in recent years on the understanding of the function of the ESCRT machinery in the degradation of ubiquitylated plasma membrane proteins in plants. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Iron-Binding Protein Degradation by Cysteine Proteases of Naegleria fowleri

    Directory of Open Access Journals (Sweden)

    Moisés Martínez-Castillo

    2015-01-01

    Full Text Available Naegleria fowleri causes acute and fulminant primary amoebic meningoencephalitis. This microorganism invades its host by penetrating the olfactory mucosa and then traveling up the mesaxonal spaces and crossing the cribriform plate; finally, the trophozoites invade the olfactory bulbs. During its invasion, the protozoan obtains nutrients such as proteins, lipids, carbohydrates, and cationic ions (e.g., iron, calcium, and sodium from the host. However, the mechanism by which these ions are obtained, particularly iron, is poorly understood. In the present study, we evaluated the ability of N. fowleri to degrade iron-binding proteins, including hololactoferrin, transferrin, ferritin, and hemoglobin. Zymography assays were performed for each substrate under physiological conditions (pH 7 at 37°C employing conditioned medium (CM and total crude extracts (TCEs of N. fowleri. Different degradation patterns with CM were observed for hololactoferrin, transferrin, and hemoglobin; however, CM did not cause ferritin degradation. In contrast, the TCEs degraded only hololactoferrin and transferrin. Inhibition assays revealed that cysteine proteases were involved in this process. Based on these results, we suggest that CM and TCEs of N. fowleri degrade iron-binding proteins by employing cysteine proteases, which enables the parasite to obtain iron to survive while invading the central nervous system.

  6. Molecular chaperones in targeting misfolded proteins for ubiquitin-dependent degradation

    DEFF Research Database (Denmark)

    Kriegenburg, Franziska; Ellgaard, Lars; Hartmann-Petersen, Rasmus

    2012-01-01

    The accumulation of misfolded proteins presents a considerable threat to the health of individual cells and has been linked to severe diseases, including neurodegenerative disorders. Considering that, in nature, cells often are exposed to stress conditions that may lead to aberrant protein...... conformational changes, it becomes clear that they must have an efficient quality control apparatus to refold or destroy misfolded proteins. In general, cells rely on molecular chaperones to seize and refold misfolded proteins. If the native state is unattainable, misfolded proteins are targeted for degradation...... via the ubiquitin-proteasome system. The specificity of this proteolysis is generally provided by E3 ubiquitin-protein ligases, hundreds of which are encoded in the human genome. However, rather than binding the misfolded proteins directly, most E3s depend on molecular chaperones to recognize...

  7. Reduced synaptic vesicle protein degradation at lysosomes curbs TBC1D24/sky-induced neurodegeneration.

    Science.gov (United States)

    Fernandes, Ana Clara; Uytterhoeven, Valerie; Kuenen, Sabine; Wang, Yu-Chun; Slabbaert, Jan R; Swerts, Jef; Kasprowicz, Jaroslaw; Aerts, Stein; Verstreken, Patrik

    2014-11-24

    Synaptic demise and accumulation of dysfunctional proteins are thought of as common features in neurodegeneration. However, the mechanisms by which synaptic proteins turn over remain elusive. In this paper, we study Drosophila melanogaster lacking active TBC1D24/Skywalker (Sky), a protein that in humans causes severe neurodegeneration, epilepsy, and DOOR (deafness, onychdystrophy, osteodystrophy, and mental retardation) syndrome, and identify endosome-to-lysosome trafficking as a mechanism for degradation of synaptic vesicle-associated proteins. In fly sky mutants, synaptic vesicles traveled excessively to endosomes. Using chimeric fluorescent timers, we show that synaptic vesicle-associated proteins were younger on average, suggesting that older proteins are more efficiently degraded. Using a genetic screen, we find that reducing endosomal-to-lysosomal trafficking, controlled by the homotypic fusion and vacuole protein sorting (HOPS) complex, rescued the neurotransmission and neurodegeneration defects in sky mutants. Consistently, synaptic vesicle proteins were older in HOPS complex mutants, and these mutants also showed reduced neurotransmission. Our findings define a mechanism in which synaptic transmission is facilitated by efficient protein turnover at lysosomes and identify a potential strategy to suppress defects arising from TBC1D24 mutations in humans. © 2014 Fernandes et al.

  8. Fractionation and evaluation of proteins in roots of Echinacea purpurea (L. Moench

    Directory of Open Access Journals (Sweden)

    Balciunaite Gabriele

    2015-12-01

    Full Text Available Echinacea purpurea (L. Moench, a member of the Asteraceae family, is a plant rich in flavonoids, essential oils, phenolic compounds, saponins, polysaccharides and glycoproteins. The aim of the study was to evaluate the protein content in dried roots of Echinacea purpurea (L. Moench after homogenization of roots with liquid nitrogen, extraction in 0.01 mol L-1 phosphate-buffered saline (PBS and purification followed by fractionation of proteins using gel filtration chromatography. Total concentration of proteins was measured using the Bradford method, and evaluation of the molecular mass of proteins was accomplished by applying the SDS-PAGE gel electrophoresis. The Bradford assay revealed that the highest concentration of proteins in fractions collected after gel filtration chomatography was 4.66–6.07 mg mL-1. Glycoproteins, alkamides and polysaccharides in roots of Echinacea purpurea (L. Moench are chemical compounds that are responsible for their immunomodulatory properties. However, information about the difference of protein contents in fresh and dried roots of E. purpurea is insufficient.

  9. Effects of the Replacement of Soybean Meal with Pea as Dietary Protein Source on the Serum Protein Fractions of Broilers

    Directory of Open Access Journals (Sweden)

    NT Bingol

    Full Text Available ABSTRACT The aim of this study was to determine the effects of the replacement of different levels of protein derived from soybean meal with that from peas in broiler diets on serum protein fractions. A corn-soybean meal basal diet was formulated as the control diet (Control=C (NRC, 1994, and then pea was added to the control diet to replace 20% (P20 or 40% (P40 of the crude protein of the control diet. The diets were randomly fed to 12 pens per treatment, each housing five birds, for 42 days. Blood samples were collected from 36 birds (3 birds x 4 pens x3 treatments and the serum protein fractions were separated. Gamma-globulin percentage was higher in group P20 compared with C and P40 groups. Total protein, beta-globulin, and gamma-globulin concentrations were significantly higher in group P20 compared with those of both control and P40 group (p<0.05.

  10. Protein composition of wheat gluten polymer fractions determined by quantitative two-dimensional gel electrophoresis and tandem mass spectrometry

    Science.gov (United States)

    Flour proteins from the US bread wheat Butte 86 were extracted in 0.5% SDS using a two-step procedure with and without sonication and further separated by size exclusion chromatography into monomeric and polymeric fractions. Proteins in each fraction were analyzed by quantitative two-dimensional gel...

  11. Anti-nutritional Factors and Ruminal Dry Matter and Crude Protein Degradability of Gamma and Microwave Irradiated Native Rapeseed

    Directory of Open Access Journals (Sweden)

    sayyed roohollah ebrahimimahmoudabad

    2016-08-01

    . The same procedure was applied to two bags to obtain the 0 h value. The residues were dried and analyzed for DM and CP to determine degradation kinetics of NRS. Digestibility of rumen undegraded CP was estimated using the three-step in vitro procedure. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE was used to monitor protein subfractions and the fate of true proteins of untreated and irradiated NRS in the rumen. Results and Discussion Irradiation had no significant effect on chemical composition, but decreased the total glucosinolate and phytic acid of NRS. This elimination of phytic acid by irradiation is probably due to chemical degradation of phytate to lower inositol phosphates and inositol, by the action of free radicals, which have lower chelating power, or cleavage of the phytate ring itself. The glucosinolate content of untreated NRS in this study, 122.8 mol/g, was reduced up to 30 kGy, but further irradiation had no effect. Major deleterious effects of glucosinolate ingestion in animals include: reduced palatability, decreased growth and reduced production. However, according to our study, gamma and microwave irradiated NRS may be fed to dairy cows at high levels without glucosinolate induced detrimental effects. Gamma and microwave irradiation decreased the washout fraction, degradation rate and effective degradability (ED of DM and CP and increased potentially degradable fraction of DM and CP of NRS. Decreasing CP degradability as a result of irradiation is due to the occurrence of cross-linking of polypeptide chains, denaturation and protein aggregation. Gamma irradiation at doses of 30 and 45 kGy and microwave irradiation increased in vitro CP digestibility of NRS. Irradiation may induce unfolding of the protein and its denaturation, thereby exposing hydrophobic amino acids (especially aromatics that are positional groups for the active sites of pepsin and trypsin enzymes. Moreover, the improvement in CP digestibility may be

  12. Protein degradation in preimplantation mouse embryos and the lethality of tritiated amino acids

    International Nuclear Information System (INIS)

    Wielbold, J.L.

    1982-01-01

    The role of protein degradation in preimplantation development in the mouse was studied. Proteins of morulae and blastocysts (M and B) cultured in vitro after labeling for 1 hour (h) in 3 H-leucine exhibit a mean half-life (t 1 / 2 ) of 8.1 h. The t 1 / 2 tends to increase (9.5 h) when 10% fetal calf serum is added to the chase medium. This decrease in protein degradation in the presence of serum is associated with an increase in the percentage of B that are hatching (P 3 H-leucine in their proteins than did Day 4 embryos remaining in culture (P<0.02), while Day 4 embryos in a Day 3 uterus retained the same amount of radioactivity as did Day 4 embryos in culture. This differential effect of uterine environment was also seen when Day 4 embryos were transferred to recipients. More fetuses developed to term when the recipient was in Day 3 of PSP (50.8%) than when the recipient was in Day 4 PSP (25.9%, P<0.001), regardless of the age of the recipient. Age of the recipient does affect the percentage of transferred embryos developing to term. Thus, protein degradation may vary with the stage of embryo development and the conditions to which the embryos are exposed. However, even low levels of incorporated tritiated leucine can have lethal effects on the embryos and compromise the validity of the protein half-lives determined

  13. Cellular Cholesterol Regulates Ubiquitination and Degradation of the Cholesterol Export Proteins ABCA1 and ABCG1*

    Science.gov (United States)

    Hsieh, Victar; Kim, Mi-Jurng; Gelissen, Ingrid C.; Brown, Andrew J.; Sandoval, Cecilia; Hallab, Jeannette C.; Kockx, Maaike; Traini, Mathew; Jessup, Wendy; Kritharides, Leonard

    2014-01-01

    The objective of this study was to examine the influence of cholesterol in post-translational control of ABCA1 and ABCG1 protein expression. Using CHO cell lines stably expressing human ABCA1 or ABCG1, we observed that the abundance of these proteins is increased by cell cholesterol loading. The response to increased cholesterol is rapid, is independent of transcription, and appears to be specific for these membrane proteins. The effect is mediated through cholesterol-dependent inhibition of transporter protein degradation. Cell cholesterol loading similarly regulates degradation of endogenously expressed ABCA1 and ABCG1 in human THP-1 macrophages. Turnover of ABCA1 and ABCG1 is strongly inhibited by proteasomal inhibitors and is unresponsive to inhibitors of lysosomal proteolysis. Furthermore, cell cholesterol loading inhibits ubiquitination of ABCA1 and ABCG1. Our findings provide evidence for a rapid, cholesterol-dependent, post-translational control of ABCA1 and ABCG1 protein levels, mediated through a specific and sterol-sensitive mechanism for suppression of transporter protein ubiquitination, which in turn decreases proteasomal degradation. This provides a mechanism for acute fine-tuning of cholesterol transporter activity in response to fluctuations in cell cholesterol levels, in addition to the longer term cholesterol-dependent transcriptional regulation of these genes. PMID:24500716

  14. The Polerovirus silencing suppressor P0 targets ARGONAUTE proteins for degradation.

    Science.gov (United States)

    Baumberger, Nicolas; Tsai, Ching-Hsui; Lie, Miranda; Havecker, Ericka; Baulcombe, David C

    2007-09-18

    Plant and animal viruses encode suppressor proteins of an adaptive immunity mechanism in which viral double-stranded RNA is processed into 21-25 nt short interfering (si)RNAs. The siRNAs guide ARGONAUTE (AGO) proteins so that they target viral RNA. Most viral suppressors bind long dsRNA or siRNAs and thereby prevent production of siRNA or binding of siRNA to AGO. The one exception is the 2b suppressor of Cucumoviruses that binds to and inhibits AGO1. Here we describe a novel suppressor mechanism in which a Polerovirus-encoded F box protein (P0) targets the PAZ motif and its adjacent upstream sequence in AGO1 and mediates its degradation. F box proteins are components of E3 ubiquitin ligase complexes that add polyubiquitin tracts on selected lysine residues and thereby mark a protein for proteasome-mediated degradation. With P0, however, the targeted degradation of AGO is insensitive to inhibition of the proteasome, indicating that the proteasome is not involved. We also show that P0 does not block a mobile signal of silencing, indicating that the signal molecule does not have AGO protein components. The ability of P0 to block silencing without affecting signal movement may contribute to the phloem restriction of viruses in the Polerovirus group.

  15. Biochemical characterization and immunolocalization studies of a Capsicum chinense Jacq. protein fraction containing DING proteins and anti-microbial activity.

    Science.gov (United States)

    Brito-Argáez, Ligia; Tamayo-Sansores, José A; Madera-Piña, Dianeli; García-Villalobos, Francisco J; Moo-Puc, Rosa E; Kú-González, Ángela; Villanueva, Marco A; Islas-Flores, Ignacio

    2016-12-01

    The DING protein family consists of proteins of great biological importance due to their ability to inhibit carcinogenic cell growth. A DING peptide with Mr ∼7.57 kDa and pI ∼5.06 was detected in G10P1.7.57, a protein fraction from Capsicum chinense Jacq. seeds. Amino acid sequencing of the peptide produced three smaller peptides showing identity to the DING protein family. G10P1.7.57 displayed a phosphatase activity capable of dephosphorylating different phosphorylated substrates and inhibited the growth of Saccharomyces cerevisiae cells. Western immunoblotting with a custom-made polyclonal antibody raised against a sequence (ITYMSPDYAAPTLAGLDDATK), derived from the ∼7.57 kDa polypeptide, immunodetected an ∼ 39 kDa polypeptide in G10P1.7.57. Purification by electroelution followed by amino acid sequencing of the ∼39 kDa polypeptide yielded seven new peptide sequences and an additional one identical to that of the initially identified peptide. Western immunoblotting of soluble proteins from C. chinense seeds and leaves revealed the presence of the ∼39 kDa polypeptide at all developmental stages, with increased accumulation when the organs reached maturity. Immunolocalization using Dabsyl chloride- or Alexa fluor 488-conjugated antibodies revealed a specific fluorescent signal in the cell cytoplasm at all developmental stages, giving support to the idea that the ∼39 kDa polypeptide is a soluble DING protein. Thus, we have identified and characterized a protein fraction with a DING protein from C. chinense. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  16. Fabrication of Flexible, Fully Organic, Degradable Energy Storage Devices Using Silk Proteins.

    Science.gov (United States)

    Pal, Ramendra K; Kundu, Subhas C; Yadavalli, Vamsi K

    2018-03-21

    Flexible and thin-film devices are of great interest in epidermal and implantable bioelectronics. The integration of energy storage and delivery devices such as supercapacitors (SCs) with properties such as flexibility, miniaturization, biocompatibility, and degradability are sought for such systems. Reducing e-waste and using sustainable materials and processes are additional desirable qualities. Herein, a silk protein-based biocompatible and degradable thin-film microSC (μSC) is reported. A protein carrier with the conducting polymer poly(3,4-ethylenedioxythiophene) polystyrene sulfonate and reduced graphene oxide dopant is used as a photopatternable biocomposite ink. Active electrodes are fabricated using photolithography under benign conditions, using only water as the solvent. These electrodes are printed on flexible protein sheets to form degradable, organic devices with a benign agarose-NaCl gel electrolyte. High capacitance, power density, cycling stability over 500 cycles, and the ability to power a light-emitting diode are shown. The device is flexible, can sustain cyclic mechanical stresses over 450 cycles, and retain capacitive properties over several days in liquid. Significantly, the μSCs are cytocompatible and completely degraded over the period of ∼1 month. By precise control of the device configuration, these silk protein-based, all-polymer organic devices can be designed to be tunably transient and provide viable alternatives for powering flexible and implantable bioelectronics.

  17. In situ ruminal crude protein degradability of by-products from cereals, oilseeds and animal origin

    NARCIS (Netherlands)

    Habib, G.; Khan, N.A.; Ali, M.; Bezabih, M.

    2013-01-01

    The aim of this study was to establish a database on in situ ruminal crude protein (CP) degradability characteristics of by-products from cereal grains, oilseeds and animal origin commonly fed to ruminants in Pakistan and South Asian Countries. The oilseed by-products were soybean meal, sunflower

  18. Delaying aging and the aging-associated decline in protein homeostasis by inhibition of tryptophan degradation

    NARCIS (Netherlands)

    van der Goot, Annemieke T.; Zhu, Wentao; Vazquez-Manrique, Rafael P.; Seinstra, Renee I.; Dettmer, Katja; Michels, Helen; Farina, Francesca; Krijnen, Jasper; Melki, Ronald; Buijsman, Rogier C.; Silva, Mariana Ruiz; Thijssen, Karen L.; Kema, Ido P.; Neri, Christian; Oefner, Peter J.; Nollen, Ellen A. A.

    2012-01-01

    Toxicity of aggregation-prone proteins is thought to play an important role in aging and age-related neurological diseases like Parkinson and Alzheimer's diseases. Here, we identify tryptophan 2,3-dioxygenase (tdo-2), the first enzyme in the kynurenine pathway of tryptophan degradation, as a

  19. Degradation and de novo synthesis of D1 protein and psbA ...

    Indian Academy of Sciences (India)

    This shows that synthesis of D1 protein is not the only component involved in the recovery process. Our events, which ... transcript levels in the green alga Chlamydomonas reinhardtii in ..... and Gaba V 1996 Accelerated degradation of the D2 ...

  20. Differential expression in Phanerochaete chrysosporium of membrane- associated proteins relevant to lignin degradation

    Science.gov (United States)

    Semarjit Shary; Alexander N. Kapich; Ellen A. Panisko; Jon K. Magnuson; Daniel Cullen; Kenneth E. Hammel

    2008-01-01

    Fungal lignin-degrading systems likely include membrane-associated proteins that participate in diverse processes such as uptake and oxidation of lignin fragments, production of ligninolytic secondary metabolites, and defense of the mycelium against ligninolytic oxidants. Little is known about the nature or regulation of these membrane-associated components. We grew...

  1. Estimation of the in situ degradation of the washout fraction of starch by using a modified in situ protocol and in vitro measurements

    NARCIS (Netherlands)

    Jonge, de L.H.; Laar, van H.; Dijkstra, J.

    2015-01-01

    The in situ degradation of the washout fraction of starch in six feed ingredients (i.e. barley, faba beans, maize, oats, peas and wheat) was studied by using a modified in situ protocol and in vitro measurements. In comparison with the washing machine method, the modified protocol comprises a milder

  2. ERManI (Endoplasmic Reticulum Class I α-Mannosidase) Is Required for HIV-1 Envelope Glycoprotein Degradation via Endoplasmic Reticulum-associated Protein Degradation Pathway.

    Science.gov (United States)

    Zhou, Tao; Frabutt, Dylan A; Moremen, Kelley W; Zheng, Yong-Hui

    2015-09-04

    Previously, we reported that the mitochondrial translocator protein (TSPO) induces HIV-1 envelope (Env) degradation via the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway, but the mechanism was not clear. Here we investigated how the four ER-associated glycoside hydrolase family 47 (GH47) α-mannosidases, ERManI, and ER-degradation enhancing α-mannosidase-like (EDEM) proteins 1, 2, and 3, are involved in the Env degradation process. Ectopic expression of these four α-mannosidases uncovers that only ERManI inhibits HIV-1 Env expression in a dose-dependent manner. In addition, genetic knock-out of the ERManI gene MAN1B1 using CRISPR/Cas9 technology disrupts the TSPO-mediated Env degradation. Biochemical studies show that HIV-1 Env interacts with ERManI, and between the ERManI cytoplasmic, transmembrane, lumenal stem, and lumenal catalytic domains, the catalytic domain plays a critical role in the Env-ERManI interaction. In addition, functional studies show that inactivation of the catalytic sites by site-directed mutagenesis disrupts the ERManI activity. These studies identify ERManI as a critical GH47 α-mannosidase in the ER-associated protein degradation pathway that initiates the Env degradation and suggests that its catalytic domain and enzymatic activity play an important role in this process. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Estimation of Relationship Between In Situ and In Vitro Rumen Protein Degradability of Extruded Full Fat Soybean

    Directory of Open Access Journals (Sweden)

    Arzu Erol Tunç

    2017-10-01

    Full Text Available The objectives of this study were to estimate the protein degradability of extruded full fat soybean (ESB by in situ (nylon bag and in vitro enzymatic method and to develop an equation in order predict in situ degradability from in vitro values. In the study enzymatic technique; hydrolysis after 1 h (INV1 and after 24 h (INV24 by a purified protease extracted from Streptomyces griseus in a borate-phosphate buffer at pH 8 was used as in vitro method. Relationship between in situ effective protein degradability (INSE and in vitro degradability after 1 and 24 hours incubations (INV1 and INV24 were determined. In situ protein degradability was measured at 0, 2, 4, 8, 16, 24, and 48 and at 72 h incubations in the rumen of 3 Holstein cows. In the study INSE, INV1 and INV24 were determined as 58.05, 20.24 and 41.46% respectively. Despite there were differences between in situ and in vitro protein degradability values, correlation coefficients between in situ and in vitro protein degradability of ESB were high and regression equations for estimation of in situ from in vitro were found significant. As conclusion in vitro enzymatic protein degradability (INV1 and INV24 can be used for estimation of in situ effective protein degradability of extruded full fat soybean.

  4. Nutrient digestibility and evaluation of protein and carbohydrate fractionation of citrus by-products

    DEFF Research Database (Denmark)

    Lashkari, Saman; Taghizadeh, Akbar

    2013-01-01

    The protein and carbohydrate fractionation and nutrient digestibility of citrus by‐products were determined. Ruminal, intestinal and total tract CP disappearance values were measured by a modified three‐step (MTSP) method and in vitro CP disappearance method (IVCP). Test feeds were orange pulp (OP...... to the results, it could be concluded that citrus by‐products have high nutritive value and also, the in vitro techniques can be easily used to determine of the nutritive value of citrus by‐products....

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

    Science.gov (United States)

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

    2013-08-14

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

  6. The central domain of yeast transcription factor Rpn4 facilitates degradation of reporter protein in human cells.

    Science.gov (United States)

    Morozov, A V; Spasskaya, D S; Karpov, D S; Karpov, V L

    2014-10-16

    Despite high interest in the cellular degradation machinery and protein degradation signals (degrons), few degrons with universal activity along species have been identified. It has been shown that fusion of a target protein with a degradation signal from mammalian ornithine decarboxylase (ODC) induces fast proteasomal degradation of the chimera in both mammalian and yeast cells. However, no degrons from yeast-encoded proteins capable to function in mammalian cells were identified so far. Here, we demonstrate that the yeast transcription factor Rpn4 undergoes fast proteasomal degradation and its central domain can destabilize green fluorescent protein and Alpha-fetoprotein in human HEK 293T cells. Furthermore, we confirm the activity of this degron in yeast. Thus, the Rpn4 central domain is an effective interspecies degradation signal. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  7. Combining proteomic tools to characterize the protein fraction of llama (Lama glama) milk.

    Science.gov (United States)

    Saadaoui, Besma; Bianchi, Leonardo; Henry, Céline; Miranda, Guy; Martin, Patrice; Cebo, Christelle

    2014-05-01

    Llamas belong to the Camelidae family along with camels. While dromedary camel milk has been broadly characterized, data on llama milk proteins are scarce. The objective of this study was thus to investigate the protein composition of llama milk. Skimmed llama milk proteins were first characterized by a 2D separation technique coupling RP-HPLC in the first dimension with SDS-PAGE in the second dimension (RP-HPLC/SDS-PAGE). Llama milk proteins, namely caseins (αs1 -, αs2 -, β-, and κ-caseins), α-lactalbumin, lactoferrin, and serum albumin, were identified using PMF. Llama milk proteins were also characterized by online LC-ESI-MS analysis. This approach allowed attributing precise molecular masses for most of the previously MS-identified llama milk proteins. Interestingly, α-lactalbumin exhibits distinct chromatographic behaviors between llama and dromedary camel milk. De novo sequencing of the llama α-lactalbumin protein by LC coupled with MS/MS (LC-MS/MS) showed the occurrence of two amino acid substitutions (R62L/I and K89L/I) that partly explained the higher hydrophobicity of llama α-lactalbumin compared with its dromedary counterpart. Taken together, these results provide for the first time a thorough description of the protein fraction of Lama glama milk. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Gamma-radiation induced damage of proteins in the thick fraction of egg white

    Directory of Open Access Journals (Sweden)

    MARIJA VUCKOVIC

    2005-11-01

    Full Text Available The thick fraction of egg white saturated with either N2O or Ar was irradiated in the dose range 1.5–45 kGy at 60Co gamma source. The gel structure decomposition and other processes accompanied with changes in protein molecular mass were followed by Sephadex G-200 exclusion chromatography, denaturing SDS-polyacrylamide gel electrophoresis, viscosity and turbidity measurements. The complex behaviour of viscosity was observed in the N2O saturated sample (where the hydrated electron was converted into the OH radical; the initial abrupt decrease that radually slows down reaching the minimum at 12 kGy (hmin = 2.7 mPa s followed by the slow rise was measured. The Ar saturated sample ([eaq-] ~ [OH] showed both the significantly faster initial decrease and lower viscosity minimum (hmin = 2.2 mPa s. The combined Sephadex G-200 exclusion chromatography and denaturing SDS-polyacrylamide gel electrophoresis data revealed that the three-dimensional egg white (hydrated gel structure was (efficiently decomposed even in the N2O saturated sample. The protein scission was detected in the entire dose range studied, while the protein agglomeration is not noticed at low doses (around 1.5 kGy; however, it dominates at higher doses. In the highest dose region studied, the loss of structure in SDS-PAGE chromatograms indicates that the agglomerates are formed from protein fragments rather than from intact proteins. The continuous linear increase in turbidity was measured. The results obtained indicate that ionizing radiation causes the breakdown of the protein network of the thick fraction of egg white via the reduction of S–S bridges by the hydrated electron and the protein fragmentation due to the direct action of ionizing radiation. The protein agglomeration is initiated by the reaction of the OH radical; its inefficiency at low doses is attributed to the glucose antioxidant properties and radical immobility.

  9. Use of electrophoretically separated serum protein fractions for the diagnosis of cardiomyopathy

    International Nuclear Information System (INIS)

    Siddiqui, Z.H.; Cheema, A.M.

    2011-01-01

    In an investigation of molecular pathogenesis in cardiovascular diseases, the blood samples of the patients diagnosed for cardiomyopathy (CMP) were obtained from the Punjab Institute of Cardiology, Lahore. Blood samples of the healthy subjects of comparable age group without any history of cardiac ailment were also collected for the control comparisons. The sera of CMP were separated and used for the study of the protein profiles with sodium dodecyle sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in first dimension. Quantification of various protein fractions done by Gene Genius Bio-imaging Gel Documentation System that provide the data of molecular weights and the percent raw volume covered by each of the fractions. The protein fractions that showed significant variation were separated by using the technique of electro blotting and electro elution and run on isoelectric focusing (IEF) in second dimension to determine their isoelectric points. The most pertinent results in the comparison were the significant increase in apolipoprotein B, Ceruloplasmin, apolipoprotein A-I and transthyretin in the sera of patients of CMP compared to healthy subjects. These results show that level of apolipoprotein B, Ceruloplasmin, apolipoprotein A-I and transthyretin are strong predictor of CMP and can also be used for the diagnosis of CMP. (author)

  10. Diagnostic use of electrophoretically separated serum protein fractions in the patients of angina pectoris

    International Nuclear Information System (INIS)

    Siddiqui, Z.H.; Cheema, A.M.

    2010-01-01

    The understanding of molecular pathogenesis of clinical states enables for diagnosis and effective management of the diseases. In an investigation of molecular pathogenesis or adaptation in cardiovascular diseases, the blood samples of the patients diagnosed for angina pectoris (AP) were obtained from the Punjab Institute of Cardiology, Lahore. Blood samples of the healthy subjects of comparable age group without any history of cardiac ailment were also collected for the control comparisons. The sera of AP were separated and used for the study of the protein profiles with sodium dodecyles sulfate polyacrylamide gel electrophoresis (SDS-PAGE)in first dimension. Quantification of various protein fractions done by Gene Genius Bio-imaging Gel Documentation System that provide the data of molecular weights and the percent raw volume covered. by each of the fractions. The protein fractions that showed significant variation were separated by using the technique of electro blotting and electro elution and run on isoelectric focusing (IEF) in second dimension to determine their isoelectric points. The most pertinent results in the comparison were the significant increase in apolipoprotein B, marked decrease in apolipoprotein A-I and high apolipoprotein B/apolipoprotein A-I ratio in the sera of patients of AP compared to healthy subjects. These results show that level of apolipoprotein A-I, apolipoprotein B and the apolipoprotein B/apolipoprotein A-I ratio are strong predictor of AP and can also be used for the diagnosis of AP. (author)

  11. Pathogenic prion protein is degraded by a manganese oxide mineral found in soils

    Science.gov (United States)

    Russo, F.; Johnson, C.J.; McKenzie, D.; Aiken, Judd M.; Pedersen, J.A.

    2009-01-01

    Prions, the aetiological agents of transmissible spongiform encephalopathies, exhibit extreme resistance to degradation. Soil can retain prion infectivity in the environment for years. Reactive soil components may, however, contribute to the inactivation of prions in soil. Members of the birnessite family of manganese oxides (MnO2) rank among the strongest natural oxidants in soils. Here, we report the abiotic degradation of pathogenic prion protein (PrPTSE) by a synthetic analogue of naturally occurring birnessite minerals. Aqueous MnO2 suspensions degraded the PrPTSE as evidenced by decreased immunoreactivity and diminished ability to seed protein misfolding cyclic amplification reactions. Birnessite-mediated PrPTSE degradation increased as a solution's pH decreased, consistent with the pH-dependence of the redox potential of MnO2. Exposure to 5.6 mg MnO2 ml-1 (PrPTSE:MnO2=1 : 110) decreased PrPTSE levels by ???4 orders of magnitude. Manganese oxides may contribute to prion degradation in soil environments rich in these minerals. ?? 2009 SGM.

  12. HIV-1 accessory proteins VPR and Vif modulate antiviral response by targeting IRF-3 for degradation

    International Nuclear Information System (INIS)

    Okumura, Atsushi; Alce, Tim; Lubyova, Barbora; Ezelle, Heather; Strebel, Klaus; Pitha, Paula M.

    2008-01-01

    The activation of IRF-3 during the early stages of viral infection is critical for the initiation of the antiviral response; however the activation of IRF-3 in HIV-1 infected cells has not yet been characterized. We demonstrate that the early steps of HIV-1 infection do not lead to the activation and nuclear translocation of IRF-3; instead, the relative levels of IRF-3 protein are decreased due to the ubiquitin-associated proteosome degradation. Addressing the molecular mechanism of this effect we show that the degradation is independent of HIV-1 replication and that virion-associated accessory proteins Vif and Vpr can independently degrade IRF-3. The null mutation of these two genes reduced the capacity of the HIV-1 virus to down modulate IRF-3 levels. The degradation was associated with Vif- and Vpr-mediated ubiquitination of IRF-3 and was independent of the activation of IRF-3. N-terminal lysine residues were shown to play a critical role in the Vif- and Vpr-mediated degradation of IRF-3. These data implicate Vif and Vpr in the disruption of the initial antiviral response and point to the need of HIV-1 to circumvent the antiviral response during the very early phase of replication

  13. A Role of Protein Degradation in Memory Consolidation after Initial Learning and Extinction Learning in the Honeybee ("Apis mellifera")

    Science.gov (United States)

    Felsenberg, Johannes; Dombrowski, Vincent; Eisenhardt, Dorothea

    2012-01-01

    Protein degradation is known to affect memory formation after extinction learning. We demonstrate here that an inhibitor of protein degradation, MG132, interferes with memory formation after extinction learning in a classical appetitive conditioning paradigm. In addition, we find an enhancement of memory formation when the same inhibitor is…

  14. The proteins of the grape (Vitis vinifera L.) seed endosperm: fractionation and identification of the major components.

    Science.gov (United States)

    Gazzola, Diana; Vincenzi, Simone; Gastaldon, Luca; Tolin, Serena; Pasini, Gabriella; Curioni, Andrea

    2014-07-15

    In the present study, grape (Vitis vinifera L.) seed endosperm proteins were characterized after sequential fractionation, according to a modified Osborne procedure. The salt-soluble fraction (albumins and globulins) comprised the majority (58.4%) of the total extracted protein. The protein fractions analysed by SDS-PAGE showed similar bands, indicating different solubility of the same protein components. SDS-PAGE in non-reducing and reducing conditions revealed the polypeptide composition of the protein bands. The main polypeptides, which were similar in all the grape varieties analysed, were identified by LC-MS/MS as homologous to the 11S globulin-like seed storage proteins of other plant species, while a monomeric 43 kDa protein presented high homology with the 7S globulins of legume seeds. The results provide new insights about the identity, structure and polypeptide composition of the grape seed storage proteins. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Degradation of the disease-associated prion protein by a serine protease from lichens

    Science.gov (United States)

    Johnson, C.J.; Bennett, J.P.; Biro, S.M.; Duque-Velasquez, J.C.; Rodriguez, C.M.; Bessen, R.A.; Rocke, T.E.; Bartz, Jason C.

    2011-01-01

    The disease-associated prion protein (PrP(TSE)), the probable etiological agent of the transmissible spongiform encephalopathies (TSEs), is resistant to degradation and can persist in the environment. Lichens, mutualistic symbioses containing fungi, algae, bacteria and occasionally cyanobacteria, are ubiquitous in the environment and have evolved unique biological activities allowing their survival in challenging ecological niches. We investigated PrP(TSE) inactivation by lichens and found acetone extracts of three lichen species (Parmelia sulcata, Cladonia rangiferina and Lobaria pulmonaria) have the ability to degrade prion protein (PrP) from TSE-infected hamsters, mice and deer. Immunoblots measuring PrP levels and protein misfolding cyclic amplification indicated at least two logs of reductions in PrP(TSE). Degradative activity was not found in closely related lichen species or in algae or a cyanobacterium that inhabit lichens. Degradation was blocked by Pefabloc SC, a serine protease inhibitor, but not inhibitors of other proteases or enzymes. Additionally, we found that PrP levels in PrP(TSE)-enriched preps or infected brain homogenates are also reduced following exposure to freshly-collected P. sulcata or an aqueous extract of the lichen. Our findings indicate that these lichen extracts efficiently degrade PrP(TSE) and suggest that some lichens could have potential to inactivate TSE infectivity on the landscape or be a source for agents to degrade prions. Further work to clone and characterize the protease, assess its effect on TSE infectivity and determine which organism or organisms present in lichens produce or influence the protease activity is warranted.

  16. HUWE1 and TRIP12 Collaborate in Degradation of Ubiquitin-Fusion Proteins and Misframed Ubiquitin

    DEFF Research Database (Denmark)

    Poulsen, Esben G; Steinhauer, Cornelia; Lees, Michael

    2012-01-01

    In eukaryotic cells an uncleavable ubiquitin moiety conjugated to the N-terminus of a protein signals the degradation of the fusion protein via the proteasome-dependent ubiquitin fusion degradation (UFD) pathway. In yeast the molecular mechanism of the UFD pathway has been well characterized...... in degradation of the UFD substrate Ub(G76V)-YFP. The most significant hits from the screen were the E3 ubiquitin-protein ligase HUWE1, as well as PSMD7 and PSMD14 that encode proteasome subunits. Accordingly, knock down of HUWE1 led to an increase in the steady state level and a retarded degradation of the UFD...... substrate. Knock down of HUWE1 also led to a stabilization of the physiological UFD substrate UBB(+1). Precipitation experiments revealed that HUWE1 is associated with both the Ub(G76V)-YFP substrate and the 26S proteasome, indicating that it functions late in the UFD pathway. Double knock down of HUWE1...

  17. Application of green fluorescent protein for monitoring phenol-degrading strains

    Directory of Open Access Journals (Sweden)

    Ana Milena Valderrama F.

    2001-07-01

    Full Text Available Several methods have been developed for detecting microorganisms in environmental samples. Some systems for incorporating reporter genes, such as lux or the green fluorescent protein (GFP gene, have been developed recently This study describes gfp gene marking of a phenol degrading strain, its evaluation and monitoring in a bioreactor containing refinery sour water. Tagged strains were obtained having the same physiological and metabolic characteristics as the parent strain. Fluorescent expression was kept stable with no selection for more than 50 consecutive generations and tagged strains were recovered from the bioreactor after forty-five days of phenol-degradation treatment.

  18. Unfolded protein response and activated degradative pathways regulation in GNE myopathy.

    Directory of Open Access Journals (Sweden)

    Honghao Li

    Full Text Available Although intracellular beta amyloid (Aβ accumulation is known as an early upstream event in the degenerative course of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE myopathy, the process by which Aβdeposits initiate various degradative pathways, and their relationship have not been fully clarified. We studied the possible secondary responses after amyloid beta precursor protein (AβPP deposition including unfolded protein response (UPR, ubiquitin proteasome system (UPS activation and its correlation with autophagy system. Eight GNE myopathy patients and five individuals with normal muscle morphology were included in this study. We performed immunofluorescence and immunoblotting to investigate the expression of AβPP, phosphorylated tau (p-tau and endoplasmic reticulum molecular chaperones. Proteasome activities were measured by cleavage of fluorogenic substrates. The expression of proteasome subunits and linkers between proteasomal and autophagy systems were also evaluated by immunoblotting and relative quantitative real-time RT-PCR. Four molecular chaperones, glucose-regulated protein 94 (GRP94, glucose-regulated protein 78 (GRP78, calreticulin and calnexin and valosin containing protein (VCP were highly expressed in GNE myopathy. 20S proteasome subunits, three main proteasome proteolytic activities, and the factors linking UPS and autophagy system were also increased. Our study suggests that AβPP deposition results in endoplasmic reticulum stress (ERS and highly expressed VCP deliver unfolded proteins from endoplasmic reticulum to proteosomal system which is activated in endoplasmic reticulum associated degradation (ERAD in GNE myopathy. Excessive ubiquitinated unfolded proteins are exported by proteins that connect UPS and autophagy to autophagy system, which is activated as an alternative pathway for degradation.

  19. Degradation of Human PDZ-Proteins by Human Alphapapillomaviruses Represents an Evolutionary Adaptation to a Novel Cellular Niche.

    Science.gov (United States)

    Van Doorslaer, Koenraad; DeSalle, Rob; Einstein, Mark H; Burk, Robert D

    2015-06-01

    In order to complete their life cycle, papillomaviruses have evolved to manipulate a plethora of cellular pathways. The products of the human Alphapapillomavirus E6 proteins specifically interact with and target PDZ containing proteins for degradation. This viral phenotype has been suggested to play a role in viral oncogenesis. To analyze the association of HPV E6 mediated PDZ-protein degradation with cervical oncogenesis, a high-throughput cell culture assay was developed. Degradation of an epitope tagged human MAGI1 isoform was visualized by immunoblot. The correlation between HPV E6-induced degradation of hMAGI1 and epidemiologically determined HPV oncogenicity was evaluated using a Bayesian approach within a phylogenetic context. All tested oncogenic types degraded the PDZ-containing protein hMAGI1d; however, E6 proteins isolated from several related albeit non-oncogenic viral types were equally efficient at degrading hMAGI1. The relationship between both traits (oncogenicity and PDZ degradation potential) is best explained by a model in which the potential to degrade PDZ proteins was acquired prior to the oncogenic phenotype. This analysis provides evidence that the ancestor of both oncogenic and non-oncogenic HPVs acquired the potential to degrade human PDZ-containing proteins. This suggests that HPV E6 directed degradation of PDZ-proteins represents an ancient ecological niche adaptation. Phylogenetic modeling indicates that this phenotype is not specifically correlated with oncogenic risk, but may act as an enabling phenotype. The role of PDZ protein degradation in HPV fitness and oncogenesis needs to be interpreted in the context of Alphapapillomavirus evolution.

  20. Degradation of Human PDZ-Proteins by Human Alphapapillomaviruses Represents an Evolutionary Adaptation to a Novel Cellular Niche

    Science.gov (United States)

    Van Doorslaer, Koenraad; DeSalle, Rob; Einstein, Mark H.; Burk, Robert D.

    2015-01-01

    In order to complete their life cycle, papillomaviruses have evolved to manipulate a plethora of cellular pathways. The products of the human Alphapapillomavirus E6 proteins specifically interact with and target PDZ containing proteins for degradation. This viral phenotype has been suggested to play a role in viral oncogenesis. To analyze the association of HPV E6 mediated PDZ-protein degradation with cervical oncogenesis, a high-throughput cell culture assay was developed. Degradation of an epitope tagged human MAGI1 isoform was visualized by immunoblot. The correlation between HPV E6-induced degradation of hMAGI1 and epidemiologically determined HPV oncogenicity was evaluated using a Bayesian approach within a phylogenetic context. All tested oncogenic types degraded the PDZ-containing protein hMAGI1d; however, E6 proteins isolated from several related albeit non-oncogenic viral types were equally efficient at degrading hMAGI1. The relationship between both traits (oncogenicity and PDZ degradation potential) is best explained by a model in which the potential to degrade PDZ proteins was acquired prior to the oncogenic phenotype. This analysis provides evidence that the ancestor of both oncogenic and non-oncogenic HPVs acquired the potential to degrade human PDZ-containing proteins. This suggests that HPV E6 directed degradation of PDZ-proteins represents an ancient ecological niche adaptation. Phylogenetic modeling indicates that this phenotype is not specifically correlated with oncogenic risk, but may act as an enabling phenotype. The role of PDZ protein degradation in HPV fitness and oncogenesis needs to be interpreted in the context of Alphapapillomavirus evolution. PMID:26086730

  1. Degradation of Human PDZ-Proteins by Human Alphapapillomaviruses Represents an Evolutionary Adaptation to a Novel Cellular Niche.

    Directory of Open Access Journals (Sweden)

    Koenraad Van Doorslaer

    2015-06-01

    Full Text Available In order to complete their life cycle, papillomaviruses have evolved to manipulate a plethora of cellular pathways. The products of the human Alphapapillomavirus E6 proteins specifically interact with and target PDZ containing proteins for degradation. This viral phenotype has been suggested to play a role in viral oncogenesis. To analyze the association of HPV E6 mediated PDZ-protein degradation with cervical oncogenesis, a high-throughput cell culture assay was developed. Degradation of an epitope tagged human MAGI1 isoform was visualized by immunoblot. The correlation between HPV E6-induced degradation of hMAGI1 and epidemiologically determined HPV oncogenicity was evaluated using a Bayesian approach within a phylogenetic context. All tested oncogenic types degraded the PDZ-containing protein hMAGI1d; however, E6 proteins isolated from several related albeit non-oncogenic viral types were equally efficient at degrading hMAGI1. The relationship between both traits (oncogenicity and PDZ degradation potential is best explained by a model in which the potential to degrade PDZ proteins was acquired prior to the oncogenic phenotype. This analysis provides evidence that the ancestor of both oncogenic and non-oncogenic HPVs acquired the potential to degrade human PDZ-containing proteins. This suggests that HPV E6 directed degradation of PDZ-proteins represents an ancient ecological niche adaptation. Phylogenetic modeling indicates that this phenotype is not specifically correlated with oncogenic risk, but may act as an enabling phenotype. The role of PDZ protein degradation in HPV fitness and oncogenesis needs to be interpreted in the context of Alphapapillomavirus evolution.

  2. Antioxidant, ACE-Inhibitory and antibacterial activities of Kluyveromyces marxianus protein hydrolysates and their peptide fractions

    Directory of Open Access Journals (Sweden)

    Mahta Mirzaeia

    2016-07-01

    Full Text Available Background: There has been evidence that proteins are potentially excellent source of antioxidants, antihypertensive and antimicrobial peptides, and that enzymatic hydrolysis is an effective method to release these peptides from protein molecules. The functional properties of protein hydrolysates depends on the protein substrate, the specificity of the enzymes, the conditions used during proteolysis, degree of hydrolysis, and the nature of peptides released including molecular weight, amino acid composition, and hydrophobicity. Context and purpose of this study: The biomass of Kluyveromyces marxianus was considered as a source of ACE inhibitory, antioxidant and antimicrobial peptides. Results: Autolysis and enzymatic hydrolysis were completed respectively, after 96 h and 5 h. Overall, trypsin (18.52% DH and chymotrypsin (21.59% DH treatments were successful in releasing antioxidant and ACE inhibitory peptides. Autolysate sample (39.51% DH demonstrated poor antioxidant and ACE inhibitory activity compared to trypsin and chymotrypsin hydrolysates. The chymotrypsin 3-5 kDa (301.6±22.81 μM TE/mg protein and trypsin < 3 kDa (280.16±39.16 μM TE/mg protein permeate peptide fractions showed the highest DPPH radical scavenging activity. The trypsin <3 kDa permeate peptide fraction showed the highest ABTS radical scavenging (1691.1±48.68 μM TE/mg protein and ACE inhibitory (IC50=0.03±0.001 mg/mL activities. The fraction (MW=5-10 kD obtained after autolysis treatment showed antibacterial activity against St. aureus and Lis. monocytogenes in well diffusion screening. The minimum inhibitory concentration (MIC value was 13.3 mg/mLagainst St. aureus and Lis. monocytogenes calculated by turbidimetric assay and it showed bactericidal activity against St. aureus at 21.3 mg/mL protein concentration. Conclusions: Altogether, the results of this study reveal that K. marxianus proteins contain specific peptides in their sequences which can be released by

  3. 'Fractional recovery' analysis of a presynaptic synaptotagmin 1-anchored endocytic protein complex.

    Directory of Open Access Journals (Sweden)

    Rajesh Khanna

    Full Text Available BACKGROUND: The integral synaptic vesicle protein and putative calcium sensor, synaptotagmin 1 (STG, has also been implicated in synaptic vesicle (SV recovery. However, proteins with which STG interacts during SV endocytosis remain poorly understood. We have isolated an STG-associated endocytic complex (SAE from presynaptic nerve terminals and have used a novel fractional recovery (FR assay based on electrostatic dissociation to identify SAE components and map the complex structure. The location of SAE in the presynaptic terminal was determined by high-resolution quantitative immunocytochemistry at the chick ciliary ganglion giant calyx-type synapse. METHODOLOGY/PRINCIPLE FINDINGS: The first step in FR analysis was to immunoprecipitate (IP the complex with an antibody against one protein component (the IP-protein. The immobilized complex was then exposed to a high salt (1150 mM stress-test that caused shedding of co-immunoprecipitated proteins (co-IP-proteins. A Fractional Recovery ratio (FR: recovery after high salt/recovery with control salt as assayed by Western blot was calculated for each co-IP-protein. These FR values reflect complex structure since an easily dissociated protein, with a low FR value, cannot be intermediary between the IP-protein and a salt-resistant protein. The structure of the complex was mapped and a blueprint generated with a pair of FR analyses generated using two different IP-proteins. The blueprint of SAE contains an AP180/X/STG/stonin 2/intersectin/epsin core (X is unknown and epsin is hypothesized, and an AP2 adaptor, H-/L-clathrin coat and dynamin scission protein perimeter. Quantitative immunocytochemistry (ICA/ICQ method at an isolated calyx-type presynaptic terminal indicates that this complex is associated with STG at the presynaptic transmitter release face but not with STG on intracellular synaptic vesicles. CONCLUSIONS/SIGNIFICANCE: We hypothesize that the SAE serves as a recognition site and also as a

  4. Phosphorylation-dependent signaling controls degradation of DNA mismatch repair protein PMS2.

    Science.gov (United States)

    Hinrichsen, Inga; Weßbecher, Isabel M; Huhn, Meik; Passmann, Sandra; Zeuzem, Stefan; Plotz, Guido; Biondi, Ricardo M; Brieger, Angela

    2017-12-01

    MutLα, a heterodimer consisting of MLH1 and PMS2, plays an important role in DNA mismatch repair and has been shown to be additionally involved in several other important cellular mechanisms. Previous work indicated that AKT could modulate PMS2 stability by phosphorylation. Still, the mechanisms of regulation of MutLα remain unclear. The stability of MutLα subunits was investigated by transiently overexpression of wild type and mutant forms of MLH1 and PMS2 using immunoblotting for measuring the protein levels after treatment. We found that treatment with the cell-permeable serine/threonine phosphatase inhibitor, Calyculin, leads to degradation of PMS2 when MLH1 or its C-terminal domain is missing or if amino acids of MLH1 essential for PMS2 interaction are mutated. In addition, we discovered that the C-terminal tail of PMS2 is relevant for this Calyculin-dependent degradation. A direct involvement of AKT, which was previously described to be responsible for PMS2 degradation, could not be detected. The multi-kinase inhibitor Sorafenib, in contrast, was able to avoid the degradation of PMS2 which postulates that cellular phosphorylation is involved in this process. Together, we show that pharmacologically induced phosphorylation by Calyculin can induce the selective proteasome-dependent degradation of PMS2 but not of MLH1 and that the PMS2 degradation could be blocked by Sorafenib treatment. Curiously, the C-terminal Lynch Syndrome-variants MLH1 L749P and MLH1 Y750X make PMS2 prone to Calyculin induced degradation. Therefore, we conclude that the specific degradation of PMS2 may represent a new mechanism to regulate MutLα. © 2017 Wiley Periodicals, Inc.

  5. Protection of protein from ruminal degradation by alkali-induced oxidation of chlorogenic acid in sunflower meal.

    Science.gov (United States)

    Bongartz, V; Böttger, C; Wilhelmy, N; Schulze-Kaysers, N; Südekum, K-H; Schieber, A

    2018-02-01

    Lactating ruminants require an adequate supply of absorbable amino acids for the synthesis of milk protein from two sources, that is crude protein (CP) synthesized microbially in the rumen and ruminally undegraded CP (RUP) from feed which can both be digested in the small intestine. Several chemical and physical methods have been identified as being effective in increasing the proportion of RUP of total CP of a feedstuff, yet there is a continuing need for developing and establishing methods which protect feed protein from ruminal degradation with acceptable expenditure of labour and other costs. The objective of this study was to identify and quantify effects of and interactions between chlorogenic acid and protein in solvent-extracted sunflower meal (SFM) as induced by alkali treatment. Response surface methodology was employed to investigate the influence of pH, reaction time and drying temperature on the resulting SFM and, subsequently, its protein value for ruminants estimated from laboratory values. For this purpose, alkali-treated SFM was subjected to a fractionation of feed CP according to the Cornell net carbohydrate and protein system as a basis for estimating RUP at different assumed ruminal passage rates (K p ). To estimate the intestinal digestibility of the treated SFM and its RUP, a three-step enzymatic in vitro procedure was applied. Alkaline treatment of SFM increased RUP values with factors ranging from approximately 3 (K p =.08/hr) to 12 (K p =.02/hr). Furthermore, the intestinal digestibility of the alkali-treated SFM was enhanced by approximately 10% compared to untreated SFM. Increasing pH and reaction time led to both increasing RUP values and intestinal digestibility. In conclusion, a targeted alkaline treatment of naturally occurring compounds in feedstuffs might be a promising approach to provide high-RUP feeds for ruminants which, at the same time, have improved intestinal digestibility values. © 2017 Blackwell Verlag GmbH.

  6. Multidimensional protein fractionation of blood proteins coupled to data-independent nanoLC-MS/MS analysis.

    Science.gov (United States)

    Levin, Yishai; Jaros, Julian A J; Schwarz, Emanuel; Bahn, Sabine

    2010-01-03

    In order to exploit human blood as a source of protein disease biomarkers, robust analytical methods are needed to overcome the inherent molecular complexity of this bio-fluid. We present the coupling of label-free SAX chromatography and IMAC to a data-independent nanoLC-MS/MS (nanoLC-MS(E)) platform for analysis of blood plasma and serum proteins. The methods were evaluated using protein standards added at different concentrations to two groups of samples. The results demonstrate that both techniques enable accurate protein quantitation using low sample volumes and a minimal number of fractions. Combining both methods, 883 unique proteins were identified, of which 423 proteins showed high reproducibility. The two approaches resulted in identification of unique molecular signatures with an overlap of approximately 30%, thus providing complimentary information on sub-proteomes. These methods are potentially useful for systems biology, biomarker discovery, and investigation of phosphoproteins in blood. (c) 2009 Elsevier B.V. All rights reserved.

  7. Generation of Oxygen Free Radicals by Proflavine: Implication in Protein Degradation

    Directory of Open Access Journals (Sweden)

    Mansour K.M. Gatasheh

    2017-07-01

    Full Text Available Proflavine, an acridine dye, is a known DNA intercalating agent. In the present study, we show that proflavine alone on photoillumination can generate reactive oxygen species (ROS. These proflavine-derived ROS cause damage to proteins, and this effect is enhanced when the divalent metal ion Cu (II is included in the reaction. Bathocuproine, a specific Cu (I sequestering agent, when present in the reaction mixture containing Cu (II, was found to inhibit the protein degradation, showing that Cu (I is an essential intermediate in the reaction. The effect of several scavengers of ROS such as superoxide dismutase, sodium azide, potassium iodide, and thiourea were examined on the protein damaging reaction. Potassium iodide was found to be the most effective in inhibiting protein damage followed by sodium azide and thiourea. Our results indicate the involvement of superoxide, singlet oxygen, triplet oxygen, and hydroxyl radicals in proflavine-induced damage to proteins.

  8. Measurement of local cerebral protein synthesis in vivo: influence of recycling of amino acids derived from protein degradation

    International Nuclear Information System (INIS)

    Smith, C.B.; Deibler, G.E.; Eng, N.; Schmidt, K.; Sokoloff, L.

    1988-01-01

    A quantitative autoradiographic method for the determination of local rates of protein synthesis in brain in vivo is being developed. The method employs L-[1- 14 C]leucine as the radiolabeled tracer. A comprehensive model has been designed that takes into account intracellular and extracellular spaces, intracellular compartmentation of leucine, and the possibility of recycling of unlabeled leucine derived from steady-state degradation of protein into the precursor pool for protein synthesis. We have evaluated the degree of recycling by measuring the ratio of the steady-state precursor pool distribution space for labeled leucine to that of unlabeled leucine. The values obtained were 0.58 in whole brain and 0.47 in liver. These results indicate that there is significant recycling of unlabeled amino acids derived from steady-state protein degradation in both tissues. Any method for the determination of rates of cerebral protein synthesis in vivo with labeled tracers that depends on estimation of precursor pool specific activity in tissue from measurements in plasma must take this recycling into account

  9. A STRUCTURAL OVERVIEW OF GH61 PROTEINS – FUNGAL CELLULOSE DEGRADING POLYSACCHARIDE MONOOXYGENASES

    Directory of Open Access Journals (Sweden)

    Leila Lo Leggio

    2012-09-01

    Full Text Available Recent years have witnessed a spurt of activities in the elucidation of the molecular function of a class of proteins with great potential in biomass degradation. GH61 proteins are of fungal origin and were originally classified in family 61 of the glycoside hydrolases. From the beginning they were strongly suspected to be involved in cellulose degradation because of their expression profiles, despite very low detectable endoglucanase activities. A major breakthrough came from structure determination of the first members, establishing the presence of a divalent metal binding site and a similarity to bacterial proteins involved in chitin degradation. A second breakthrough came from the identification of cellulase boosting activity dependent on the integrity of the metal binding site. Finally very recently GH61 proteins were demonstrated to oxidatively cleave crystalline cellulose in a Cu and reductant dependant manner. This mini-review in particular focuses on the contribution that structure elucidation has made in the understanding of GH61 molecular function and reviews the currently known structures and the challenges remaining ahead for exploiting this new class of enzymes to the full.

  10. A structural overview of GH61 proteins – fungal cellulose degrading polysaccharide monooxygenases

    Directory of Open Access Journals (Sweden)

    Leila Lo Leggio

    2012-09-01

    Full Text Available Recent years have witnessed a spurt of activities in the elucidation of the molecular function of a class of proteins with great potential in biomass degradation. GH61 proteins are of fungal origin and were originally classified in family 61 of the glycoside hydrolases. From the beginning they were strongly suspected to be involved in cellulose degradation because of their expression profiles, despite very low detectable endoglucanase activities. A major breakthrough came from structure determination of the first members, establishing the presence of a divalent metal binding site and a similarity to bacterial proteins involved in chitin degradation. A second breakthrough came from the identification of cellulase boosting activity dependent on the integrity of the metal binding site. Finally very recently GH61 proteins were demonstrated to oxidatively cleave crystalline cellulose in a Cu and reductant dependant manner. This mini-review in particular focuses on the contribution that structure elucidation has made in the understanding of GH61 molecular function and reviews the currently known structures and the challenges remaining ahead for exploiting this new class of enzymes to the full.

  11. HUWE1 and TRIP12 collaborate in degradation of ubiquitin-fusion proteins and misframed ubiquitin.

    Directory of Open Access Journals (Sweden)

    Esben G Poulsen

    Full Text Available In eukaryotic cells an uncleavable ubiquitin moiety conjugated to the N-terminus of a protein signals the degradation of the fusion protein via the proteasome-dependent ubiquitin fusion degradation (UFD pathway. In yeast the molecular mechanism of the UFD pathway has been well characterized. Recently the human E3 ubiquitin-protein ligase TRIP12 was connected with the UFD pathway, but little is otherwise known about this system in mammalian cells. In the present work, we utilized high-throughput imaging on cells transfected with a targeted siRNA library to identify components involved in degradation of the UFD substrate Ub(G76V-YFP. The most significant hits from the screen were the E3 ubiquitin-protein ligase HUWE1, as well as PSMD7 and PSMD14 that encode proteasome subunits. Accordingly, knock down of HUWE1 led to an increase in the steady state level and a retarded degradation of the UFD substrate. Knock down of HUWE1 also led to a stabilization of the physiological UFD substrate UBB(+1. Precipitation experiments revealed that HUWE1 is associated with both the Ub(G76V-YFP substrate and the 26S proteasome, indicating that it functions late in the UFD pathway. Double knock down of HUWE1 and TRIP12 resulted in an additive stabilization of the substrate, suggesting that HUWE1 and TRIP12 function in parallel during UFD. However, even when both HUWE1 and TRIP12 are downregulated, ubiquitylation of the UFD substrate was still apparent, revealing functional redundancy between HUWE1, TRIP12 and yet other ubiquitin-protein ligases.

  12. Protection of a protein against irradiation-induced degradation by additives in the solid state

    International Nuclear Information System (INIS)

    Shalaev, E.; Reddy, R.; Kimball, R.N.; Weinschenk, M.F.; Guinn, M.; Margulis, L.

    2003-01-01

    The impact of ionizing radiation on a globular protein (porcine somatotropin, pST) in the solid state was studied using rate of dissolution, high-performance liquid chromatography, and Electron spin resonance (ESR) in the presence of different additives. o-Vanillin stabilized pST against irradiation-induced degradation whereas effects of trolox and isopropyl alcohol were less significant. Stabilization effect of o-vanillin has been related to the energy transfer from pST molecules to the additive which was facilitated by formation of covalent bonds between o-vanillin and pST molecules. Anticorrelation between the level of free radicals and chemical degradation (i.e. degradation increased with decrease in a free radical level) was observed in the presence of o-vanillin

  13. Gold nanoparticles enhance the X-ray-induced degradation of human centrin 2 protein

    Energy Technology Data Exchange (ETDEWEB)

    Brun, Emilie [Laboratoire de Chimie Physique, CNRS UMR 8000, Universite Paris-Sud 11, Bat. 350, 91405 Orsay Cedex (France); Duchambon, Patricia; Blouquit, Yves [INSERM U759, Imagerie Integrative, Campus Universitaire d' Orsay, Bat. 112, Institut Curie, Centre de Recherche, Laboratoire R. Latarjet, Campus Universitaire d' Orsay, 91405 Orsay Cedex (France); Keller, Gerard [UMR CNRS 8612, Physico-Chimie-Pharmacotechnie-Biopharmacie, Universite Paris 11, Faculte de Pharmacie, 5 rue Jean-Baptiste Clement, 92296 Chatenay-Malabry (France); Sanche, Leon [Groupe en Sciences des Radiations, Departement de Medecine Nucleaire et Radiobiologie, Faculte de Medecine, Universite de Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4 (Canada); Sicard-Roselli, Cecile [Laboratoire de Chimie Physique, CNRS UMR 8000, Universite Paris-Sud 11, Bat. 350, 91405 Orsay Cedex (France)], E-mail: cecile.sicard@u-psud.fr

    2009-03-15

    In the war against cancer, radiotherapy is a prominent tool but counterbalanced by the fact that it also induces damages in healthy tissues. Nanotechnologies could open a new possibility to decrease these side effects. In particular, gold nanoparticles (GNPs) could be used as radio-sensitizers. As the role of proteins in the processes leading to cell death cannot be neglected, their radio-sensitization by GNPs is of great interest. This is particularly true in the case of the human centrin 2 protein, which has been proposed to be involved in DNA repair processes. To investigate this effect, we quantified for the first time the degradation of this protein in a gold colloidal solution when submitted to X-rays. We showed that the X-ray-induced degradation of the human centrin 2 protein is enhanced 1.5-fold in the presence of GNPs, even though no covalent bond exists between protein and GNPs. Among the conditions tested, the maximum enhancement was found with the higher GNP:protein ratio of 2x10{sup -4} and with the higher X-ray energy of 49 keV.

  14. Gold nanoparticles enhance the X-ray-induced degradation of human centrin 2 protein

    International Nuclear Information System (INIS)

    Brun, Emilie; Duchambon, Patricia; Blouquit, Yves; Keller, Gerard; Sanche, Leon; Sicard-Roselli, Cecile

    2009-01-01

    In the war against cancer, radiotherapy is a prominent tool but counterbalanced by the fact that it also induces damages in healthy tissues. Nanotechnologies could open a new possibility to decrease these side effects. In particular, gold nanoparticles (GNPs) could be used as radio-sensitizers. As the role of proteins in the processes leading to cell death cannot be neglected, their radio-sensitization by GNPs is of great interest. This is particularly true in the case of the human centrin 2 protein, which has been proposed to be involved in DNA repair processes. To investigate this effect, we quantified for the first time the degradation of this protein in a gold colloidal solution when submitted to X-rays. We showed that the X-ray-induced degradation of the human centrin 2 protein is enhanced 1.5-fold in the presence of GNPs, even though no covalent bond exists between protein and GNPs. Among the conditions tested, the maximum enhancement was found with the higher GNP:protein ratio of 2x10 -4 and with the higher X-ray energy of 49 keV

  15. Blood plasma proteins and protein fractions in roe deer Capreolus capreolus L.

    Directory of Open Access Journals (Sweden)

    Dorota CYGAN-SZCZEGIELNIAK

    2015-09-01

    Full Text Available The aim of the research was to investigate some selected biochemical blood parameters in roe deer (Capreolus capreolus L.. The experiment covered 15 from 2 to 3-year-old bucks from Kuyavian-Pomeranian Voivodeship. The animals were shot by individual hunters on the shooting grounds during the hunting season of 2008/2009 (in the accordance with the Journal of Laws No 48. The material for the research was blood plasma obtained after centrifuging full, nonhemolyzed blood. The blood was collected from the zygomatic vein directly to the test tubes with EDTA and transported in cooling conditions to the laboratory. After transporting the samples of blood to a certified analytical laboratory, the following elements of the obtained blood plasma were examined: ceruloplasmin . using turbidimetric method; transferrin . using immunoturbimetric method; troponin- using a third generation assay on an Elecsys; total protein, albumin, globulin . using spectrophotometric method and total iron . using colorimetric method. The results were statistically analyzed, i.e. the correlation between the parameters was measured by means of Pearsonfs correlation coefficient. The analysis of the results revealed a number of statistically significant relations between the parameters under the investigation, especially among the compounds directly responsible for metabolism of iron and copper. A statistically important positive correlation was observed between ceruloplasmin and ferritin (r = 0.563; P.0.05 and a negative one between transferrin and troponin (r = -0.609; P.0.05. Moreover, the content of transferrin . an iron-binding protein . was 0.17 g/l, while the concentration of iron was 58 ƒĘmol/l. The content of ceruloplasmin . a protein responsible for metabolism of copper . was very low (0.036 g/l. The level of proteins in the blood plasma of the animals under the research was approximately 72 g/l, with the share of albumins about 46%. The albumin-globulin ratio was 0.86.

  16. Qualitative and quantitative multiplexed proteomic analysis of complex yeast protein fractions that modulate the assembly of the yeast prion Sup35p.

    Directory of Open Access Journals (Sweden)

    Virginie Redeker

    Full Text Available BACKGROUND: The aggregation of the baker's yeast prion Sup35p is at the origin of the transmissible [PSI(+] trait. We and others have shown that molecular chaperones modulate Sup35p aggregation. However, other protein classes might be involved in [PSI(+] formation. RESULTS: We designed a functional proteomic study that combines two techniques to identify modulators of Sup35p aggregation and describe the changes associated to [PSI(+] formation. The first allows measuring the effect of fractionated Saccharomyces cerevisiae cytosolic extracts from [PSI(+] and [psi(-] yeast cells on Sup35p assembly. The second is a multiplex qualitative and quantitative comparison of protein composition of active and inactive fractions using a gel-free and label-free LC-MS approach. We identify changes in proteins involved in translation, folding, degradation, oxido-reduction and metabolic processes. CONCLUSION: Our functional proteomic study provides the first inventory list of over 300 proteins that directly or indirectly affect Sup35p aggregation and [PSI(+] formation. Our results highlight the complexity of the cellular changes accompanying [PSI(+] formation and pave the way for in vitro studies aimed to document the effect of individual and/or combinations of proteins identified here, susceptible of affecting Sup35p assembly.

  17. Effects of different fractions of whey protein on postprandial lipid and hormone responses in type 2 diabetes

    DEFF Research Database (Denmark)

    Mortensen, L.S.; Holmer-Jensen, Jens; Hartvigsen, Merete

    2012-01-01

    Background/Objectives:Exacerbated postprandial lipid responses are associated with an increased cardiovascular risk. Dietary proteins influence postprandial lipemia differently, and whey protein has a preferential lipid-lowering effect. We compared the effects of different whey protein fractions .......European Journal of Clinical Nutrition advance online publication, 16 May 2012; doi:10.1038/ejcn.2012.48....

  18. Komposisi Kimiawi dan Fraksinasi Protein Susu Kuda Sumba (THE CHEMICAL COMPOSITION AND PROTEIN FRACTIONATION OF SUMBA MARE’S MILK

    Directory of Open Access Journals (Sweden)

    Annytha Ina Rohi Detha

    2015-05-01

    Full Text Available The aims of this study were to determine both chemical composition and fraction of the proteincompounds of sumba mare’s milk. Determination of the chemical compositions of sumba mare’s milk havedone by analyzing protein content using the Kjeldahl method, fat content using Gerber method, lactosecontent and the total solids content. Identification of antimicrobial compounds of whey proteins in milkusing high performance liquid chromatography (HPLC method. The results showed that the average ofsumba mare’s milk contained protein, fat, lactose and total solids were; 1.82%, 1.67%, 6.48% and 11.37%respectively. The average value of protein and fat in sumba mare’s milk was decrease significantly at fifthmonth of lactation period. Based on identification of antimicrobial compounds using HPLC method, thereare six main peaks with different polarities and retention times. In conclusion, sumba mare’s milk havea balance composition that can be used as a source of nutritious food and the milk likely also has six mainantimicrobial compounds in its whey protein.

  19. Ruminal and intestinal protein degradability of various seaweed species measured in situ in dairy cows

    DEFF Research Database (Denmark)

    Tayyab, Usama; Novoa-Garrido, Margarita; Roleda, Michael Y.

    2016-01-01

    The use of seaweeds in animal diets is not new. However, little is known about the feed value of seaweed, both in terms of chemical composition and protein digestibility, and regarding variation between species and season. In this study, eight seaweed species of the genus Acrosiphonia, Alaria......, Laminaria, Mastocarpus, Palmaria, Pelvetia, Porphyra, and Ulva were sampled in spring (March) and autumn (October and November) 2014 at the coast of Bodø in Northern Norway, and were analysed for chemical composition, in situ rumen degradability and total tract crude protein (CP) digestibility. Ash content...... for Pelvetia (90 g/kg DM). Spring samples were higher in CP than autumn samples. The effective degradability estimated at 5% rumen passage rate (ED5) of CP varied between species (P Ulva (240 g...

  20. Requirements for the selective degradation of CD4 receptor molecules by the human immunodeficiency virus type 1 Vpu protein in the endoplasmic reticulum

    Directory of Open Access Journals (Sweden)

    Halawani Dalia

    2007-10-01

    Full Text Available Abstract Background HIV-1 Vpu targets newly synthesized CD4 receptor for rapid degradation by a process reminiscent of endoplasmic reticulum (ER-associated protein degradation (ERAD. Vpu is thought to act as an adaptor protein, connecting CD4 to the ubiquitin (Ub-proteasome degradative system through an interaction with β-TrCP, a component of the SCFβ-TrCP E3 Ub ligase complex. Results Here, we provide direct evidence indicating that Vpu promotes trans-ubiquitination of CD4 through recruitment of SCFβ-TrCP in human cells. To examine whether Ub conjugation occurs on the cytosolic tail of CD4, we substituted all four Ub acceptor lysine residues for arginines. Replacement of cytosolic lysine residues reduced but did not prevent Vpu-mediated CD4 degradation and ubiquitination, suggesting that Vpu-mediated CD4 degradation is not entirely dependent on the ubiquitination of cytosolic lysines and as such might also involve ubiquitination of other sites. Cell fractionation studies revealed that Vpu enhanced the levels of ubiquitinated forms of CD4 detected in association with not only the ER membrane but also the cytosol. Interestingly, significant amounts of membrane-associated ubiquitinated CD4 appeared to be fully dislocated since they could be recovered following sodium carbonate salt treatment. Finally, expression of a transdominant negative mutant of the AAA ATPase Cdc48/p97 involved in the extraction of ERAD substrates from the ER membrane inhibited Vpu-mediated CD4 degradation. Conclusion Taken together, these results are consistent with a model whereby HIV-1 Vpu targets CD4 for degradation by an ERAD-like process involving most likely poly-ubiquitination of the CD4 cytosolic tail by SCFβ-TrCP prior to dislocation of receptor molecules across the ER membrane by a process that depends on the AAA ATPase Cdc48/p97.

  1. Dma1-dependent degradation of SIN proteins during meiosis in Schizosaccharomyces pombe.

    Science.gov (United States)

    Krapp, Andrea; Simanis, Viesturs

    2014-07-15

    The Schizosaccharomyces pombe septation initiation network (SIN) is required for cytokinesis during vegetative growth and for spore formation during meiosis. Regulation of the SIN during mitosis has been studied extensively, but less is known about its meiotic regulation. Here, we show that several aspects of SIN regulation differ between mitosis and meiosis. First, the presence of GTP-bound Spg1p is not the main determinant of the timing of Cdc7p and Sid1p association with the spindle pole body (SPB) during meiosis. Second, the localisation dependencies of SIN proteins differ from those in mitotic cells, suggesting a modified functional organisation of the SIN during meiosis. Third, there is stage-specific degradation of SIN components in meiosis; Byr4p is degraded after meiosis I, whereas the degradation of Cdc7p, Cdc11p and Sid4p occurs after the second meiotic division and depends upon the ubiquitin ligase Dma1p. Finally, Dma1p-dependent degradation is not restricted to the SIN, as we show that Dma1p is needed for the degradation of Mcp6p (also known as Hrs1p) during meiosis I. Taken together, these data suggest that stage-specific targeted proteolysis plays an important role in regulating meiotic progression. © 2014. Published by The Company of Biologists Ltd.

  2. Protein oxidation and degradation during proliferative senescence of human MRC-5 fibroblasts.

    Science.gov (United States)

    Sitte, N; Merker, K; von Zglinicki, T; Grune, T

    2000-03-01

    One of the highlights of age-related changes of cellular metabolism is the accumulation of oxidized proteins. The aging process on a cellular level can be treated either as the ongoing proliferation until a certain number of cell divisions is reached (the Hayflick limit) or as the aging of nondividing cells, that is, the age-related changes in cells without proliferation. The present investigation was undertaken to reveal the changes in protein turnover, proteasome activity, and protein oxidation status during proliferative senescence. We were able to demonstrate that the activity of the cytosolic proteasomal system declines dramatically during the proliferative senescence of human MRC-5 fibroblasts. Regardless of the loss in activity, it could be demonstrated that there are no changes in the transcription and translation of proteasomal subunits. This decline in proteasome activity was accompanied by an increased concentration of oxidized proteins. Cells at higher proliferation stages were no longer able to respond with increased degradation of endogenous [(35)S]-Met-radiolabeled proteins after hydrogen peroxide- or quinone-induced oxidative stress. It could be demonstrated that oxidized proteins in senescent human MRC-5 fibroblasts are not as quickly removed as they are in young cells. Therefore, our study demonstrates that the accumulation of oxidized proteins and decline in protein turnover and activity of the proteasomal system are not only a process of postmitotic aging but also occur during proliferative senescence and result in an increased half-life of oxidized proteins.

  3. Determination of degradability of germinated and heated soybean seeds and its proteins fractions

    Directory of Open Access Journals (Sweden)

    Behruz Alizadeh

    2016-09-01

    Full Text Available A study on the sensitivity of watermelon variety Bojura to mutagenic agents was carried out in 2013-2014. The goal was to establish effective doses for mutagenic treatment of dry seeds with 60Co gamma rays (80, 100, 200, 250, 350 and 450 Gy and swollen seeds with water for 24 hours were treated with ethyl methanesulfonate (EMS at a concentration of 2%. Dominant mutations were not observed in the M1 generation. Morphological changes in 14 of 1395 M2 plants were observed. Phenotypic variations changes were the colour of the seed coat, chlorophyll disorders of cotyledons, leaves, petals, and alterations of the location of the fruit set in the central stem. Visible changes of the morphological characteristics of the fruit were not observed. The doses induced certain morphological changes, however, higher doses or combined gamma rays 60Co and EMS treatments would induce mutations more efficiently. Subsequent experiments are required to obtain mutants with changes that affect flowers and fruits. The results are important for increasing mutation efficiency in watermelon breeding.

  4. New strategy for renal fibrosis: Targeting Smad3 proteins for ubiquitination and degradation.

    Science.gov (United States)

    Wang, Xin; Feng, Shaozhen; Fan, Jinjin; Li, Xiaoyan; Wen, Qiong; Luo, Ning

    2016-09-15

    Smad3 is a critical signaling protein in renal fibrosis. Proteolysis targeting chimeric molecules (PROTACs) are small molecules designed to degrade target proteins via ubiquitination. They have three components: (1) a recognition motif for E3 ligase; (2) a linker; and (3) a ligand for the target protein. We aimed to design a new PROTAC to prevent renal fibrosis by targeting Smad3 proteins and using hydroxylated pentapeptide of hypoxia-inducible factor-1α as the recognition motif for von Hippel-Lindau (VHL) ubiquitin ligase (E3). Computer-aided drug design was used to find a specific ligand targeting Smad3. Surface plasmon resonance (SPR) was used to verify and optimize screening results. Synthesized PROTAC was validated by two-stage mass spectrometry. The PROTAC's specificity for VHL (E3 ligase) was proved with two human renal carcinoma cell lines, 786-0 (VHL(-)) and ACHN (VHL(+)), and its anti-fibrosis effect was tested in renal fibrosis cell models. Thirteen small molecular compounds (SMCs) were obtained from the Enamine library using GLIDE molecular docking program. SPR results showed that #8 SMC (EN300-72284) combined best with Smad3 (KD=4.547×10(-5)M). Mass spectrometry showed that synthesized PROTAC had the correct peptide molecular weights. Western blot showed Smad3 was degraded by PROTAC with whole-cell lysate of ACHN but not 786-0. Degradation, but not ubiquitination, of Smad3 was inhibited by proteasome inhibitor MG132. The upregulation of fibronectin and Collagen I induced by TGF-β1 in both renal fibroblast and mesangial cells were inhibited by PROTAC. The new PROTAC might prevent renal fibrosis by targeting Smad3 for ubiquitination and degradation. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Fractionation of hairless canary seed (Phalaris canariensis) into starch, protein, and oil.

    Science.gov (United States)

    Abdel-Aal, El-Sayed M; Hucl, Pierre; Patterson, Carol Ann; Gray, Danielle

    2010-06-09

    Canary seed is an important specialty crop in Canada. The current market for this true cereal (i.e., belonging to the family Poaceae as wheat) is limited to feed for caged birds. However, canary seed holds a promise for many food and industrial applications based on its composition. Three wet milling procedures based on ethanol (E), water (W), and alkaline (A) extractions used in different order were investigated to determine extraction efficiency and purity of starch, protein, oil, and fiber separated from hairless canary seed, a variety developed for human consumption. Highest extraction efficiencies were obtained when canary seed was defatted with ethanol and then extracted with alkali and water (EAW process). Using this process, approximately 92% pure starch, 75% pure protein, and oil were recovered from canary seed groats. The highest purity of protein, however, was obtained when canary seed was fractionated by the EWA process, that is, defatted and then extracted with water followed by alkali. Fiber component separated prior to alkaline extraction contained high amounts of nonfiber components as indicated by its yield. The EAW extraction process seems to be more promising in canary seed fractionation based on recovery and purity of components.

  6. Nck adaptor proteins link Tks5 to invadopodia actin regulation and ECM degradation.

    Science.gov (United States)

    Stylli, Stanley S; Stacey, T T I; Verhagen, Anne M; Xu, San San; Pass, Ian; Courtneidge, Sara A; Lock, Peter

    2009-08-01

    Invadopodia are actin-based projections enriched with proteases, which invasive cancer cells use to degrade the extracellular matrix (ECM). The Phox homology (PX)-Src homology (SH)3 domain adaptor protein Tks5 (also known as SH3PXD2A) cooperates with Src tyrosine kinase to promote invadopodia formation but the underlying pathway is not clear. Here we show that Src phosphorylates Tks5 at Y557, inducing it to associate directly with the SH3-SH2 domain adaptor proteins Nck1 and Nck2 in invadopodia. Tks5 mutants unable to bind Nck show reduced matrix degradation-promoting activity and recruit actin to invadopodia inefficiently. Conversely, Src- and Tks5-driven matrix proteolysis and actin assembly in invadopodia are enhanced by Nck1 or Nck2 overexpression and inhibited by Nck1 depletion. We show that clustering at the plasma membrane of the Tks5 inter-SH3 region containing Y557 triggers phosphorylation at this site, facilitating Nck recruitment and F-actin assembly. These results identify a Src-Tks5-Nck pathway in ECM-degrading invadopodia that shows parallels with pathways linking several mammalian and pathogen-derived proteins to local actin regulation.

  7. Lysine-Less Variants of Spinal Muscular Atrophy SMN and SMNΔ7 Proteins Are Degraded by the Proteasome Pathway

    Directory of Open Access Journals (Sweden)

    Raúl Sánchez-Lanzas

    2017-12-01

    Full Text Available Spinal muscular atrophy is due to mutations affecting the SMN1 gene coding for the full-length protein (survival motor neuron; SMN and the SMN2 gene that preferentially generates an exon 7-deleted protein (SMNΔ7 by alternative splicing. To study SMN and SMNΔ7 degradation in the cell, we have used tagged versions at the N- (Flag or C-terminus (V5 of both proteins. Transfection of those constructs into HeLa cells and treatment with cycloheximide showed that those protein constructs were degraded. Proteasomal degradation usually requires prior lysine ubiquitylation. Surprisingly, lysine-less variants of both proteins tagged either at N- (Flag or C-terminus (V5 were also degraded. The degradation of the endogenous SMN protein, and the protein constructs mentioned above, was mediated by the proteasome, as it was blocked by lactacystin, a specific and irreversible proteasomal inhibitor. The results obtained allowed us to conclude that SMN and SMNΔ7 proteasomal degradation did not absolutely require internal ubiquitylation nor N-terminal ubiquitylation (prevented by N-terminal tagging. While the above conclusions are firmly supported by the experimental data presented, we discuss and justify the need of deep proteomic techniques for the study of SMN complex components (orphan and bound turn-over to understand the physiological relevant mechanisms of degradation of SMN and SMNΔ7 in the cell.

  8. Degradation of brown adipocyte purine nucleotides regulates uncoupling protein 1 activity

    Directory of Open Access Journals (Sweden)

    Tobias Fromme

    2018-02-01

    Full Text Available Objective: Non-shivering thermogenesis in mammalian brown adipose tissue depends on thermogenic uncoupling protein 1. Its activity is triggered by free fatty acids while purine nucleotides mediate inhibition. During activation, it is thought that free fatty acids overcome purine-mediated inhibition. We measured the cellular concentration and the release of purine nucleotide metabolites to uncover a possible role of purine nucleotide degradation in uncoupling protein 1 activation. Methods: With mass spectrometry, purine nucleotide metabolites were quantified in cellular homogenates and supernatants of cultured primary brown adipocytes. We also determined oxygen consumption in response to a β-adrenergic agonist. Results: Upon adrenergic activation, brown adipocytes decreased the intracellular concentration of inhibitory nucleotides (ATP, ADP, GTP and GDP and released the respective degradation products. At the same time, an increase in cellular calcium occurred. None of these phenomena occurred in white adipocytes or myotubes. The brown adipocyte expression of enzymes implicated in purine metabolic remodeling is altered upon cold exposure. Pharmacological and genetic interference of purine metabolism altered uncoupling protein 1 mediated uncoupled respiration. Conclusion: Adrenergic stimulation of brown adipocytes lowers the intracellular concentration of purine nucleotides, thereby contributing to uncoupling protein 1 activation. Keywords: Purine nucleotides, Uncoupling protein 1, Brown adipose tissue, Non-shivering thermogenesis, HILIC-MS/MS, Guanosine monophosphate reductase

  9. Methods for the visualization and analysis of extracellular matrix protein structure and degradation.

    Science.gov (United States)

    Leonard, Annemarie K; Loughran, Elizabeth A; Klymenko, Yuliya; Liu, Yueying; Kim, Oleg; Asem, Marwa; McAbee, Kevin; Ravosa, Matthew J; Stack, M Sharon

    2018-01-01

    This chapter highlights methods for visualization and analysis of extracellular matrix (ECM) proteins, with particular emphasis on collagen type I, the most abundant protein in mammals. Protocols described range from advanced imaging of complex in vivo matrices to simple biochemical analysis of individual ECM proteins. The first section of this chapter describes common methods to image ECM components and includes protocols for second harmonic generation, scanning electron microscopy, and several histological methods of ECM localization and degradation analysis, including immunohistochemistry, Trichrome staining, and in situ zymography. The second section of this chapter details both a common transwell invasion assay and a novel live imaging method to investigate cellular behavior with respect to collagen and other ECM proteins of interest. The final section consists of common electrophoresis-based biochemical methods that are used in analysis of ECM proteins. Use of the methods described herein will enable researchers to gain a greater understanding of the role of ECM structure and degradation in development and matrix-related diseases such as cancer and connective tissue disorders. © 2018 Elsevier Inc. All rights reserved.

  10. Effects of high hydrostatic pressure on the functional and rheological properties of the protein fraction extracted from pine nuts.

    Science.gov (United States)

    Cao, Baiying; Fang, Li; Liu, Chunlei; Min, Weihong; Liu, Jingsheng

    2018-01-01

    High hydrostatic pressure treatments could increase the protein solubility (200 MPa), water holding capacity (400 MPa), and oil holding capacity (400 MPa) of pine nuts protein fractions, respectively. The exposed sufhydryl content for albumin was highest at 100 MPa while for other fractions it was 400 MPa, contrary for total sufhydryl content-generally it was at 100 MPa, except glutelin (400 MPa). Pine nuts protein fractions demonstrated the typical behavior of weak gels (G' > G″). After the treatments of high hydrostatic pressure the specific surface area of pine nuts protein particle was increased upon pressure, and the surface of protein became rough which increased the particle size. The functional groups of protein were found to be unchanged, but the characteristic peaks of pine nuts protein moved to a low-band displacement and the value of peaks was amplified accordingly to the pressure. The high hydrostatic pressure treatments were found to improve the functional properties of pine nuts protein isolates by enhancing the heat-induced gel strength of pine nuts protein isolates which make proteins more stretchable. These results suggest that high hydrostatic pressure treatments can increase the functional properties and alter the rheological properties of pine nuts protein fractions which will broaden its applications in food industry.

  11. Modulation of intracellular protein degradation by SSB1-SIS1 chaperon system in yeast S. cerevisiae.

    Science.gov (United States)

    Ohba, M

    1997-06-09

    In prokaryotes, DnaK-DnaJ chaperon is involved in the protein degradation catalyzed by proteases La and ClpA/B complex as shown in E. coli. To extend this into eukaryotic cells, we examined the effects of hsp70 genes, SSA1 and SSB1, and DnaJ genes, SIS1 and YDJ1, on the growth of proteasome subunit mutants of the yeast S. cerevisiae. The results identified SSB1 and SIS1 as a pair of chaperon genes specifically involved in efficient protein turnover in the yeast, whose overexpression suppressed the growth defects caused by the proteasome mutations. Moreover, a single amino acid substitution in the putative peptide-binding site of SSB1 protein profoundly enhanced the suppression activity, indicating that the activity is mediated by the peptide-binding activity of this chaperon. Thus SSB1, with its partner DnaJ, SIS1, modulates the efficiency of protein turnover through its chaperon activity.

  12. Retinoblastoma protein co-purifies with proteasomal insulin-degrading enzyme: Implications for cell proliferation control

    Energy Technology Data Exchange (ETDEWEB)

    Radulescu, Razvan T., E-mail: ratura@gmx.net [Molecular Concepts Research (MCR), Muenster (Germany); Duckworth, William C. [Department of Medicine, Phoenix VA Health Care System, Phoenix, AZ (United States); Levy, Jennifer L. [Research Service, Phoenix VA Health Care System, Phoenix, AZ (United States); Fawcett, Janet, E-mail: janet.fawcett@va.gov [Research Service, Phoenix VA Health Care System, Phoenix, AZ (United States)

    2010-04-30

    Previous investigations on proteasomal preparations containing insulin-degrading enzyme (IDE; EC 3.4.24.56) have invariably yielded a co-purifying protein with a molecular weight of about 110 kDa. We have now found both in MCF-7 breast cancer and HepG2 hepatoma cells that this associated molecule is the retinoblastoma tumor suppressor protein (RB). Interestingly, the amount of RB in this protein complex seemed to be lower in HepG2 vs. MCF-7 cells, indicating a higher (cytoplasmic) protein turnover in the former vs. the latter cells. Moreover, immunofluorescence showed increased nuclear localization of RB in HepG2 vs. MCF-7 cells. Beyond these subtle differences between these distinct tumor cell types, our present study more generally suggests an interplay between RB and IDE within the proteasome that may have important growth-regulatory consequences.

  13. Life and death of proteins after protease cleavage: protein degradation by the N-end rule pathway.

    Science.gov (United States)

    Dissmeyer, Nico; Rivas, Susana; Graciet, Emmanuelle

    2018-05-01

    Contents Summary 929 I. conservation and diversity of N-end rule pathways 929 II. Defensive functions of the N-end rule pathway in plants 930 III. Proteases and degradation by the N-end rule pathway 930 IV. New proteomics approaches for the identification of N-end rule substrates 932 V. Concluding remarks 932 Acknowledgements 934 References 934 SUMMARY: The N-end rule relates the stability of a protein to the identity of its N-terminal residue and some of its modifications. Since its discovery in the 1980s, the repertoire of N-terminal degradation signals has expanded, leading to a diversity of N-end rule pathways. Although some of these newly discovered N-end rule pathways remain largely unexplored in plants, recent discoveries have highlighted roles of N-end rule-mediated protein degradation in plant defense against pathogens and in cell proliferation during organ growth. Despite this progress, a bottleneck remains the proteome-wide identification of N-end rule substrates due to the prerequisite for endoproteolytic cleavage and technical limitations. Here, we discuss the recent diversification of N-end rule pathways and their newly discovered functions in plant defenses, stressing the role of proteases. We expect that novel proteomics techniques (N-terminomics) will be essential for substrate identification. We review these methods, their limitations and future developments. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  14. INFLUENCE OF NATURAL IMMUNOMODULATORS ON PROTEIN FRACTIONS AND CORTISOL CONTENT IN RABBIT BLOOD UNDER STRESS

    Directory of Open Access Journals (Sweden)

    Grabovskyi S.

    2015-08-01

    Full Text Available The results of determination of protein fractions, cortisol content in blood of rabbits, which further added to the feed of natural origin biologically active substances are presented in the article. As an antistressors and immunomodulators in pre-slaughter period are using of spleen extract biologically active substances were obtained with ultrasound application. The purpose of research — determination of changes of protein fractions, cortisol content in rabbits blood before slaughter and their correction of natural origin biologically active substances (spleen extract. Object and research methods. The experiment was conducted on 15 rabbits with standard diet. Three groups of rabbits five month of age (5 rabbits each was formed for research. The spleen extract were using as an biologically active substances to the feed rabbits in pre-slaughter period (five days before slaughter. The extracts were applied to feed by aerosol method (70 °alcohol solution of spleen extract volume of 1.4 ml per rabbit (group I. The rabbits (group II received to the feed in the same way of 70 °alcohol solution in the same volume. The control group rabbits received the standard feed in the same volume. The feed eating by rabbits was exercised daily. The rabbits ate food completely. The rabbits slaughter was carried out in the morning. The blood plasma protein fractions separation was carried out by horizontal electrophoresis in polyacrylamide gel (PAAG. Mathematical treatment of the research results worked statistically using the software package Statistica 6.0 and Microsoft Excel for Windows XP. Probability differences was assessed by Student t-test and results considered likely at P ≤ 0.05. Results and discussion. We measured the ratio of blood plasma protein fractions of rabbits, which in addition to the feed fed of natural origin biologically active substances. As a result of research was found that aerosol introduction of the spleen extract to the rabbits

  15. Coupling detergent lysis/clean-up methodology with intact protein fractionation for enhanced proteome characterization

    Energy Technology Data Exchange (ETDEWEB)

    Sharma, Ritin [ORNL; Dill, Brian [ORNL; Chourey, Karuna [ORNL; Shah, Manesh B [ORNL; Verberkmoes, Nathan C [ORNL; Hettich, Robert {Bob} L [ORNL

    2012-01-01

    The expanding use of surfactants for proteome sample preparations has prompted the need to systematically optimize the application and removal of these MS-deleterious agents prior to proteome measurements. Here we compare four different detergent clean-up methods (Trichloroacetic acid (TCA) precipitation, Chloroform/Methanol/Water (CMW) extraction, commercial detergent removal spin column method (DRS) and filter-aided sample preparation(FASP)) with respect to varying amounts of protein biomass in the samples, and provide efficiency benchmarks with respect to protein, peptide, and spectral identifications for each method. Our results show that for protein limited samples, FASP outperforms the other three clean-up methods, while at high protein amount all the methods are comparable. This information was used in a dual strategy of comparing molecular weight based fractionated and unfractionated lysates from three increasingly complex samples (Escherichia coli, a five microbial isolate mixture, and a natural microbial community groundwater sample), which were all lysed with SDS and cleaned up using FASP. The two approaches complemented each other by enhancing the number of protein identifications by 8%-25% across the three samples and provided broad pathway coverage.

  16. Ubiquitination is absolutely required for the degradation of hypoxia-inducible factor - 1 alpha protein in hypoxic conditions

    International Nuclear Information System (INIS)

    Wang, Ronghai; Zhang, Ping; Li, Jinhang; Guan, Hongzai; Shi, Guangjun

    2016-01-01

    The hypoxia-inducible factor (HIF) is recognized as the master regulator of hypoxia response. HIF-α subunits expression are tightly regulated. In this study, our data show that ts20 cells still expressed detectable E1 protein even at 39.5° C for 12 h, and complete depletion of E1 protein expression at 39.5° C by siRNA enhanced HIF-1α and P53 protein expression. Further inhibition of E1 at 39.5 °C by siRNA, or E1 inhibitor Ube1-41 completely blocked HIF-1α degradation. Moreover, immunoprecipitations of co-transfection of HA-ubiquitin and FLAG–HIF–1α plasmids directly confirmed the involvement of ubiquitin in the hypoxic degradation of HIF-1α. Additionally, hypoxic HIF-1 α degradation is independent of HAF, RACK1, sumoylation or nuclear/cytoplasmic localization. Taken together, our data suggest that constitutive HIF-1α protein degradation in hypoxia is absolutely ubiquitination-dependent, and unidentified E3 ligase may exist for this degradation pathway. - Highlights: • HIF-1α protein is constitutively degraded in hypoxic conditions. • Requirement of ubiquitination for HIF-1α degradation in hypoxia. • Hypoxic HIF-1α degradation is independent of HAF, RACK1, sumoylation or nuclear/cytoplasmic localization.

  17. Ubiquitination is absolutely required for the degradation of hypoxia-inducible factor - 1 alpha protein in hypoxic conditions

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Ronghai [Department of Urology, Linzi District People' s Hospital, Zibo, 255400 (China); Zhang, Ping, E-mail: zpskx001@163.com [Department of Gynecology, Qingdao Municipal Hospital, Qingdao, 266011 (China); Li, Jinhang [Department of Gynecology, Qingdao Municipal Hospital, Qingdao, 266011 (China); Guan, Hongzai [Laboratory Department, School of Medicine, Qingdao University, Qingdao, 266071 (China); Shi, Guangjun, E-mail: qdmhshigj@yahoo.com [Department of Hepatobiliary Surgery, Qingdao Municipal Hospital, Qingdao, 266071 (China)

    2016-01-29

    The hypoxia-inducible factor (HIF) is recognized as the master regulator of hypoxia response. HIF-α subunits expression are tightly regulated. In this study, our data show that ts20 cells still expressed detectable E1 protein even at 39.5° C for 12 h, and complete depletion of E1 protein expression at 39.5° C by siRNA enhanced HIF-1α and P53 protein expression. Further inhibition of E1 at 39.5 °C by siRNA, or E1 inhibitor Ube1-41 completely blocked HIF-1α degradation. Moreover, immunoprecipitations of co-transfection of HA-ubiquitin and FLAG–HIF–1α plasmids directly confirmed the involvement of ubiquitin in the hypoxic degradation of HIF-1α. Additionally, hypoxic HIF-1 α degradation is independent of HAF, RACK1, sumoylation or nuclear/cytoplasmic localization. Taken together, our data suggest that constitutive HIF-1α protein degradation in hypoxia is absolutely ubiquitination-dependent, and unidentified E3 ligase may exist for this degradation pathway. - Highlights: • HIF-1α protein is constitutively degraded in hypoxic conditions. • Requirement of ubiquitination for HIF-1α degradation in hypoxia. • Hypoxic HIF-1α degradation is independent of HAF, RACK1, sumoylation or nuclear/cytoplasmic localization.

  18. Epinephrine ameliorating response of serum proteins and protein fractions to whole body gamma irradiation in albino rats

    International Nuclear Information System (INIS)

    Mohamed, M.A.; Saada, H.N.; Roushdy, H.M.; Awad, O.M.; El-Sayed, M.M.; Azab, Kh.Sh.

    1997-01-01

    The present study was carried out to investigate the role of epinephrine in modifying the radiation induced effects on serum protein as presented by total protein, protein fractions and albumin/globulin (A/G) ratio in adult albino rats. Epinephrine was intraperitoneally injected at a concentration of 200 M/g body weight, 15 min, pre-9 or just after 0 whole body gamma-irradiation of rats at a dose of 6 Gy (single dose). Studies have been undertaken at periods of 1 hr, 4 hrs, 1,3 and 7 days after irradiation. Data of the present study revealed that whole body gamma-irradiation induced significant decreased in the total content of serum protein and albumin at 1,3 and 7 days post radiation exposure alpha 1-globulin significantly increased only on the 1 st hr post-irradiation, however alpha 1-globulin significantly increased along all the experimental periods. B-globulin insignificantly changed after irradiation but gamma-globulin significantly decreased during the experimental periods. These changes were associated with significant decreases in A/G ratio at 3 and 7 days post-irradiation. Administration of epinephrine pre-or after radiation exposure produced some amelioration in the radiation induced changes in the studied parameters. So, it could be concluded that epinephrine plays a beneficial radioprotective role through its pharmacologic properties

  19. Effect of pre-treatment on in vitro gastric digestion of quinoa protein (Chenopodium quinoa Willd.) obtained by wet and dry fractionation

    NARCIS (Netherlands)

    Opazo-Navarrete, M.; Schutyser, M.A.I.; Boom, R.M.; Janssen, A.E.M.

    2018-01-01

    Quinoa protein was isolated from quinoa seeds using wet fractionation that resulted in a protein isolate (QPI) with a high protein purity of 87.1% (w/dw) and a protein yield of around 54%, and a dry fractionation method delivered a quinoa protein concentrate (QPC) with a purity of 27.8% (w/dw)

  20. The effect of starch, inulin, and degradable protein on ruminal fermentation and microbial growth in rumen simulation technique

    Directory of Open Access Journals (Sweden)

    Xiang H. Zhao

    2014-03-01

    Full Text Available A rumen simulation technique apparatus with eight 800 mL fermentation vessels was used to investigate the effects of rumen degradable protein (RDP level and non-fibre carbohydrate (NFC type on ruminal fermentation, microbial growth, and populations of ruminal cellulolytic bacteria. Treatments consisted of two NFC types (starch and inulin supplemented with 0 g/d (low RDP or 1.56 g/d (high RDP sodium caseinate. No significant differences existed among dietary treatments in the apparent disappearance of dietary nutrients except for dietary N, which increased with increased dietary RDP (P<0.001. Compared with starch, inulin treatments reduced the molar proportion of acetate (P<0.001, the acetate:propionate ratio (P<0.001, and methane production (P=0.006, but increased the butyrate proportion (P<0.001. Increased dietary RDP led to increases in production of total volatile fatty acid (P=0.014 and methane (P=0.050, various measures of N (P≤0.046, and 16s rDNA copy numbers of Ruminococcus flavefaciens (P≤0.010. Non-fibre carbohydrate source did not affect daily microbial N flow regardless of dietary RDP, but ammonia N production was lower for inulin than for starch treatments under high RDP conditions (P<0.001. Compared with starch treatments, inulin depressed the copy numbers of Fibrobacter succinogenes in solid fraction (P=0.023 and R. flavefaciens in liquid (P=0.017 and solid fractions (P=0.007, but it increased the carboxymethylcellulase activity in solid fraction (P=0.045. Current results suggest that starch and inulin differ in ruminal volatile fatty acid fermentation but have similar effects on ruminal digestion and microbial synthesis in vitro, although inulin suppressed the growth of partial ruminal cellulolytic bacteria.

  1. Impact of repeated two-phase olive mill waste application on phosphorus fractionation in a degraded olive grove soil

    International Nuclear Information System (INIS)

    Lopez-Pineiro, A.; Albarran, A.; Flores, S.; Rato, J. M.; Munoz, A.; Cabrera, D.; Pena, D.; Fernandez, S.

    2009-01-01

    Loss of organic matter is one of the main forms of soil degradation in Mediterranean agricultural soils, and external sources of organic matter are required to improve soil properties. the two-phase centrifugation system in the olive-oil extraction industry produces a large amount of olive mill waste sludge (TPOMW) which can be used to add organic C to degraded soils. (Author)

  2. The treatment of fish meal with gamma radiation - effect on protein fraction

    International Nuclear Information System (INIS)

    Uchman, W.; Konieczny, P.; Zabielski, J.

    1996-01-01

    The aim of this study was determination of the gamma radiation effect on changes of protein fraction of fish meal. For determination of the changes seven dose levels (0-20 kGy) were applied. The doses up to 5 kGy did not influence significantly on solubility, aminoacids composition, content of amino-nitrogen and enzymatic digestibility of proteins in vitro. For 5-10 kGy doses an insignificant decrease of the content of tryptophan was observed. The dose of 5 kGy is quite satisfactory in elimination of vegetative forms of most pathogens. For elimination of yeasts and moulds, the doses up to 10 kGy have to be applied. In this situation supplementation the fish meal with tryptophan is recommended

  3. Fractionation separation of human plasma proteins using HPLC with a homemade iron porphyrin based monolithic column.

    Science.gov (United States)

    Zhang, Doudou; Zhao, Yu; Lan, Dandan; Pang, Xiaomin; Bai, Ligai; Liu, Haiyan; Yan, Hongyuan

    2017-11-15

    In this work a polymer monolithic column was fabricated within the confines of a stainless steel column (50×4.6mm i.d.) via radical polymerization by using iron porphyrin and butyl methacrylate as co-monomers, ethylene glycol dimethacrylate as crosslinking agent, ethylene glycol, isopropyl alcohol and N, N-dimethylformamide as tri-porogens, benzoyl peroxide and N,N-dimethylaniline as initiators. The resulting monolithic column was characterized by elemental analysis, scanning electron microscopy, nitrogen adsorption BET surface area, and mercury intrusion porosimetry, respectively. Results showed that the homemade monolith occupied relatively uniform pore structure, low back pressure, and enhanced selectivity for proteins in complex bio-samples. The present work described a simple and efficient method for "fractionation separation" of human plasma proteins, and it is a promising separation method for complex bio-samples in proteomic research. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Studies to Prevent Degradation of Recombinant Fc-Fusion Protein Expressed in Mammalian Cell Line and Protein Characterization

    Directory of Open Access Journals (Sweden)

    Sanjukta Chakrabarti

    2016-06-01

    Full Text Available Clipping of recombinant proteins is a major issue in animal cell cultures. A recombinant Fc-fusion protein, VEGFR1(D1–D3-Fc expressed in CHOK1SV GS-KO cells was observed to be undergoing clippings in lab scale cultures. Partial cleaving of expressed protein initiated early on in cell culture and was observed to increase over time in culture and also on storage. In this study, a few parameters were explored in a bid to inhibit clipping in the fusion protein The effects of culture temperature, duration of culture, the addition of an anti-clumping agent, ferric citrate and use of protease inhibitor cocktail on inhibition of proteolysis of the Fc fusion were studied. Lowering of culture temperature from 37 to 30 °C alone appears to be the best solution for reducing protein degradation from the quality, cost and regulatory points of view. The obtained Fc protein was characterized and found to be in its stable folded state, exhibiting a high affinity for its ligand and also biological and functional activities.

  5. Copper-mediated oxidative degradation of catecholamines and oxidative damage of protein

    Energy Technology Data Exchange (ETDEWEB)

    Goncalves, P.R.; Harria, M.I.N.; Felix, J.M.; Hoffmann, M.E. [Universidade Estadual de Campinas, SP (Brazil). Inst. de Biologia

    1997-12-31

    Full text. Degradative oxidation of catecholamines has been a matter of large interest in recent years due to the evidences associating their autoxidation with the etiology of neurotoxic and cardiotoxic processes. In this work we present data on the degradative oxidation of catecholamines of physiological importance: isoproterenol (IP), epinephrine (EP), norepinephrine (NEP), deoxyepinephrine (DEP) and dopamine (DA). The degradative oxidation of the catecholamines was followed by measurement of spectral changes and oxygen consumption by neutral aqueous solutions. The data show that Cu{sup 2+} strongly accelerated the rate of catecholamine oxidation, following the decreasing order; EP>DEP>IP>NEP>DA. The production of superoxide anion radical during catecholamine oxidation was very slow, even in the presence of Cu{sup 2+}. The ability of IP to induce damages on bovine serum albumin (BSA) was determined by measuring the formation of carbonyl-groups in the protein, detected by reduction with tritiated Na BH{sub 4}. The incubation of BSA with IP (50-500{mu}M), in the presence of 100{mu}M Cu{sup 2+} leaded to an increased and dose dependent {sup 3} H-incorporation by the oxidized protein. The production of oxidative damage by IP/Cu{sup 2+} was accompanied by marked BSA fragmentation, detected by SDS-polyacrylamide gel dependent (25-400{mu}M IP) des appearance of the original BSA band and appearance of smaller fragments spread in the gel, when incubation has been done in the presence of 100{mu}M Cu{sup 2+}. These results suggest that copper-catalysed oxidative degradation of proteins induced by catecholamines might be critically involved in the toxic action of these molecules

  6. Quantifying protein synthesis and degradation in Arabidopsis by dynamic 13CO2 labeling and analysis of enrichment in individual amino acids in their free pools and in protein.

    Science.gov (United States)

    Ishihara, Hirofumi; Obata, Toshihiro; Sulpice, Ronan; Fernie, Alisdair R; Stitt, Mark

    2015-05-01

    Protein synthesis and degradation represent substantial costs during plant growth. To obtain a quantitative measure of the rate of protein synthesis and degradation, we supplied (13)CO2 to intact Arabidopsis (Arabidopsis thaliana) Columbia-0 plants and analyzed enrichment in free amino acids and in amino acid residues in protein during a 24-h pulse and 4-d chase. While many free amino acids labeled slowly and incompletely, alanine showed a rapid rise in enrichment in the pulse and a decrease in the chase. Enrichment in free alanine was used to correct enrichment in alanine residues in protein and calculate the rate of protein synthesis. The latter was compared with the relative growth rate to estimate the rate of protein degradation. The relative growth rate was estimated from sequential determination of fresh weight, sequential images of rosette area, and labeling of glucose in the cell wall. In an 8-h photoperiod, protein synthesis and cell wall synthesis were 3-fold faster in the day than at night, protein degradation was slow (3%-4% d(-1)), and flux to growth and degradation resulted in a protein half-life of 3.5 d. In the starchless phosphoglucomutase mutant at night, protein synthesis was further decreased and protein degradation increased, while cell wall synthesis was totally inhibited, quantitatively accounting for the inhibition of growth in this mutant. We also investigated the rates of protein synthesis and degradation during leaf development, during growth at high temperature, and compared synthesis rates of Rubisco large and small subunits of in the light and dark. © 2015 American Society of Plant Biologists. All Rights Reserved.

  7. Delaying aging and the aging-associated decline in protein homeostasis by inhibition of tryptophan degradation

    Science.gov (United States)

    van der Goot, Annemieke T.; Zhu, Wentao; Vázquez-Manrique, Rafael P.; Seinstra, Renée I.; Dettmer, Katja; Michels, Helen; Farina, Francesca; Krijnen, Jasper; Melki, Ronald; Buijsman, Rogier C.; Ruiz Silva, Mariana; Thijssen, Karen L.; Kema, Ido P.; Neri, Christian; Oefner, Peter J.; Nollen, Ellen A. A.

    2012-01-01

    Toxicity of aggregation-prone proteins is thought to play an important role in aging and age-related neurological diseases like Parkinson and Alzheimer’s diseases. Here, we identify tryptophan 2,3-dioxygenase (tdo-2), the first enzyme in the kynurenine pathway of tryptophan degradation, as a metabolic regulator of age-related α-synuclein toxicity in a Caenorhabditis elegans model. Depletion of tdo-2 also suppresses toxicity of other heterologous aggregation-prone proteins, including amyloid-β and polyglutamine proteins, and endogenous metastable proteins that are sensors of normal protein homeostasis. This finding suggests that tdo-2 functions as a general regulator of protein homeostasis. Analysis of metabolite levels in C. elegans strains with mutations in enzymes that act downstream of tdo-2 indicates that this suppression of toxicity is independent of downstream metabolites in the kynurenine pathway. Depletion of tdo-2 increases tryptophan levels, and feeding worms with extra l-tryptophan also suppresses toxicity, suggesting that tdo-2 regulates proteotoxicity through tryptophan. Depletion of tdo-2 extends lifespan in these worms. Together, these results implicate tdo-2 as a metabolic switch of age-related protein homeostasis and lifespan. With TDO and Indoleamine 2,3-dioxygenase as evolutionarily conserved human orthologs of TDO-2, intervening with tryptophan metabolism may offer avenues to reducing proteotoxicity in aging and age-related diseases. PMID:22927396

  8. Comparison of composition and whey protein fractions of human, camel, donkey, goat and cow milk

    Directory of Open Access Journals (Sweden)

    Halima El-Hatmi

    2015-07-01

    Full Text Available The aim of this study was to compare the physicochemical parameters of milk samples of five different species: cow, goat, donkey, camel and human. Also the analysis of whey protein profile in different milk samples was performed by anion-exchange fast protein liquid chromatography (FPLC while polyacrylamide gel electrophoresis was used to identify a single fraction. Camel milk was the most acid (pH 6.460±0.005 and the richest in total proteins (3.41±0.31 % and ash (0.750±0.102 %, whereas donkey milk had a neutral pH (7.03±0.02 and characterised by low proteins (1.12±0.40 % and fat (0.97±0.03 % content, being very close to human milk. Proteomic analysis of cow, goat, donkey, camel and human milk highlighted significant interspecies differences. Camel milk was similar to human milk in lacking of β-lactoglobulin and richness of α-lactalbumin. The knowledge gained from the proteomic comparison of the milk samples analysed within this study might be of relevance, both, in terms of identifying sources of hypoallergenic alternatives to bovine milk and detection of adulteration of milk samples and products.

  9. HECTD3 Mediates an HSP90-Dependent Degradation Pathway for Protein Kinase Clients

    Directory of Open Access Journals (Sweden)

    Zhaobo Li

    2017-06-01

    Full Text Available Inhibition of the ATPase cycle of the HSP90 chaperone promotes ubiquitylation and proteasomal degradation of its client proteins, which include many oncogenic protein kinases. This provides the rationale for HSP90 inhibitors as cancer therapeutics. However, the mechanism by which HSP90 ATPase inhibition triggers ubiquitylation is not understood, and the E3 ubiquitin ligases involved are largely unknown. Using a siRNA screen, we have identified components of two independent degradation pathways for the HSP90 client kinase CRAF. The first requires CUL5, Elongin B, and Elongin C, while the second requires the E3 ligase HECTD3, which is also involved in the degradation of MASTL and LKB1. HECTD3 associates with HSP90 and CRAF in cells via its N-terminal DOC domain, which is mutationally disrupted in tumor cells with activated MAP kinase signaling. Our data implicate HECTD3 as a tumor suppressor modulating the activity of this important oncogenic signaling pathway.

  10. Particulate matter air pollution disrupts endothelial cell barrier via calpain-mediated tight junction protein degradation

    Directory of Open Access Journals (Sweden)

    Wang Ting

    2012-08-01

    Full Text Available Abstract Background Exposure to particulate matter (PM is a significant risk factor for increased cardiopulmonary morbidity and mortality. The mechanism of PM-mediated pathophysiology remains unknown. However, PM is proinflammatory to the endothelium and increases vascular permeability in vitro and in vivo via ROS generation. Objectives We explored the role of tight junction proteins as targets for PM-induced loss of lung endothelial cell (EC barrier integrity and enhanced cardiopulmonary dysfunction. Methods Changes in human lung EC monolayer permeability were assessed by Transendothelial Electrical Resistance (TER in response to PM challenge (collected from Ft. McHenry Tunnel, Baltimore, MD, particle size >0.1 μm. Biochemical assessment of ROS generation and Ca2+ mobilization were also measured. Results PM exposure induced tight junction protein Zona occludens-1 (ZO-1 relocation from the cell periphery, which was accompanied by significant reductions in ZO-1 protein levels but not in adherens junction proteins (VE-cadherin and β-catenin. N-acetyl-cysteine (NAC, 5 mM reduced PM-induced ROS generation in ECs, which further prevented TER decreases and atteneuated ZO-1 degradation. PM also mediated intracellular calcium mobilization via the transient receptor potential cation channel M2 (TRPM2, in a ROS-dependent manner with subsequent activation of the Ca2+-dependent protease calpain. PM-activated calpain is responsible for ZO-1 degradation and EC barrier disruption. Overexpression of ZO-1 attenuated PM-induced endothelial barrier disruption and vascular hyperpermeability in vivo and in vitro. Conclusions These results demonstrate that PM induces marked increases in vascular permeability via ROS-mediated calcium leakage via activated TRPM2, and via ZO-1 degradation by activated calpain. These findings support a novel mechanism for PM-induced lung damage and adverse cardiovascular outcomes.

  11. In Vitro Ruminal Degradability of Soybean Meal Protein Protected with Natural Tannin

    Science.gov (United States)

    Prasetiyono, B. W. H. E.; Subrata, A.; Tampoebolon, B. I. M.; Surono; Widiyanto

    2018-02-01

    The influence of tannin from tea waste and gambier as natural tannin sources on ruminal protein degradability was studied in this investigation. The soybean meal was used as protein source in this investigation. There were three treatments in this investigation mainly without protection (NT); protection with tea waste (Tt); and protection with gambier (Tg). The measured parameters consisted of in vitro dry matter digestibility (IVDMD), in vitro organic matter digestibility (IVOMD), and the ruminal fermentation characteristics. Results of this experiment showed that protection with tannin from tea waste as well as gambier increased (pRUP) in Tt and Tg group was higher than that in NT group (66.29 and 69.20 vs 51.10%). The ruminal protozoa population decreased (pRUP. The ruminal protozoa population and ammonia concentration, on the other hand, were decreased by tannin protection from those tannin natural sources. The natural tannin from gambier was the most effective protection agent for soybean meal protein.

  12. Ruminal and intestinal protein degradability of various seaweed species measured in situ in dairy cows

    DEFF Research Database (Denmark)

    Tayyab, Usama; Novoa-Garrido, Margarita; Roleda, Michael Y.

    2016-01-01

    , Laminaria, Mastocarpus, Palmaria, Pelvetia, Porphyra, and Ulva were sampled in spring (March) and autumn (October and November) 2014 at the coast of Bodø in Northern Norway, and were analysed for chemical composition, in situ rumen degradability and total tract crude protein (CP) digestibility. Ash content......The use of seaweeds in animal diets is not new. However, little is known about the feed value of seaweed, both in terms of chemical composition and protein digestibility, and regarding variation between species and season. In this study, eight seaweed species of the genus Acrosiphonia, Alaria....../kg CP). Digestible rumen escape protein (DEP) varied significantly between species (P Laminaria, Mastocarpus and Palmaria can supply...

  13. Differential Regulation of Telomerase Reverse Transcriptase Promoter Activation and Protein Degradation by Histone Deacetylase Inhibition.

    Science.gov (United States)

    Qing, Hua; Aono, Jun; Findeisen, Hannes M; Jones, Karrie L; Heywood, Elizabeth B; Bruemmer, Dennis

    2016-06-01

    Telomerase reverse transcriptase (TERT) maintains telomeres and is rate limiting for replicative life span. While most somatic tissues silence TERT transcription resulting in telomere shortening, cells derived from cancer or cardiovascular diseases express TERT and activate telomerase. In the present study, we demonstrate that histone deacetylase (HDAC) inhibition induces TERT transcription and promoter activation. At the protein level in contrast, HDAC inhibition decreases TERT protein abundance through enhanced degradation, which decreases telomerase activity and induces senescence. Finally, we demonstrate that HDAC inhibition decreases TERT expression during vascular remodeling in vivo. These data illustrate a differential regulation of TERT transcription and protein stability by HDAC inhibition and suggest that TERT may constitute an important target for the anti-proliferative efficacy of HDAC inhibitors. © 2015 Wiley Periodicals, Inc.

  14. L-Alanylglutamine inhibits signaling proteins that activate protein degradation, but does not affect proteins that activate protein synthesis after an acute resistance exercise.

    Science.gov (United States)

    Wang, Wanyi; Choi, Ran Hee; Solares, Geoffrey J; Tseng, Hung-Min; Ding, Zhenping; Kim, Kyoungrae; Ivy, John L

    2015-07-01

    Sustamine™ (SUS) is a dipeptide composed of alanine and glutamine (AlaGln). Glutamine has been suggested to increase muscle protein accretion; however, the underlying molecular mechanisms of glutamine on muscle protein metabolism following resistance exercise have not been fully addressed. In the present study, 2-month-old rats climbed a ladder 10 times with a weight equal to 75 % of their body mass attached at the tail. Rats were then orally administered one of four solutions: placebo (PLA-glycine = 0.52 g/kg), whey protein (WP = 0.4 g/kg), low dose of SUS (LSUS = 0.1 g/kg), or high dose of SUS (HSUS = 0.5 g/kg). An additional group of sedentary (SED) rats was intubated with glycine (0.52 g/kg) at the same time as the ladder-climbing rats. Blood samples were collected immediately after exercise and at either 20 or 40 min after recovery. The flexor hallucis longus (FHL), a muscle used for climbing, was excised at 20 or 40 min post exercise and analyzed for proteins regulating protein synthesis and degradation. All supplements elevated the phosphorylation of FOXO3A above SED at 20 min post exercise, but only the SUS supplements significantly reduced the phosphorylation of AMPK and NF-kB p65. SUS supplements had no effect on mTOR signaling, but WP supplementation yielded a greater phosphorylation of mTOR, p70S6k, and rpS6 compared with PLA at 20 min post exercise. However, by 40 min post exercise, phosphorylation of mTOR and rpS6 in PLA had risen to levels not different than WP. These results suggest that SUS blocks the activation of intracellular signals for MPB, whereas WP accelerates mRNA translation.

  15. Ruminal degradation and intestinal digestibility of protein and amino acids in high-protein feedstuffs commonly used in dairy diets.

    Science.gov (United States)

    Paz, H A; Klopfenstein, T J; Hostetler, D; Fernando, S C; Castillo-Lopez, E; Kononoff, P J

    2014-10-01

    A study was conducted to determine the rumen degradation and intestinal digestibility of crude protein (CP) and AA, and AA composition of the rumen-undegradable protein (RUP) from 3 sources of blood meal (BM1, BM2, and BM3), canola meal (CM), low-fat distillers dried grains with solubles (LFDG), soybean meal (SBM), and expeller soybean meal (ESBM). Two Holstein cows fitted with ruminal and proximal duodenal cannulas were used for in situ incubation of 16h and for the mobile bag technique. To correct for bacterial contamination of the RUP, 2 methods were used: purines and DNA as bacterial markers. Ruminal degradations of CP were 85.3, 29.8, 40.7, 75.7, 76.9, 68.8, and 37.0 ± 3.93% for BM1, BM2, BM3, CM, LFDG, SBM, and ESBM, respectively. Ruminal degradation of both total essential AA and nonessential AA followed a similar pattern to that of CP across feedstuffs. Based on the ratio of AA concentration in the RUP to AA concentration in the original feedstuff, ruminal incubation decreased (ratio 1) the concentrations of Ile and Met across feedstuffs. Compared with purines, the use of DNA as bacterial marker resulted in a higher estimate of bacterial CP contamination for CM and lower estimates for LFDG and ESBM. Intestinal digestibility of RUP could not be estimated for BM1, BM3, and SBM due to insufficient recovery of residue. For the remaining feedstuffs, intestinal digestibility of RUP was highest for ESBM, followed by BM2 and LFDG, and lowest for CM: 98.8, 87.9, 89.7, and 72.4 ± 1.40%, respectively. Intestinal absorbable dietary protein was higher for BM2 compared with CM and LFDG, at 61.7, 17.9, and 20.7 ± 2.73% CP, respectively. As prices fluctuate, intestinal absorbable protein or AA may be used as a tool to aid in the selection among feedstuffs with different protein quality. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  16. Enzymic and structural studies on processed proteins from the vacuolar (lutoid-body) fraction of latex of Hevea brasiliensis

    NARCIS (Netherlands)

    Subroto, T; de Vries, H; Schuringa, JJ; Soedjanaatmadja, UMS; Hofsteenge, J; Jekel, PA; Beintema, JJ

    2001-01-01

    The lutoid-body (bottom) fraction of latex from the rubber tree (Hevea brasiliensis) contains a limited number of major proteins. These are the chitin-binding protein hevein, its precursor and C-terminal fragment of the precursor, a basic chitinase/lysozyme, and a beta-1,3-glucanase. The content and

  17. CNPY2 inhibits MYLIP-mediated AR protein degradation in prostate cancer cells.

    Science.gov (United States)

    Ito, Saya; Ueno, Akihisa; Ueda, Takashi; Nakagawa, Hideo; Taniguchi, Hidefumi; Kayukawa, Naruhiro; Fujihara-Iwata, Atsuko; Hongo, Fumiya; Okihara, Koji; Ukimura, Osamu

    2018-04-03

    The androgen receptor (AR) is a ligand-dependent transcription factor that promotes prostate cancer (PC) cell growth through control of target gene expression. This report suggests that Canopy FGF signaling regulator 2 (CNPY2) controls AR protein levels in PC cells. We found that AR was ubiquitinated by an E3 ubiquitin ligase, myosin regulatory light chain interacting protein (MYLIP) and then degraded through the ubiquitin-proteasome pathway. CNPY2 decreased the ubiquitination activity of MYLIP by inhibition of interaction between MYLIP and UBE2D1, an E2 ubiquitin ligase. CNPY2 up-regulated gene expression of AR target genes such as KLK3 gene which encodes the prostate specific antigen (PSA) and promoted cell growth of PC cells. The cell growth inhibition by CNPY2 knockdown was rescued by AR overexpression. Furthermore, positive correlation of expression levels between CNPY2 and AR/AR target genes was observed in tissue samples from human prostate cancer patients. Together, these results suggested that CNPY2 promoted cell growth of PC cells by inhibition of AR protein degradation through MYLIP-mediated AR ubiquitination.

  18. Saturated fatty acid palmitate negatively regulates autophagy by promoting ATG5 protein degradation in meniscus cells.

    Science.gov (United States)

    Mallik, Aritra; Yammani, Raghunatha R

    2018-07-20

    Obesity and associated metabolic factors are major risk factors for the development of osteoarthritis. Previously, we have shown that the free fatty acid palmitate induces endoplasmic reticulum (ER) stress and induces apoptosis in meniscus cells. However, the molecular mechanisms involved in these effects are not clearly understood. In our current study, we found that palmitate inhibits autophagy by modulating the protein levels of autophagy-related genes-5 (ATG5) that is associated with decreased lipidation of LC3 and increased activation of cleaved caspase 3. Pretreatment of meniscus cells with 4-phenyl butyric acid, a small molecule chemical chaperone that alleviates ER stress, or with MG-132, a proteasome inhibitor, restored normal levels of ATG5 and autophagosome formation, and decreased expression of cleaved caspase 3. Thus, our data suggest that palmitate downregulates autophagy in meniscus cells by degrading ATG5 protein via ER-associated protein degradation, and thus promotes apoptosis. This is the first study to demonstrate that palmitate-induced endoplasmic reticulum stress negatively regulates autophagy. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  19. NAD(P)H quinone oxidoreductase 1 inhibits the proteasomal degradation of homocysteine-induced endoplasmic reticulum protein

    Energy Technology Data Exchange (ETDEWEB)

    Maeda, Tomoji, E-mail: t-maeda@nichiyaku.ac.jp [Department of Neuroscience, School of Pharmacy, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba-Cho, Shiwagun, Iwate, 028-3603 (Japan); Tanabe-Fujimura, Chiaki; Fujita, Yu; Abe, Chihiro; Nanakida, Yoshino; Zou, Kun; Liu, Junjun; Liu, Shuyu [Department of Neuroscience, School of Pharmacy, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba-Cho, Shiwagun, Iwate, 028-3603 (Japan); Nakajima, Toshihiro [Institute of Medical Science, Tokyo Medical University, 6-1-1 Shinjyuku, Shinjyuku, Tokyo, Tokyo, 160-8402 (Japan); Komano, Hiroto, E-mail: hkomano@iwate-med.ac.jp [Department of Neuroscience, School of Pharmacy, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba-Cho, Shiwagun, Iwate, 028-3603 (Japan)

    2016-05-13

    Homocysteine-induced endoplasmic reticulum (ER) protein (Herp) is an ER stress-inducible key regulatory component of ER-associated degradation (ERAD) that has been implicated in insulin hypersecretion in diabetic mouse models. Herp expression is tightly regulated. Additionally, Herp is a highly labile protein and interacts with various proteins, which are characteristic features of ubiquitinated protein. Previously, we reported that ubiquitination is not required for Herp degradation. In addition, we found that the lysine residues of Herp (which are ubiquitinated by E3 ubiquitin ligase) are not sufficient for regulation of Herp degradation. In this study, we found that NAD(P)H quinone oxidoreductase 1 (NQO1)-mediated targeting of Herp to the proteasome was involved in Herp degradation. In addition, we found that Herp protein levels were markedly elevated in synoviolin-null cells. The E3 ubiquitin ligase synoviolin is a central component of ERAD and is involved in the degradation of nuclear factor E2-related factor-2 (Nrf2), which regulates cellular reactive oxygen species. Additionally, NQO1 is a target of Nrf2. Thus, our findings indicated that NQO1 could stabilize Herp protein expression via indirect regulation of synoviolin. -- Highlights: •Herp interacts with NQO1. •NQO1 regulates Herp degradation.

  20. NAD(P)H quinone oxidoreductase 1 inhibits the proteasomal degradation of homocysteine-induced endoplasmic reticulum protein

    International Nuclear Information System (INIS)

    Maeda, Tomoji; Tanabe-Fujimura, Chiaki; Fujita, Yu; Abe, Chihiro; Nanakida, Yoshino; Zou, Kun; Liu, Junjun; Liu, Shuyu; Nakajima, Toshihiro; Komano, Hiroto

    2016-01-01

    Homocysteine-induced endoplasmic reticulum (ER) protein (Herp) is an ER stress-inducible key regulatory component of ER-associated degradation (ERAD) that has been implicated in insulin hypersecretion in diabetic mouse models. Herp expression is tightly regulated. Additionally, Herp is a highly labile protein and interacts with various proteins, which are characteristic features of ubiquitinated protein. Previously, we reported that ubiquitination is not required for Herp degradation. In addition, we found that the lysine residues of Herp (which are ubiquitinated by E3 ubiquitin ligase) are not sufficient for regulation of Herp degradation. In this study, we found that NAD(P)H quinone oxidoreductase 1 (NQO1)-mediated targeting of Herp to the proteasome was involved in Herp degradation. In addition, we found that Herp protein levels were markedly elevated in synoviolin-null cells. The E3 ubiquitin ligase synoviolin is a central component of ERAD and is involved in the degradation of nuclear factor E2-related factor-2 (Nrf2), which regulates cellular reactive oxygen species. Additionally, NQO1 is a target of Nrf2. Thus, our findings indicated that NQO1 could stabilize Herp protein expression via indirect regulation of synoviolin. -- Highlights: •Herp interacts with NQO1. •NQO1 regulates Herp degradation.

  1. Multidimensional protein fractionation using ProteomeLab PF 2D™ for profiling amyotrophic lateral sclerosis immunity: A preliminary report

    Directory of Open Access Journals (Sweden)

    Mosley R Lee

    2008-09-01

    Full Text Available Abstract Background The ProteomeLab™ PF 2D platform is a relatively new approach to global protein profiling. Herein, it was used for investigation of plasma proteome changes in amyotrophic lateral sclerosis (ALS patients before and during immunization with glatiramer acetate (GA in a clinical trial. Results The experimental design included immunoaffinity depletion of 12 most abundant proteins from plasma samples with the ProteomeLab™ IgY-12 LC10 column kit as first dimension separation, also referred to as immuno-partitioning. Second and third dimension separations of the enriched proteome were performed on the PF 2D platform utilizing 2D isoelectric focusing and RP-HPLC with the resulting fractions collected for analysis. 1D gel electrophoresis was added as a fourth dimension when sufficient protein was available. Protein identification from collected fractions was performed using nano-LC-MS/MS approach. Analysis of differences in the resulting two-dimensional maps of fractions obtained from the PF 2D and the ability to identify proteins from these fractions allowed sensitivity threshold measurements. Masked proteins in the PF 2D fractions are discussed. Conclusion We offer some insight into the strengths and limitations of this emerging proteomic platform.

  2. Novel function of the endoplasmic reticulum degradation-enhancing α-mannosidase-like proteins in the human hepatitis B virus life cycle, mediated by the middle envelope protein.

    Science.gov (United States)

    Lazar, Catalin; Uta, Mihaela; Petrescu, Stefana Maria; Branza-Nichita, Norica

    2017-02-01

    Cells replicating the human hepatitis B virus (HBV) express high levels of degradation-enhancing α-mannosidase-like proteins (EDEMs), a family of proteins involved in the endoplasmic reticulum associated degradation, one of the pathways activated during the unfolded protein response. Owing to their α-1,2 mannosidase activity, the EDEM1-3 proteins are able to process the N-linked glycans of misfolded or incompletely folded proteins, providing the recognition signal for their subsequent degradation. The HBV small (S), medium (M), and large (L) surface proteins bear an N-linked glycosylation site in the common S domain that is partially occupied in all proteins. The M protein contains an additional site in its preS2 domain, which is always functional. Here, we report that these oligosaccharides are processed by EDEMs, more efficiently by EDEM3, which induces degradation of L and S proteins, accompanied by a reduction of subviral particles production. In striking contrast, M not only is spared from degradation but its trafficking is also accelerated leading to an improved secretion. This unusual behavior of the M protein requires strictly the mannose trimming of the preS2 N-linked glycan. Furthermore, we show that HBV secretion is significantly inhibited under strong endoplasmic reticulum stress conditions when M expression is prevented by mutagenesis of the viral genome. These observations unfold unique properties of the M protein in the HBV life cycle during unfolded protein response and point to alternative mechanisms employed by EDEMs to alleviate this stress in case of necessity by promoting glycoprotein trafficking rather than degradation. © 2016 John Wiley & Sons Ltd.

  3. Simvastatin Ameliorates Radiation Enteropathy Development After Localized, Fractionated Irradiation by a Protein C-Independent Mechanism

    International Nuclear Information System (INIS)

    Wang Junru; Boerma, Marjan; Fu Qiang; Kulkarni, Ashwini; Fink, Louis M.; Hauer-Jensen, Martin

    2007-01-01

    Purpose: Microvascular injury plays a key role in normal tissue radiation responses. Statins, in addition to their lipid-lowering effects, have vasculoprotective properties that may counteract some effects of radiation on normal tissues. We examined whether administration of simvastatin ameliorates intestinal radiation injury, and whether the effect depends on protein C activation. Methods and Materials: Rats received localized, fractionated small bowel irradiation. The animals were fed either regular chow or chow containing simvastatin from 2 weeks before irradiation until termination of the experiment. Groups of rats were euthanized at 2 weeks and 26 weeks for assessment of early and delayed radiation injury by quantitative histology, morphometry, and quantitative immunohistochemistry. Dependency on protein C activation was examined in thrombomodulin (TM) mutant mice with deficient ability to activate protein C. Results: Simvastatin administration was associated with lower radiation injury scores (p < 0.0001), improved mucosal preservation (p = 0.0009), and reduced thickening of the intestinal wall and subserosa (p = 0.008 and p = 0.004), neutrophil infiltration (p = 0.04), and accumulation of collagen I (p = 0.0003). The effect of simvastatin was consistently more pronounced for delayed than for early injury. Surprisingly, simvastatin reduced intestinal radiation injury in TM mutant mice, indicating that the enteroprotective effect of simvastatin after localized irradiation is unrelated to protein C activation. Conclusions: Simvastatin ameliorates the intestinal radiation response. The radioprotective effect of simvastatin after localized small bowel irradiation does not appear to be related to protein C activation. Statins should undergo clinical testing as a strategy to minimize side effects of radiation on the intestine and other normal tissues

  4. Effective rumen degradation of dry matter, crude protein and neutral detergent fibre in forage determined by near infrared reflectance spectroscopy

    DEFF Research Database (Denmark)

    Ohlsson, C; Houmøller, L P; Weisbjerg, Martin Riis

    2007-01-01

    The objective of the present study was to examine if near infrared reflectance spectroscopy (NIRS) could be used to predict degradation parameters and effective degradation from scans of original forage samples. Degradability of dry matter (DM), crude protein (CP) and neutral detergent fibre (NDF......) of 61 samples of perennial ryegrass (Lolium perenne L.) and orchardgrass (Dactylis glomerata L.) was tested by using the in situ technique. The grass samples were harvested at three different stages, early vegetative growth, early reproductive growth and late reproductive growth. Degradability...

  5. Predicting beta-turns in proteins using support vector machines with fractional polynomials.

    Science.gov (United States)

    Elbashir, Murtada; Wang, Jianxin; Wu, Fang-Xiang; Wang, Lusheng

    2013-11-07

    β-turns are secondary structure type that have essential role in molecular recognition, protein folding, and stability. They are found to be the most common type of non-repetitive structures since 25% of amino acids in protein structures are situated on them. Their prediction is considered to be one of the crucial problems in bioinformatics and molecular biology, which can provide valuable insights and inputs for the fold recognition and drug design. We propose an approach that combines support vector machines (SVMs) and logistic regression (LR) in a hybrid prediction method, which we call (H-SVM-LR) to predict β-turns in proteins. Fractional polynomials are used for LR modeling. We utilize position specific scoring matrices (PSSMs) and predicted secondary structure (PSS) as features. Our simulation studies show that H-SVM-LR achieves Qtotal of 82.87%, 82.84%, and 82.32% on the BT426, BT547, and BT823 datasets respectively. These values are the highest among other β-turns prediction methods that are based on PSSMs and secondary structure information. H-SVM-LR also achieves favorable performance in predicting β-turns as measured by the Matthew's correlation coefficient (MCC) on these datasets. Furthermore, H-SVM-LR shows good performance when considering shape strings as additional features. In this paper, we present a comprehensive approach for β-turns prediction. Experiments show that our proposed approach achieves better performance compared to other competing prediction methods.

  6. Determination of degradation rates of organic substances in the unsaturated soil zone depending on the grain size fractions of various soil types

    Science.gov (United States)

    Fichtner, Thomas; Stefan, Catalin; Goersmeyer, Nora

    2015-04-01

    Rate and extent of the biological degradation of organic substances during transport through the unsaturated soil zone is decisively influenced by the chemical and physical properties of the pollutants such as water solubility, toxicity and molecular structure. Furthermore microbial degradation processes are also influenced by soil-specific properties. An important parameter is the soil grain size distribution on which the pore volume and the pore size depends. Changes lead to changes in air and water circulation as well as preferred flow paths. Transport capacity of water inclusive nutrients is lower in existing bad-drainable fine pores in soils with small grain size fractions than in well-drainable coarse pores in a soil with bigger grain size fractions. Because fine pores are saturated with water for a longer time than the coarse pores and oxygen diffusion in water is ten thousand times slower than in air, oxygen is replenished much slower in soils with small grain size fractions. As a result life and growth conditions of the microorganisms are negatively affected. This leads to less biological activity, restricted degradation/mineralization of pollutants or altered microbial processes. The aim of conducted laboratory column experiments was to study the correlation between the grain size fractions respectively pore sizes, the oxygen content and the biodegradation rate of infiltrated organic substances. Therefore two columns (active + sterile control) were filled with different grain size fractions (0,063-0,125 mm, 0,2-0,63 mm and 1-2 mm) of soils. The sterile soil was inoculated with a defined amount of a special bacteria culture (sphingobium yanoikuae). A solution with organic substances glucose, oxalic acid, sinaphylic alcohol and nutrients was infiltrated from the top in intervals. The degradation of organic substances was controlled by the measurement of dissolved organic carbon in the in- and outflow of the column. The control of different pore volumes

  7. The Major Chromophore Arising from Glucose Degradation and Oxidative Stress Occurrence during Lens Proteins Glycation Induced by Glucose

    Directory of Open Access Journals (Sweden)

    Felipe Ávila

    2017-12-01

    Full Text Available Glucose autoxidation has been proposed as a key reaction associated with deleterious effects induced by hyperglycemia in the eye lens. Little is known about chromophores generated during glucose autoxidation. In this study, we analyzed the effect of oxidative and dicarbonyl stress in the generation of a major chromophore arising from glucose degradation (GDC and its association with oxidative damage in lens proteins. Glucose (5 mM was incubated with H2O2 (0.5–5 mM, Cu2+ (5–50 μM, glyoxal (0.5–5 mM or methylglyoxal (0.5–5 mM at pH 7.4, 5% O2, 37 °C, from 0 to 30 days. GDC concentration increased with incubation time, as well as when incubated in the presence of H2O2 and/or Cu2+, which were effective even at the lowest concentrations. Dicarbonylic compounds did not increase the levels of GDC during incubations. 1H, 13C and FT-IR spectra from the purified fraction containing the chromophore (detected by UV/vis spectroscopy showed oxidation products of glucose, including gluconic acid. Lens proteins solutions (10 mg/mL incubated with glucose (30 mM presented increased levels of carboxymethyl-lysine and hydrogen peroxide that were associated with GDC increase. Our results suggest a possible use of GDC as a marker of autoxidative reactions occurring during lens proteins glycation induced by glucose.

  8. Ubiquitin Ligase gp78 Targets Unglycosylated Prion Protein PrP for Ubiquitylation and Degradation

    OpenAIRE

    Shao, Jia; Choe, Vitnary; Cheng, Haili; Tsai, Yien Che; Weissman, Allan M.; Luo, Shiwen; Rao, Hai

    2014-01-01

    Prion protein PrP is a central player in several devastating neurodegenerative disorders, including mad cow disease and Creutzfeltd-Jacob disease. Conformational alteration of PrP into an aggregation-prone infectious form PrPSc can trigger pathogenic events. How levels of PrP are regulated is poorly understood. Human PrP is known to be degraded by the proteasome, but the specific proteolytic pathway responsible for PrP destruction remains elusive. Here, we demonstrate that the ubiquitin ligas...

  9. Two waves of proteasome-dependent protein degradation in the hippocampus are required for recognition memory consolidation.

    Science.gov (United States)

    Figueiredo, Luciana S; Dornelles, Arethuza S; Petry, Fernanda S; Falavigna, Lucio; Dargél, Vinicius A; Köbe, Luiza M; Aguzzoli, Cristiano; Roesler, Rafael; Schröder, Nadja

    2015-04-01

    Healthy neuronal function and synaptic modification require a concert of synthesis and degradation of proteins. Increasing evidence indicates that protein turnover mediated by proteasome activity is involved in long-term synaptic plasticity and memory. However, its role in different phases of memory remains debated, and previous studies have not examined the possible requirement of protein degradation in recognition memory. Here, we show that the proteasome inhibitor, lactacystin (LAC), infused into the CA1 area of the hippocampus at two specific time points during consolidation, impairs 24-retention of memory for object recognition in rats. Administration of LAC after retrieval did not affect retention. These findings provide the first evidence for a requirement of proteasome activity in recognition memory, indicate that protein degradation in the hippocampus is necessary during selective time windows of memory consolidation, and further our understanding of the role of protein turnover in memory formation. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Clofibrate-induced increases in peroxisomal proteins: effect on synthesis, degradation, and mRNA activity

    International Nuclear Information System (INIS)

    Mortensen, R.M.

    1983-01-01

    The effect of clofibrate on the polypeptide composition of peroxisomes was determined. A simple method was developed for the isolation of peroxisomes with a purity of 90-95% using sedimentation in a metrizamide gradient. The specific activities of HD did not change with clofibrate treatment so that the increases in enzyme activities are solely due to increases in protein amounts. The hepatic concentration of HD increased 63 times. The HD synthesis rate, as measured by the incorporation of [ 3 H]leucine, increased 74 times, so that the increase in the synthesis was sufficient to account for the increase in protein. Clofibrate caused no discernible change in the degradation rate of HD labeled with [ 14 C]bicarbonate. The half-life of HD was approximately 2 days. The translatable mRBA coding for HD increased 55 times. This value is not significantly different from the increase in HD protein or in HD synthesis. This observation was also true for several other peroxisomal proteins. Therefore, clofibrate causes an increase in the mRNA activity, which increases the synthesis of HD leading to an accumulation of protein and enzyme activity. The kinetics of the clofibrate-induced changes in HD synthesis rate, protein level, and enzymatic activity was analyzed using a simple model which included the half-lives of the drug, mRNA, and protein. The best fit of the model to the data gave an mRNA half-life of 10 hours and a protein half-life of 1.8 days, with no significant change by clofibrate

  11. Metabolic and productive response to ruminal protein degradability in early lactation cows fed untreated or xylose-treated soybean meal-based diets.

    Science.gov (United States)

    Jahani-Moghadam, M; Amanlou, H; Nikkhah, A

    2009-12-01

    Effects of different dietary rumen undegradable (RUP) to degradable (RDP) protein ratios on ruminal nutrient degradation, feed intake, blood metabolites and milk production were determined in early lactation cows. Four multiparous (43 ± 5 days in milk) and four primiparous (40 ± 6 days in milk) tie-stall-housed Holstein cows were used in a duplicated 4 × 4 Latin square design with four 21-day periods. Each period had 14-day of adaptation and 7-day of sampling. Diets contained on a dry matter (DM) basis, 23.3% alfalfa hay, 20% corn silage and 56.7% concentrate. Cows were first offered alfalfa hay at 7:00, 15:00 and 23:00 hours, and 30 min after each alfalfa hay delivery were offered a mixture of corn silage and concentrate. Treatments were diets with RUP:RDP ratios of (i) 5.2:11.6 (control), (ii) 6.1:10.6, (iii) 7.1:9.5 and (iv) 8.1:8.5, on a dietary DM% basis. Different RUP:RDP ratios were obtained by partial and total replacement of untreated soybean meal (SBM) with xylose-treated SBM (XSBM). In situ study using three rumen-cannulated non-lactating cows showed that DM and crude protein (CP) of SBM had greater rapidly degradable fractions. The potentially degradable fractions were degraded more slowly in XSBM. Treatment cows produced greater milk, protein, lactose, solids-non-fat and total solids than control cows. Increasing RUP:RDP reduced blood urea linearly. Feed costs dropped at RUP:RDP ratios of 6.1:10.6 and 7.1:9.5, but not at 8.1:8.5, compared with the 5.2:11.6 ratio. Intake of DM and CP, rumen pH, blood glucose, albumin and total protein, faecal and urine pH, changes in body weight and body condition score, and milk lactose and solids-non-fat percentages did not differ among treatments. Results provide evidence that increasing dietary RUP:RDP ratio from 5.2:11.6 to 7.1:9.5 optimizes nitrogen metabolism and milk production and reduces feed costs in early lactation cows. Reduced blood urea suggests reprodutive benefits.

  12. Hydrothermal degradation of cellulosic matter to sugars and their fermentative conversion to protein

    International Nuclear Information System (INIS)

    Bobleter, O.; Niesner, R.; Roehr, M.

    1976-01-01

    For the hydrothermal degradation of cellulosic matter, an apparatus was developed in which water is used as extraction medium. Samples, 0.15 g each, of pure cellulose (filter paper), natural straw, and 14 C-labeled straw were treated at temperatures of between 200 and 275 0 C. Of the inserted cellulose, 65.7 percent was recovered at the optimum temperature as sugars and hydroxymethylfurfural. It was possible to degrade the straw selectively: at lower temperatures, the hemicellulose part of the plant matter was converted to xylose and arabinose; and then at higher temperatures, the cellulose was converted to glucose and cellobiose. At the same time, a certain amount of the sugars was transformed to furfural compounds. The growth behavior of the yeast Candida utilis (strain Weissenbach) was analyzed, using cellobiose, xylose, and glucose (standard) as carbon sources. The growth curves applying cellobiose were nearly identical to those of glucose. Xylose showed lower productivity than the hexoses. The main products of the hydrothermal degradation can, therefore, be used favorably as nutritive substances for this protein-producing yeast

  13. Postmortem muscle protein degradation in humans as a tool for PMI delimitation.

    Science.gov (United States)

    Pittner, Stefan; Ehrenfellner, Bianca; Monticelli, Fabio C; Zissler, Angela; Sänger, Alexandra M; Stoiber, Walter; Steinbacher, Peter

    2016-11-01

    Forensic estimation of time since death relies on diverse approaches, including measurement and comparison of environmental and body core temperature and analysis of insect colonization on a dead body. However, most of the applied methods have practical limitations or provide insufficient results under certain circumstances. Thus, new methods that can easily be implemented into forensic routine work are required to deliver more and discrete information about the postmortem interval (PMI). Following a previous work on skeletal muscle degradation in the porcine model, we analyzed human postmortem skeletal muscle samples of 40 forensic cases by Western blotting and casein zymography. Our results demonstrate predictable protein degradation processes in human muscle that are distinctly associated with temperature and the PMI. We provide information on promising degradation markers for certain periods of time postmortem, which can be useful tools for time since death delimitation. In addition, we discuss external influencing factors such as age, body mass index, sex, and cause of death that need to be considered in future routine application of the method in humans.

  14. Indian hedgehog signaling triggers Nkx3.2 protein degradation during chondrocyte maturation

    Science.gov (United States)

    Choi, Seung-Won; Jeong, Da-Un; Kim, Jeong-Ah; Lee, Boyoung; Joeng, Kyu Sang; Long, Fanxin; Kim, Dae-Won

    2015-01-01

    The Indian hedgehog (Ihh) pathway plays an essential role in facilitating chondrocyte hypertrophy and bone formation during skeletal development. Nkx3.2 is initially induced in chondrocyte precursor cells, maintained in early-stage chondrocytes, and down-regulated in terminal-stage chondrocytes. Consistent with these expression patterns, Nkx3.2 has been shown to enhance chondrocyte differentiation and cell survival, while inhibiting chondrocyte hypertrophy and apoptosis. Thus, in this work, we investigate whether Nkx3.2, an early stage chondrogenic factor, can be regulated by Ihh, a key regulator for chondrocyte hypertrophy. Here, we show that Ihh signaling can induce proteasomal degradation of Nkx3.2. In addition, we found that Ihh can suppress levels of Lrp (Wnt co-receptor) and Sfrp (Wnt antagonist) expression, which, in turn, may selectively enhance Lrp-independent non-canonical Wnt pathways in chondrocyte. In agreement with these findings, Ihh-induced Nkx3.2 degradation requires Wnt5a, which is capable of triggering Nkx3.2 degradation. Finally, we found that Nkx3.2 protein levels in chondrocytes are remarkably elevated in mice defective in Ihh signaling by deletion of either Ihh or Smoothened. Thus, these results suggest that Ihh/Wnt5a signaling may play a role in negative regulation of Nkx3.2 for appropriate progression of chondrocyte hypertrophy during chondrogenesis. PMID:22507129

  15. Hepatitis C virus induces E6AP-dependent degradation of the retinoblastoma protein.

    Directory of Open Access Journals (Sweden)

    Tsubasa Munakata

    2007-09-01

    Full Text Available Hepatitis C virus (HCV is a positive-strand RNA virus that frequently causes persistent infections and is uniquely associated with the development of hepatocellular carcinoma. While the mechanism(s by which the virus promotes cancer are poorly defined, previous studies indicate that the HCV RNA-dependent RNA polymerase, nonstructural protein 5B (NS5B, forms a complex with the retinoblastoma tumor suppressor protein (pRb, targeting it for degradation, activating E2F-responsive promoters, and stimulating cellular proliferation. Here, we describe the mechanism underlying pRb regulation by HCV and its relevance to HCV infection. We show that the abundance of pRb is strongly downregulated, and its normal nuclear localization altered to include a major cytoplasmic component, following infection of cultured hepatoma cells with either genotype 1a or 2a HCV. We further demonstrate that this is due to NS5B-dependent ubiquitination of pRb and its subsequent degradation via the proteasome. The NS5B-dependent ubiquitination of pRb requires the ubiquitin ligase activity of E6-associated protein (E6AP, as pRb abundance was restored by siRNA knockdown of E6AP or overexpression of a dominant-negative E6AP mutant in cells containing HCV RNA replicons. E6AP also forms a complex with pRb in an NS5B-dependent manner. These findings suggest a novel mechanism for the regulation of pRb in which the HCV NS5B protein traps pRb in the cytoplasm, and subsequently recruits E6AP to this complex in a process that leads to the ubiquitination of pRb. The disruption of pRb/E2F regulatory pathways in cells infected with HCV is likely to promote hepatocellular proliferation and chromosomal instability, factors important for the development of liver cancer.

  16. Fractionation of whey protein isolate with supercritical carbon dioxide to produce enriched alpha-lactalbumin and beta-lactoglobulin food ingredients

    Science.gov (United States)

    A potentially economical and environmentally friendly whey protein fractionation process was developed using supercritical carbon dioxide (SCO2) as an acid to produce enriched fractions of alpha-lactalbumin (a-LA) and beta-lactoglobulin (b-LG) from whey protein isolate. To prepare the fractions, so...

  17. Chitin Degradation Proteins Produced by the Marine Bacterium Vibrio harveyi Growing on Different Forms of Chitin.

    Science.gov (United States)

    Svitil, A L; Chadhain, S; Moore, J A; Kirchman, D L

    1997-02-01

    Relatively little is known about the number, diversity, and function of chitinases produced by bacteria, even though chitin is one of the most abundant polymers in nature. Because of the importance of chitin, especially in marine environments, we examined chitin-degrading proteins in the marine bacterium Vibrio harveyi. This bacterium had a higher growth rate and more chitinase activity when grown on (beta)-chitin (isolated from squid pen) than on (alpha)-chitin (isolated from snow crab), probably because of the more open structure of (beta)-chitin. When exposed to different types of chitin, V. harveyi excreted several chitin-degrading proteins into the culture media. Some chitinases were present with all of the tested chitins, while others were unique to a particular chitin. We cloned and identified six separate chitinase genes from V. harveyi. These chitinases appear to be unique based on DNA restriction patterns, immunological data, and enzyme activity. This marine bacterium and probably others appear to synthesize separate chitinases for efficient utilization of different forms of chitin and chitin by-products.

  18. LINGO-1 promotes lysosomal degradation of amyloid-β protein precursor

    Directory of Open Access Journals (Sweden)

    Rian de Laat

    2015-03-01

    Full Text Available Sequential proteolytic cleavages of amyloid-β protein precursor (AβPP by β-secretase and γ-secretase generate amyloid β (Aβ peptides, which are thought to contribute to Alzheimer's disease (AD. Much of this processing occurs in endosomes following endocytosis of AβPP from the plasma membrane. However, this pathogenic mode of processing AβPP may occur in competition with lysosomal degradation of AβPP, a common fate of membrane proteins trafficking through the endosomal system. Following up on published reports that LINGO-1 binds and promotes the amyloidogenic processing of AβPP we have examined the consequences of LINGO-1/AβPP interactions. We report that LINGO-1 and its paralogs, LINGO-2 and LINGO-3, decrease processing of AβPP in the amyloidogenic pathway by promoting lysosomal degradation of AβPP. We also report that LINGO-1 levels are reduced in AD brain, representing a possible pathogenic mechanism stimulating the generation of Aβ peptides in AD.

  19. Activity dependent protein degradation is critical for the formation and stability of fear memory in the amygdala.

    Directory of Open Access Journals (Sweden)

    Timothy J Jarome

    Full Text Available Protein degradation through the ubiquitin-proteasome system [UPS] plays a critical role in some forms of synaptic plasticity. However, its role in memory formation in the amygdala, a site critical for the formation of fear memories, currently remains unknown. Here we provide the first evidence that protein degradation through the UPS is critically engaged at amygdala synapses during memory formation and retrieval. Fear conditioning results in NMDA-dependent increases in degradation-specific polyubiquitination in the amygdala, targeting proteins involved in translational control and synaptic structure and blocking the degradation of these proteins significantly impairs long-term memory. Furthermore, retrieval of fear memory results in a second wave of NMDA-dependent polyubiquitination that targets proteins involved in translational silencing and synaptic structure and is critical for memory updating following recall. These results indicate that UPS-mediated protein degradation is a major regulator of synaptic plasticity necessary for the formation and stability of long-term memories at amygdala synapses.

  20. A novel mosquito ubiquitin targets viral envelope protein for degradation and reduces virion production during dengue virus infection.

    Science.gov (United States)

    Troupin, Andrea; Londono-Renteria, Berlin; Conway, Michael J; Cloherty, Erin; Jameson, Samuel; Higgs, Stephen; Vanlandingham, Dana L; Fikrig, Erol; Colpitts, Tonya M

    2016-09-01

    Dengue virus (DENV) is a mosquito-borne flavivirus that causes significant human disease and mortality in the tropics and subtropics. By examining the effects of virus infection on gene expression, and interactions between virus and vector, new targets for prevention of infection and novel treatments may be identified in mosquitoes. We previously performed a microarray analysis of the Aedes aegypti transcriptome during infection with DENV and found that mosquito ubiquitin protein Ub3881 (AAEL003881) was specifically and highly down-regulated. Ubiquitin proteins have multiple functions in insects, including marking proteins for proteasomal degradation, regulating apoptosis and mediating innate immune signaling. We used qRT-PCR to quantify gene expression and infection, and RNAi to reduce Ub3881 expression. Mosquitoes were infected with DENV through blood feeding. We transfected DENV protein expression constructs to examine the effect of Ub3881 on protein degradation. We used site-directed mutagenesis and transfection to determine what amino acids are involved in Ub3881-mediated protein degradation. Immunofluorescence, Co-immunoprecipitation and Western blotting were used to examine protein interactions and co-localization. The overexpression of Ub3881, but not related ubiquitin proteins, decreased DENV infection in mosquito cells and live Ae. aegypti. The Ub3881 protein was demonstrated to be involved in DENV envelope protein degradation and reduce the number of infectious virions released. We conclude that Ub3881 has several antiviral functions in the mosquito, including specific viral protein degradation. Our data highlights Ub3881 as a target for future DENV prevention strategies in the mosquito transmission vector. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  1. Leucine supplementation stimulates protein synthesis and reduces degradation signal activation in muscle of newborn pigs during acute endotoxemia

    Science.gov (United States)

    Sepsis disrupts skeletal muscle proteostasis and mitigates the anabolic response to leucine (Leu) in muscle of mature animals. We have shown that Leu stimulates muscle protein synthesis (PS) in healthy neonatal piglets. To determine if supplemental Leu can stimulate PS and reduce protein degradation...

  2. Interplay between Molecular Chaperones and the Ubiquitin-Proteasome System in Targeting of Misfolded Proteins for Degradation

    DEFF Research Database (Denmark)

    Poulsen, Esben Guldahl

    interacting with purified 26S proteasomes, and the subsequent characterization of two novel proteasome interacting proteins. The third study was aimed at analyzing the chaperone-assisted pathway leading to degradation of misfolded kinetochore proteins in S. pombe. In this study chaperones, E2s, E3s and DUBs...

  3. Alteration of cardiac glycoside positive inotropic action by modulators of protein synthesis and degradation

    International Nuclear Information System (INIS)

    Nosek, T.M.; Adams, R.J.

    1986-01-01

    Numerous membrane bound and cytoplasmic proteins participate in the cardiac expression of the positive inotropic action (PIA) of digitalis glycosides including the Na,K-ATPase (NKA). Exposure of the myocardium to an inhibitor of protein synthesis (cycloheximide, CYC) or of protein degradation (leupeptin, LEU) alters the PIA of ouabain in isolated, paced guinea pig papillary muscles (PM) in opposite ways. In vivo exposure to CYC for 3 hr resulted in a 30% depression of the in vitro PIA of ouabain at 1.7μM compared to control. In vivo exposure to LEU for 1 hr resulted in a 47% enhancement of the in vitro PIA of 1.7μM ouabain. Neither drug had an apparent effect on the ouabain PIA ED50. Neither CYC nor LEU exposure to PM in vitro affect resting or developed tension or the response of skinned PM to calcium. The mechanisms of the PIA alterations by CYC or LEU do not involve a direct effect on the digitalis receptor. Exposure of isolated cardiac sarcolemma enriched in NKA to 10-100μM CYC or LEU did not affect NKA activity or 3 H-ouabain binding. Although direct physicochemical effects of CYC or LEU may be involved in the alterations of the ouabain PIA, it is possible that modulation of the cellular levels or turnover rate of short-lived proteins may affect cardiac regulation of the digitalis PIA

  4. Ciliopathy proteins regulate paracrine signaling by modulating proteasomal degradation of mediators

    Science.gov (United States)

    Liu, Yangfan P.; Tsai, I-Chun; Morleo, Manuela; Oh, Edwin C.; Leitch, Carmen C.; Massa, Filomena; Lee, Byung-Hoon; Parker, David S.; Finley, Daniel; Zaghloul, Norann A.; Franco, Brunella; Katsanis, Nicholas

    2014-01-01

    Cilia are critical mediators of paracrine signaling; however, it is unknown whether proteins that contribute to ciliopathies converge on multiple paracrine pathways through a common mechanism. Here, we show that loss of cilopathy-associated proteins Bardet-Biedl syndrome 4 (BBS4) or oral-facial-digital syndrome 1 (OFD1) results in the accumulation of signaling mediators normally targeted for proteasomal degradation. In WT cells, several BBS proteins and OFD1 interacted with proteasomal subunits, and loss of either BBS4 or OFD1 led to depletion of multiple subunits from the centrosomal proteasome. Furthermore, overexpression of proteasomal regulatory components or treatment with proteasomal activators sulforaphane (SFN) and mevalonolactone (MVA) ameliorated signaling defects in cells lacking BBS1, BBS4, and OFD1, in morphant zebrafish embryos, and in induced neurons from Ofd1-deficient mice. Finally, we tested the hypothesis that other proteasome-dependent pathways not known to be associated with ciliopathies are defective in the absence of ciliopathy proteins. We found that loss of BBS1, BBS4, or OFD1 led to decreased NF-κB activity and concomitant IκBβ accumulation and that these defects were ameliorated with SFN treatment. Taken together, our data indicate that basal body proteasomal regulation governs paracrine signaling pathways and suggest that augmenting proteasomal function might benefit ciliopathy patients. PMID:24691443

  5. Degradability of dry matter and crude protein of dry grains and wet grain silages from different processing corn hybrids (Zea mays

    Directory of Open Access Journals (Sweden)

    Wagner dos Reis

    2013-03-01

    Full Text Available The objective of this work was to evaluate the effect of processing two corn hybrids conserved, dry and humid grains, the dry matter (DM and crude protein (CP degradability in situ. The particle size was determined and difference was verified in MGD (Medium Geometric Diameter of processed ingredients. Three sheep were used with rumen canulated, in a completely randomized design, using a factorial outline 2 x 2 x 3, being two corn hybrid, two conservation methods and three processing forms (whole, coarsely and finely ground, with five times of incubation (3, 6, 12, 24 and 48 hours. The fraction A in SDC (silage of dent corn of DM was superior to GDC (grain of dent corn in all of the particles size. The ensiling process increased the DM solubility, reducing the fraction B in comparison to dry grain. The values regarding the fractions DP and DE the 5% per hour of the protein, were larger for SDC and GDC, it presents a decreasing when the incubation time advances. The fermentation rate was superior for SDC and GDC. The ensiling process has positive effect in the decreasing of DM and CP in comparison to GDC.

  6. Human Adenovirus Infection Causes Cellular E3 Ubiquitin Ligase MKRN1 Degradation Involving the Viral Core Protein pVII.

    Science.gov (United States)

    Inturi, Raviteja; Mun, Kwangchol; Singethan, Katrin; Schreiner, Sabrina; Punga, Tanel

    2018-02-01

    Human adenoviruses (HAdVs) are common human pathogens encoding a highly abundant histone-like core protein, VII, which is involved in nuclear delivery and protection of viral DNA as well as in sequestering immune danger signals in infected cells. The molecular details of how protein VII acts as a multifunctional protein have remained to a large extent enigmatic. Here we report the identification of several cellular proteins interacting with the precursor pVII protein. We show that the cellular E3 ubiquitin ligase MKRN1 is a novel precursor pVII-interacting protein in HAdV-C5-infected cells. Surprisingly, the endogenous MKRN1 protein underwent proteasomal degradation during the late phase of HAdV-C5 infection in various human cell lines. MKRN1 protein degradation occurred independently of the HAdV E1B55K and E4orf6 proteins. We provide experimental evidence that the precursor pVII protein binding enhances MKRN1 self-ubiquitination, whereas the processed mature VII protein is deficient in this function. Based on these data, we propose that the pVII protein binding promotes MKRN1 self-ubiquitination, followed by proteasomal degradation of the MKRN1 protein, in HAdV-C5-infected cells. In addition, we show that measles virus and vesicular stomatitis virus infections reduce the MKRN1 protein accumulation in the recipient cells. Taken together, our results expand the functional repertoire of the HAdV-C5 precursor pVII protein in lytic virus infection and highlight MKRN1 as a potential common target during different virus infections. IMPORTANCE Human adenoviruses (HAdVs) are common pathogens causing a wide range of diseases. To achieve pathogenicity, HAdVs have to counteract a variety of host cell antiviral defense systems, which would otherwise hamper virus replication. In this study, we show that the HAdV-C5 histone-like core protein pVII binds to and promotes self-ubiquitination of a cellular E3 ubiquitin ligase named MKRN1. This mutual interaction between the pVII and

  7. C1q protein binds to the apoptotic nucleolus and causes C1 protease degradation of nucleolar proteins.

    Science.gov (United States)

    Cai, Yitian; Teo, Boon Heng Dennis; Yeo, Joo Guan; Lu, Jinhua

    2015-09-11

    In infection, complement C1q recognizes pathogen-congregated antibodies and elicits complement activation. Among endogenous ligands, C1q binds to DNA and apoptotic cells, but whether C1q binds to nuclear DNA in apoptotic cells remains to be investigated. With UV irradiation-induced apoptosis, C1q initially bound to peripheral cellular regions in early apoptotic cells. By 6 h, binding concentrated in the nuclei to the nucleolus but not the chromatins. When nucleoli were isolated from non-apoptotic cells, C1q also bound to these structures. In vivo, C1q exists as the C1 complex (C1qC1r2C1s2), and C1q binding to ligands activates the C1r/C1s proteases. Incubation of nucleoli with C1 caused degradation of the nucleolar proteins nucleolin and nucleophosmin 1. This was inhibited by the C1 inhibitor. The nucleoli are abundant with autoantigens. C1q binding and C1r/C1s degradation of nucleolar antigens during cell apoptosis potentially reduces autoimmunity. These findings help us to understand why genetic C1q and C1r/C1s deficiencies cause systemic lupus erythematosus. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    Directory of Open Access Journals (Sweden)

    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.

  9. Calpain activation by ROS mediates human ether-a-go-go-related gene protein degradation by intermittent hypoxia.

    Science.gov (United States)

    Wang, N; Kang, H S; Ahmmed, G; Khan, S A; Makarenko, V V; Prabhakar, N R; Nanduri, J

    2016-03-01

    Human ether-a-go-go-related gene (hERG) channels conduct delayed rectifier K(+) current. However, little information is available on physiological situations affecting hERG channel protein and function. In the present study we examined the effects of intermittent hypoxia (IH), which is a hallmark manifestation of sleep apnea, on hERG channel protein and function. Experiments were performed on SH-SY5Y neuroblastoma cells, which express hERG protein. Cells were exposed to IH consisting of alternating cycles of 30 s of hypoxia (1.5% O2) and 5 min of 20% O2. IH decreased hERG protein expression in a stimulus-dependent manner. A similar reduction in hERG protein was also seen in adrenal medullary chromaffin cells from IH-exposed neonatal rats. The decreased hERG protein was associated with attenuated hERG K(+) current. IH-evoked hERG protein degradation was not due to reduced transcription or increased proteosome/lysomal degradation. Rather it was mediated by calcium-activated calpain proteases. Both COOH- and NH2-terminal sequences of the hERG protein were the targets of calpain-dependent degradation. IH increased reactive oxygen species (ROS) levels, intracellular Ca(2+) concentration ([Ca(2+)]i), calpain enzyme activity, and hERG protein degradation, and all these effects were prevented by manganese-(111)-tetrakis-(1-methyl-4-pyridyl)-porphyrin pentachloride, a membrane-permeable ROS scavenger. These results demonstrate that activation of calpains by ROS-dependent elevation of [Ca(2+)]i mediates hERG protein degradation by IH. Copyright © 2016 the American Physiological Society.

  10. Masseter muscle myofibrillar protein synthesis and degradation in an experimental critical illness myopathy model.

    Directory of Open Access Journals (Sweden)

    Hazem Akkad

    Full Text Available Critical illness myopathy (CIM is a debilitating common consequence of modern intensive care, characterized by severe muscle wasting, weakness and a decreased myosin/actin (M/A ratio. Limb/trunk muscles are primarily affected by this myopathy while cranial nerve innervated muscles are spared or less affected, but the mechanisms underlying these muscle-specific differences remain unknown. In this time-resolved study, the cranial nerve innervated masseter muscle was studied in a unique experimental rat intensive care unit (ICU model, where animals were exposed to sedation, neuromuscular blockade (NMB, mechanical ventilation, and immobilization for durations varying between 6 h and 14d. Gel electrophoresis, immunoblotting, RT-PCR and morphological staining techniques were used to analyze M/A ratios, myofiber size, synthesis and degradation of myofibrillar proteins, and levels of heat shock proteins (HSPs. Results obtained in the masseter muscle were compared with previous observations in experimental and clinical studies of limb muscles. Significant muscle-specific differences were observed, i.e., in the masseter, the decline in M/A ratio and muscle fiber size was small and delayed. Furthermore, transcriptional regulation of myosin and actin synthesis was maintained, and Akt phosphorylation was only briefly reduced. In studied degradation pathways, only mRNA, but not protein levels of MuRF1, atrogin-1 and the autophagy marker LC3b were activated by the ICU condition. The matrix metalloproteinase MMP-2 was inhibited and protective HSPs were up-regulated early. These results confirm that the cranial nerve innervated masticatory muscles is less affected by the ICU-stress response than limb muscles, in accordance with clinical observation in ICU patients with CIM, supporting the model' credibility as a valid CIM model.

  11. Bioactive protein fraction DLBS1033 containing lumbrokinase isolated from Lumbricus rubellus: ex vivo, in vivo, and pharmaceutic studies

    Directory of Open Access Journals (Sweden)

    Tjandrawinata RR

    2014-09-01

    Full Text Available Raymond R Tjandrawinata,1 Jessica Trisina,1 Puji Rahayu,1 Lorentius Agung Prasetya,1 Aang Hanafiah,2 Heni Rachmawati3 1Dexa Laboratories of Biomolecular Sciences, Dexa Medica, Cikarang, Indonesia; 2National Nuclear Energy Agency, Bandung, Indonesia; 3School of Pharmacy, Bandung Institute of Technology, Bandung, Indonesia Abstract: DLBS1033 is a bioactive protein fraction isolated from Lumbricus rubellus that tends to be unstable when exposed to the gastrointestinal environment. Accordingly, appropriate pharmaceutical development is needed to maximize absorption of the protein fraction in the gastrointestinal tract. In vitro, ex vivo, and in vivo stability assays were performed to study the stability of the bioactive protein fraction in gastric conditions. The bioactive protein fraction DLBS1033 was found to be unstable at low pH and in gastric fluid. The “enteric coating” formulation showed no leakage in gastric fluid–like medium and possessed a good release profile in simulated intestinal medium. DLBS1033 was absorbed through the small intestine in an intact protein form, confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE analysis. This result confirmed that an enteric coating formula using methacrylic acid copolymer could protect DLBS1033 from the acidic condition of the stomach by preventing the release of DLBS1033 in the stomach, while promoting its release when reaching the intestine. From the blood concentration–versus-time curve, 99mTc-DLBS1033 showed a circulation half-life of 70 minutes. This relatively long biological half-life supports its function as a thrombolytic protein. Thus, an enteric delivery system is considered the best approach for DLBS1033 as an oral thrombolytic agent. Keywords: bioactive protein fraction, enteric coated tablet, pharmacodynamic

  12. Chemical biology based on target-selective degradation of proteins and carbohydrates using light-activatable organic molecules.

    Science.gov (United States)

    Toshima, Kazunobu

    2013-05-01

    Proteins and carbohydrates play crucial roles in a wide range of biological processes, including serious diseases. The development of novel and innovative methods for selective control of specific proteins and carbohydrates functions has attracted much attention in the field of chemical biology. In this account article, the development of novel chemical tools, which can degrade target proteins and carbohydrates by irradiation with a specific wavelength of light under mild conditions without any additives, is introduced. This novel class of photochemical agents promise bright prospects for finding not only molecular-targeted bioprobes for understanding of the structure-activity relationships of proteins and carbohydrates but also novel therapeutic drugs targeting proteins and carbohydrates.

  13. Tau protein degradation is catalyzed by the ATP/ubiquitin-independent 20S proteasome under normal cell conditions

    OpenAIRE

    Grune, Tilman; Botzen, Diana; Engels, Martina; Voss, Peter; Kaiser, Barbara; Jung, Tobias; Grimm, Stefanie; Ermak, Gennady; Davies, Kelvin J. A.

    2010-01-01

    Tau is the major protein exhibiting intracellular accumulation in Alzheimer disease. The mechanisms leading to its accumulation are not fully understood. It has been proposed that the proteasome is responsible for degrading tau but, since proteasomal inhibitors block both the ubiquitin-dependent 26S proteasome and the ubiqutin-independent 20S proteasome pathways, it is not clear which of these pathways is involved in tau degradation. Some involvement of the ubiquitin ligase, CHIP in tau degra...

  14. Storage stability of cauliflower soup powder: The effect of lipid oxidation and protein degradation reactions.

    Science.gov (United States)

    Raitio, Riikka; Orlien, Vibeke; Skibsted, Leif H

    2011-09-15

    Soups based on cauliflower soup powders, prepared by dry mixing of ingredients and rapeseed oil, showed a decrease in quality, as evaluated by a sensory panel, during the storage of the soup powder in the dark for up to 12weeks under mildly accelerated conditions of 40°C and 75% relative humidity. Antioxidant, shown to be effective in protecting the rapeseed bulk oil, used for the powder preparation, had no effect on storage stability of the soup powder. The freshly prepared soup powder had a relatively high concentration of free radicals, as measured by electron spin resonance spectroscopy, which decreased during storage, and most remarkably during the first two weeks of storage, with only marginal increase in lipid hydroperoxides as primary lipid oxidation products, and without any increase in secondary lipid oxidation products. Analyses of volatiles by SPME-GC-MS revealed a significant increase in concentrations of 2-methyl- and 3-methyl butanals, related to Maillard reactions, together with an increase in 2-acetylpyrrole concentration. The soup powders became more brown during storage, as indicated by a decreasing Hunter L-value, in accord with non-enzymatic browning reactions. A significant increase in the concentrations of dimethyl disulfide in soup powder headspace indicated free radical-initiated protein oxidation. Protein degradation, including Maillard reactions and protein oxidation, is concluded to be more important than lipid oxidation in determining the shelf-life of dry cauliflower soup powder. Copyright © 2011 Elsevier Ltd. All rights reserved.

  15. Measurement of Muscle Protein Fractional Synthetic Rate by Capillary Gas Chromatography/Combustion Isotope Ratio Mass Spectrometry

    OpenAIRE

    Yarasheski, Kevin E.; Smith, Kenneth; Rennie, Michael J.; Bier, Dennis M.

    1992-01-01

    The measurement of skeletal muscle protein fractional synthetic rate using an infusion of (1-13C)leucine and measuring the isotopic abundance of the tracer in skeletal muscle protein by preparative gas chromatography (GC)/ninhydrin isotope ratio mass spectrometry (IRMS) is laborious and subject to errors owing to contamination by 12C. The purpose of this study was to compare muscle (13C)leucine enrichment measured with the conventional preparative GC/ninhydrin IRMS approach to a new, continuo...

  16. A maize spermine synthase 1 PEST sequence fused to the GUS reporter protein facilitates proteolytic degradation.

    Science.gov (United States)

    Maruri-López, Israel; Rodríguez-Kessler, Margarita; Rodríguez-Hernández, Aída Araceli; Becerra-Flora, Alicia; Olivares-Grajales, Juan Elías; Jiménez-Bremont, Juan Francisco

    2014-05-01

    Polyamines are low molecular weight aliphatic compounds involved in various biochemical, cellular and physiological processes in all organisms. In plants, genes involved in polyamine biosynthesis and catabolism are regulated at transcriptional, translational, and posttranslational level. In this research, we focused on the characterization of a PEST sequence (rich in proline, glutamic acid, serine, and threonine) of the maize spermine synthase 1 (ZmSPMS1). To this aim, 123 bp encoding 40 amino acids of the C-terminal region of the ZmSPMS1 enzyme containing the PEST sequence were fused to the GUS reporter gene. This fusion was evaluated in Arabidopsis thaliana transgenic lines and onion monolayers transient expression system. The ZmSPMS1 PEST sequence leads to specific degradation of the GUS reporter protein. It is suggested that the 26S proteasome may be involved in GUS::PEST fusion degradation in both onion and Arabidopsis. The PEST sequences appear to be present in plant spermine synthases, mainly in monocots. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  17. Bidirectional gene sequences with similar homology to functional proteins of alkane degrading bacterium pseudomonas fredriksbergensis DNA

    International Nuclear Information System (INIS)

    Megeed, A.A.

    2011-01-01

    The potential for two overlapping fragments of DNA from a clone of newly isolated alkanes degrading bacterium Pseudomonas frederiksbergensis encoding sequences with similar homology to two parts of functional proteins is described. One strand contains a sequence with high homology to alkanes monooxygenase (alkB), a member of the alkanes hydroxylase family, and the other strand contains a sequence with some homology to alcohol dehydrogenase gene (alkJ). Overlapping of the genes on opposite strands has been reported in eukaryotic species, and is now reported in a bacterial species. The sequence comparisons and ORFS results revealed that the regulation and the genes organization involved in alkane oxidation represented in Pseudomonas frederiksberghensis varies among the different known alkane degrading bacteria. The alk gene cluster containing homologues to the known alkane monooxygenase (alkB), and rubredoxin (alkG) are oriented in the same direction, whereas alcohol dehydrogenase (alkJ) is oriented in the opposite direction. Such genomes encode messages on both strands of the DNA, or in an overlapping but different reading frames, of the same strand of DNA. The possibility of creating novel genes from pre-existing sequences, known as overprinting, which is a widespread phenomenon in small viruses. Here, the origin and evolution of the gene overlap to bacteriophages belonging to the family Microviridae have been investigated. Such a phenomenon is most widely described in extremely small genomes such as those of viruses or small plasmids, yet here is a unique phenomenon. (author)

  18. Recombinant protein production facility for fungal biomass-degrading enzymes using the yeast Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Mireille eHaon

    2015-09-01

    Full Text Available Filamentous fungi are the predominant source of lignocellulolytic enzymes used in industry for the transformation of plant biomass into high-value molecules and biofuels. The rapidity with which new fungal genomic and post-genomic data are being produced is vastly outpacing functional studies. This underscores the critical need for developing platforms dedicated to the recombinant expression of enzymes lacking confident functional annotation, a prerequisite to their functional and structural study. In the last decade, the yeast Pichia pastoris has become increasingly popular as a host for the production of fungal biomass-degrading enzymes, and particularly carbohydrate-active enzymes (CAZymes. This study aimed at setting-up a platform to easily and quickly screen the extracellular expression of biomass-degrading enzymes in Pichia pastoris. We first used three fungal glycoside hydrolases that we previously expressed using the protocol devised by Invitrogen to try different modifications of the original protocol. Considering the gain in time and convenience provided by the new protocol, we used it as basis to set-up the facility and produce a suite of fungal CAZymes (glycoside hydrolases, carbohydrate esterases and auxiliary activity enzyme families out of which more than 70% were successfully expressed. The platform tasks range from gene cloning to automated protein purifications and activity tests, and is open to the CAZyme users’ community.

  19. RING finger protein 121 facilitates the degradation and membrane localization of voltage-gated sodium channels

    Science.gov (United States)

    Ogino, Kazutoyo; Low, Sean E.; Yamada, Kenta; Saint-Amant, Louis; Zhou, Weibin; Muto, Akira; Asakawa, Kazuhide; Nakai, Junichi; Kawakami, Koichi; Kuwada, John Y.; Hirata, Hiromi

    2015-01-01

    Following their synthesis in the endoplasmic reticulum (ER), voltage-gated sodium channels (NaV) are transported to the membranes of excitable cells, where they often cluster, such as at the axon initial segment of neurons. Although the mechanisms by which NaV channels form and maintain clusters have been extensively examined, the processes that govern their transport and degradation have received less attention. Our entry into the study of these processes began with the isolation of a new allele of the zebrafish mutant alligator, which we found to be caused by mutations in the gene encoding really interesting new gene (RING) finger protein 121 (RNF121), an E3-ubiquitin ligase present in the ER and cis-Golgi compartments. Here we demonstrate that RNF121 facilitates two opposing fates of NaV channels: (i) ubiquitin-mediated proteasome degradation and (ii) membrane localization when coexpressed with auxiliary NaVβ subunits. Collectively, these results indicate that RNF121 participates in the quality control of NaV channels during their synthesis and subsequent transport to the membrane. PMID:25691753

  20. High-throughput fractionation of human plasma for fast enrichment of low- and high-abundance proteins.

    Science.gov (United States)

    Breen, Lucas; Cao, Lulu; Eom, Kirsten; Srajer Gajdosik, Martina; Camara, Lila; Giacometti, Jasminka; Dupuy, Damian E; Josic, Djuro

    2012-05-01

    Fast, cost-effective and reproducible isolation of IgM from plasma is invaluable to the study of IgM and subsequent understanding of the human immune system. Additionally, vast amounts of information regarding human physiology and disease can be derived from analysis of the low abundance proteome of the plasma. In this study, methods were optimized for both the high-throughput isolation of IgM from human plasma, and the high-throughput isolation and fractionation of low abundance plasma proteins. To optimize the chromatographic isolation of IgM from human plasma, many variables were examined including chromatography resin, mobile phases, and order of chromatographic separations. Purification of IgM was achieved most successfully through isolation of immunoglobulin from human plasma using Protein A chromatography with a specific resin followed by subsequent fractionation using QA strong anion exchange chromatography. Through these optimization experiments, an additional method was established to prepare plasma for analysis of low abundance proteins. This method involved chromatographic depletion of high-abundance plasma proteins and reduction of plasma proteome complexity through further chromatographic fractionation. Purification of IgM was achieved with high purity as confirmed by SDS-PAGE and IgM-specific immunoblot. Isolation and fractionation of low abundance protein was also performed successfully, as confirmed by SDS-PAGE and mass spectrometry analysis followed by label-free quantitative spectral analysis. The level of purity of the isolated IgM allows for further IgM-specific analysis of plasma samples. The developed fractionation scheme can be used for high throughput screening of human plasma in order to identify low and high abundance proteins as potential prognostic and diagnostic disease biomarkers.

  1. Comparison of the protein and fatty acid fraction of Balkan donkey and human milk

    Directory of Open Access Journals (Sweden)

    Jasmina Gubić

    2015-07-01

    Full Text Available The aim of this study was to compare the protein and fatty acid fractions of Balkan donkey and human milk in the early lactation stage (40 and 90 day. This study revealed that donkey milk contains αs1-casein (1.38-1.89 g/L and higher concentration of β-casein (0.1-0.55 g/L in comparison to human milk. The concentration of α-lactalbumin increased during the lactation phases from 40 to 90 days in both types of milk. Donkey milk contained β-lactoglobulin in low concentrations which decreased to 90th day of lactation. Donkey milk was particularly rich in two whey proteins, lactoferrin and lysozyme, which were found to have molecular weight of approximately 76 kDa and 14.9-15.4 kDa, respectively. The content of lysozyme in donkey milk ranged from 2.39 to 2.97 g/L, while human milk contained 30-50 times lower concentrations of lysozyme in comparison to donkey milk. Thus, donkey milk contained also a higher concentration of lactoferrin (0.012-0.25 g/L than it was found in the human milk. Lysozyme and lactoferrin content in donkey milk increased during the period from 40th to 90th day of lactation. The percentage of total SFA, MUFA and PUFA was similar in donkey and human milk. The content of essential fatty acids increased during 40-90 days of lactation and was approximately 2.5 times higher in comparison to human milk.

  2. The balance of protein expression and degradation: an ESCRTs point of view.

    Science.gov (United States)

    Babst, Markus; Odorizzi, Greg

    2013-08-01

    Endosomal sorting complexes required for transport (ESCRTs) execute the biogenesis of late endosomal multivesicular bodies (MVBs). The ESCRT pathway has traditionally been viewed as a means by which transmembrane proteins are degraded in vacuoles/lysosomes. More recent studies aimed at understanding the broader functions of ESCRTs have uncovered unexpected links with pathways that control cellular metabolism. Central to this communication is TORC1, the kinase complex that controls many of the catabolic and anabolic systems. The connection between TORC1 activity and ESCRTs allows cells to quickly adapt to the stress of nutrient limitations until the longer-term autophagic pathway is activated. Increasing evidence also points to ESCRTs regulating RNA interference (RNAi) pathways that control translation. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. Effect of conventional and extrusion pelleting on in situ ruminal degradability of starch, protein, and fibre in cattle

    DEFF Research Database (Denmark)

    Razzaghi, Ali; Larsen, Mogens; Lund, Peter

    2016-01-01

    +50% sugar beet pulp (SBP), or 50% maize+50% SBP. Meals were pelleted by either conventional pelleting, or by cooking extrusion using two distinct settings giving pellets with either high density (HD) or low density (LD). Ruminal degradation of starch, crude protein (CP) and NDF, and intestinal...... affected ruminal degradability of starch, protein, and NDF differently depending on both type of cereal and composition of the concentrate mixture.......>Pelleting>Meal). In contradiction, ESD for pure wheat and wheat mixtures was reduced, though differences were minor. Conventional pelleting reduced the effective protein degradability (EPD) for pure wheat, but extrusion did not further affect the EPD. In contrast, the most intense processing with extrusion LD increased EPD...

  4. Comparative Effects of Biodynes, Tocotrienol-Rich Fraction, and Tocopherol in Enhancing Collagen Synthesis and Inhibiting Collagen Degradation in Stress-Induced Premature Senescence Model of Human Diploid Fibroblasts

    Science.gov (United States)

    Jam, Faidruz Azura; Ismail, Zahariah; Wan Ngah, Wan Zurinah

    2013-01-01

    Biodynes, tocotrienol-rich fraction (TRF), and tocopherol have shown antiaging properties. However, the combined effects of these compounds on skin aging are yet to be investigated. This study aimed to elucidate the skin aging effects of biodynes, TRF, and tocopherol on stress-induced premature senescence (SIPS) model of human diploid fibroblasts (HDFs) by determining the expression of collagen and MMPs at gene and protein levels. Primary HDFs were treated with biodynes, TRF, and tocopherol prior to hydrogen peroxide (H2O2) exposure. The expression of COL1A1, COL3A1, MMP1, MMP2, MMP3, and MMP9 genes was determined by qRT-PCR. Type I and type III procollagen proteins were measured by Western blotting while the activities of MMPs were quantified by fluorometric Sensolyte MMP Kit. Our results showed that biodynes, TRF, and tocopherol upregulated collagen genes and downregulated MMP genes (P < 0.05). Type I procollagen and type III procollagen protein levels were significantly increased in response to biodynes, TRF, and tocopherol treatment (P < 0.05) with reduction in MMP-1, MMP-2, MMP-3, and MMP-9 activities (P < 0.05). These findings indicated that biodynes, TRF, and tocopherol effectively enhanced collagen synthesis and inhibited collagen degradation and therefore may protect the skin from aging. PMID:24396567

  5. Comparative Effects of Biodynes, Tocotrienol-Rich Fraction, and Tocopherol in Enhancing Collagen Synthesis and Inhibiting Collagen Degradation in Stress-Induced Premature Senescence Model of Human Diploid Fibroblasts

    Directory of Open Access Journals (Sweden)

    Suzana Makpol

    2013-01-01

    Full Text Available Biodynes, tocotrienol-rich fraction (TRF, and tocopherol have shown antiaging properties. However, the combined effects of these compounds on skin aging are yet to be investigated. This study aimed to elucidate the skin aging effects of biodynes, TRF, and tocopherol on stress-induced premature senescence (SIPS model of human diploid fibroblasts (HDFs by determining the expression of collagen and MMPs at gene and protein levels. Primary HDFs were treated with biodynes, TRF, and tocopherol prior to hydrogen peroxide (H2O2 exposure. The expression of COL1A1, COL3A1, MMP1, MMP2, MMP3, and MMP9 genes was determined by qRT-PCR. Type I and type III procollagen proteins were measured by Western blotting while the activities of MMPs were quantified by fluorometric Sensolyte MMP Kit. Our results showed that biodynes, TRF, and tocopherol upregulated collagen genes and downregulated MMP genes (P<0.05. Type I procollagen and type III procollagen protein levels were significantly increased in response to biodynes, TRF, and tocopherol treatment (P<0.05 with reduction in MMP-1, MMP-2, MMP-3, and MMP-9 activities (P<0.05. These findings indicated that biodynes, TRF, and tocopherol effectively enhanced collagen synthesis and inhibited collagen degradation and therefore may protect the skin from aging.

  6. Effects of Dietary Supplementation with Hainanmycin on Protein Degradation and Populations of Ammonia-producing Bacteria

    Directory of Open Access Journals (Sweden)

    Z. B. Wang

    2013-05-01

    Full Text Available An in vitro fermentation was conducted to determine the effects of hainanmycin on protein degradation and populations of ammonia-producing bacteria. The substrates (DM basis for in vitro fermentation consisted of alfalfa hay (31.7%, Chinese wild rye grass hay (28.3%, ground corn grain (24.5%, soybean meal (15.5% with a forage: concentrate of 60:40. Treatments were the control (no additive and hainanmycin supplemented at 0.1 (H0.1, 1 (H1, 10 (H10, and 100 mg/kg (H100 of the substrates. After 24 h of fermentation, the highest addition level of hainanmycin decreased total VFA concentration and increased the final pH. The high addition level of hainanmycin (H1, H10, and H100 reduced (p0.05. After 24 h of fermentation, H10 and H100 increased (p<0.05 concentrations of peptide nitrogen and AA nitrogen and proteinase activity, and decreased (p<0.05 NH3-N concentration and deaminase activity compared with control. Peptidase activitives were not affected by hainanmycin. Hainanmycin supplementation only inhibited the growth of Butyrivibrio fibrisolvens, which is one of the species of low deaminative activity. Hainanmycin supplementation also decreased (p<0.05 relative population sizes of hyper-ammonia-producing species, except for H0.1 on Clostridium aminophilum. It was concluded that dietary supplementation with hainanmycin could improve ruminal fermentation and modify protein degradation by changing population size of ammonia-producing bacteria in vitro; and the addition level of 10 mg/kg appeared to achieve the best results.

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

    Directory of Open Access Journals (Sweden)

    G. Vonghia

    2011-03-01

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

  8. Ubiquitination and degradation of the hominoid-specific oncoprotein TBC1D3 is regulated by protein palmitoylation

    Energy Technology Data Exchange (ETDEWEB)

    Kong, Chen; Lange, Jeffrey J.; Samovski, Dmitri [Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110 (United States); Su, Xiong [Department of Internal Medicine, Center for Human Nutrition Washington University School of Medicine, St. Louis, MO 63110 (United States); Liu, Jialiu [Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110 (United States); Sundaresan, Sinju [Department of Internal Medicine, Center for Human Nutrition Washington University School of Medicine, St. Louis, MO 63110 (United States); Stahl, Philip D., E-mail: pstahl@wustl.edu [Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110 (United States)

    2013-05-03

    Highlights: •Hominoid-specific oncogene TBC1D3 is targeted to plasma membrane by palmitoylation. •TBC1D3 is palmitoylated on two cysteine residues: 318 and 325. •TBC1D3 palmitoylation governs growth factors-induced TBC1D3 degradation. •Post-translational modifications may regulate oncogenic properties of TBC1D3. -- Abstract: Expression of the hominoid-specific oncoprotein TBC1D3 promotes enhanced cell growth and proliferation by increased activation of signal transduction through several growth factors. Recently we documented the role of CUL7 E3 ligase in growth factors-induced ubiquitination and degradation of TBC1D3. Here we expanded our study to discover additional molecular mechanisms that control TBC1D3 protein turnover. We report that TBC1D3 is palmitoylated on two cysteine residues: 318 and 325. The expression of double palmitoylation mutant TBC1D3:C318/325S resulted in protein mislocalization and enhanced growth factors-induced TBC1D3 degradation. Moreover, ubiquitination of TBC1D3 via CUL7 E3 ligase complex was increased by mutating the palmitoylation sites, suggesting that depalmitoylation of TBC1D3 makes the protein more available for ubiquitination and degradation. The results reported here provide novel insights into the molecular mechanisms that govern TBC1D3 protein degradation. Dysregulation of these mechanisms in vivo could potentially result in aberrant TBC1D3 expression and promote oncogenesis.

  9. Proteolytic degradation of regulator of G protein signaling 2 facilitates temporal regulation of Gq/11 signaling and vascular contraction.

    Science.gov (United States)

    Kanai, Stanley M; Edwards, Alethia J; Rurik, Joel G; Osei-Owusu, Patrick; Blumer, Kendall J

    2017-11-24

    Regulator of G protein signaling 2 (RGS2) controls signaling by receptors coupled to the G q/11 class heterotrimeric G proteins. RGS2 deficiency causes several phenotypes in mice and occurs in several diseases, including hypertension in which a proteolytically unstable RGS2 mutant has been reported. However, the mechanisms and functions of RGS2 proteolysis remain poorly understood. Here we addressed these questions by identifying degradation signals in RGS2, and studying dynamic regulation of G q/11 -evoked Ca 2+ signaling and vascular contraction. We identified a novel bipartite degradation signal in the N-terminal domain of RGS2. Mutations disrupting this signal blunted proteolytic degradation downstream of E3 ubiquitin ligase binding to RGS2. Analysis of RGS2 mutants proteolyzed at various rates and the effects of proteasome inhibition indicated that proteolytic degradation controls agonist efficacy by setting RGS2 protein expression levels, and affecting the rate at which cells regain agonist responsiveness as synthesis of RGS2 stops. Analyzing contraction of mesenteric resistance arteries supported the biological relevance of this mechanism. Because RGS2 mRNA expression often is strikingly and transiently up-regulated and then down-regulated upon cell stimulation, our findings indicate that proteolytic degradation tightly couples RGS2 transcription, protein levels, and function. Together these mechanisms provide tight temporal control of G q/11 -coupled receptor signaling in the cardiovascular, immune, and nervous systems. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Ubiquitination is absolutely required for the degradation of hypoxia-inducible factor--1 alpha protein in hypoxic conditions.

    Science.gov (United States)

    Wang, Ronghai; Zhang, Ping; Li, Jinhang; Guan, Hongzai; Shi, Guangjun

    2016-01-29

    The hypoxia-inducible factor (HIF) is recognized as the master regulator of hypoxia response. HIF-α subunits expression are tightly regulated. In this study, our data show that ts20 cells still expressed detectable E1 protein even at 39.5° C for 12 h, and complete depletion of E1 protein expression at 39.5° C by siRNA enhanced HIF-1α and P53 protein expression. Further inhibition of E1 at 39.5 °C by siRNA, or E1 inhibitor Ube1-41 completely blocked HIF-1α degradation. Moreover, immunoprecipitations of co-transfection of HA-ubiquitin and FLAG-HIF-1α plasmids directly confirmed the involvement of ubiquitin in the hypoxic degradation of HIF-1α. Additionally, hypoxic HIF-1 α degradation is independent of HAF, RACK1, sumoylation or nuclear/cytoplasmic localization. Taken together, our data suggest that constitutive HIF-1α protein degradation in hypoxia is absolutely ubiquitination-dependent, and unidentified E3 ligase may exist for this degradation pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. The stimulating effects of polyphenol and protein fractions from jelly fig (Ficus awkeotsang Makino achenes against proliferation of leukemia cells

    Directory of Open Access Journals (Sweden)

    Yi-Zhen Shih

    2017-10-01

    Full Text Available This study aimed to investigate the direct and immune-stimulated antiproliferative activities of jelly fig achenes fractions including pectinesterase inhibitors, crude polyphenols extract, and purified polyphenols extract (PP. Beside the measurement of cell viability of U937, the quantity of cytokines in conditioned medium and morphologic changes in leukemia were observed. After surveying all fractions in jelly fig, the obtained fractions of polyphenol exhibited the highest stimulating effects and directly cytotoxic effects against leukemia with the lowest effect found in protein fractions. The leukemia treated by our PP fraction showed dose-dependent response between the concentration and G2/M cell numbers of the U937 cells. The PP fraction had more pronounced effect on immune-stimulated than direct antiproliferative activities. The finding was also supported by morphological analysis by showing the formation of apoptotic bodies and differentiation from immature U937 cells into mature monocytes/macrophages on cells cultured with PP-conditioned medium. In conclusion, polyphenol fraction of pectinesterase inhibitors from jelly fig showed the immune-stimulated antiproliferative activities against U937 cell.

  12. Rift Valley fever virus NSs inhibits host transcription independently of the degradation of dsRNA-dependent Protein Kinase PKR

    OpenAIRE

    Kalveram, Birte; Lihoradova, Olga; Indran, Sabarish V.; Lokugamage, Nandadeva; Head, Jennifer A.; Ikegami, Tetsuro

    2012-01-01

    Rift Valley fever virus (RVFV) encodes one major virulence factor, the NSs protein. NSs suppresses host general transcription, including interferon (IFN)-β mRNA synthesis, and promotes degradation of the dsRNA-dependent protein kinase (PKR). We generated a novel RVFV mutant (rMP12-NSsR173A) specifically lacking the function to promote PKR degradation. rMP12-NSsR173A infection induces early phosphorylation of eIF2α through PKR activation, while retaining the function to inhibit host general tr...

  13. Nitrogen 15 abundance in protein fractions of beans fertilized with (15NH4)2SO4

    International Nuclear Information System (INIS)

    Chaud, Saula Goulart; Oliveira, Admar Costa de; Trivelin, Paulo Cesar Ocheuze

    2002-01-01

    Studies evaluating the protein nutritive value of beans labelled with 15 N, using nitrogen balance and the quantitation of faecal and urinary endogenous nitrogen, determined by isotopic dilution, have been extensively used. The objective of this research was to verify if the isotopic labelling of raw, freeze dried beans (Phaseolus vulgaris L., cultivar Pirata 1) with 1.394 atoms % 15 N, resulted in the same abundance of the whole flour and of the protein fractions extracted from the beans with 0.5 mol L -1 NaCl. The isotopic abundance found in the whole bean flour, in the protein extract, in the globulin and albumin fractions were respectively: 1.394 +- 0.011; 1.403 +- 0.012; 1.399 +- 0.007 and 1.399 +- 0.028 atoms % of 15 N, presenting no difference (P > 0.05). However, a difference was found (P < 0.05) between the above mentioned abundances and the isotopic abundance found in the nitrogen of the proteins in the extraction residue, which was 0.969 +- 0.084. Since the abundances did not differ, the protein nutritive indexes, such as digestibility and biological value, determined from the nitrogen balance and corrected for isotopic dilution, would not be affected by extracting the proteins from the beans with 0.5 mol L 1 NaCl. If working with the nitrogen balance of the residual proteins after extraction and even with the whole flours, these indexes could present incorrect values, since the isotopic labelling of the residual proteins was less than that of the protein fractions. (author)

  14. Memory formation for trace fear conditioning requires ubiquitin-proteasome mediated protein degradation in the prefrontal cortex.

    Directory of Open Access Journals (Sweden)

    David S Reis

    2013-10-01

    Full Text Available The cellular mechanisms supporting plasticity during memory consolidation have been a subject of considerable interest. De novo protein and mRNA synthesis in several brain areas are critical, and more recently protein degradation, mediated by the ubiquitin-proteasome system (UPS, has been shown to be important. Previous work clearly establishes a relationship between protein synthesis and protein degradation in the amygdala, but it is unclear whether cortical mechanisms of memory consolidation are similar to those in the amygdala. Recent work demonstrating a critical role for prefrontal cortex (PFC in the acquisition and consolidation of fear memory allows us to address this question. Here we use a PFC-dependent fear conditioning protocol to determine whether UPS mediated protein degradation is necessary for memory consolidation in PFC. Groups of rats were trained with auditory delay or trace fear conditioning and sacrificed 60 min after training. PFC tissue was then analyzed to quantify the amount of polyubiquinated protein. Other animals were trained with similar procedures but were infused with either a proteasome inhibitor (clasto-lactacystin β-lactone or a translation inhibitor (anisomycin in the PFC immediately after training. Our results show increased UPS-mediated protein degradation in the PFC following trace but not delay fear conditioning. Additionally, post-training proteasome or translation inhibition significantly impaired trace but not delay fear memory when tested the next day. Our results further support the idea that the PFC is critical for trace but not delay fear conditioning highlight the role of UPS-mediated degradation as critical for synaptic plasticity.

  15. Neuroprotective mechanism of Kai Xin San: upregulation of hippocampal insulin-degrading enzyme protein expression and acceleration of amyloid-beta degradation

    Directory of Open Access Journals (Sweden)

    Na Wang

    2017-01-01

    Full Text Available Kai Xin San is a Chinese herbal formula composed of Radix Ginseng , Poria , Radix Polygalae and Acorus Tatarinowii Rhizome . It has been used in China for many years for treating amnesia. Kai Xin San ameliorates amyloid-β (Aβ-induced cognitive dysfunction and is neuroprotective in vivo , but its precise mechanism remains unclear. Expression of insulin-degrading enzyme (IDE, which degrades Aβ, is strongly correlated with cognitive function. Here, we injected rats with exogenous Aβ42 (200 μM, 5 μL into the hippocampus and subsequently administered Kai Xin San (0.54 or 1.08 g/kg/d intragastrically for 21 consecutive days. Hematoxylin-eosin and Nissl staining revealed that Kai Xin San protected neurons against Aβ-induced damage. Furthermore, enzyme-linked immunosorbent assay, western blot and polymerase chain reaction results showed that Kai Xin San decreased Aβ42 protein levels and increased expression of IDE protein, but not mRNA, in the hippocampus. Our findings reveal that Kai Xin San facilitates hippocampal Aβ degradation and increases IDE expression, which leads, at least in part, to the alleviation of hippocampal neuron injury in rats.

  16. Decomposition of insoluble and hard-to-degrade animal proteins by enzyme E77 and its potential applications.

    Science.gov (United States)

    Zhao, Hui; Mitsuiki, Shinji; Takasugi, Mikako; Sakai, Masashi; Goto, Masatoshi; Kanouchi, Hiroaki; Oka, Tatsuzo

    2012-04-01

    Insoluble and hard-to-degrade animal proteins are group of troublesome proteins, such as collagen, elastin, keratin, and prion proteins that are largely generated by the meat industry and ultimately converted to industrial wastes. We analyzed the ability of the abnormal prion protein-degrading enzyme E77 to degrade insoluble and hard-to-degrade animal proteins including keratin, collagen, and elastin. The results indicate that E77 has a much higher keratinolytic activity than proteinase K and subtilisin. Maximal E77 keratinolytic activity was observed at pH 12.0 and 65 °C. E77 was also adsorbed by keratin in a pH-independent manner. E77 showed lower collagenolytic and elastinolytic specificities than proteinase K and subtilisin. Moreover, E77 treatment did not damage collagens in ovine small intestines but did almost completely remove the muscles. We consider that E77 has the potential ability for application in the processing of animal feedstuffs and sausages.

  17. Effect of boiled oil as dietary supplements for Japanese Quail on serum protein fractions and intestinal and hepatic cells

    International Nuclear Information System (INIS)

    Faramawy, A.A.; Soliman, S.M.; Fahmy, Y.M.O.

    2006-01-01

    This study was designed to examine the levels of serum protein fractions and testosterone, in addition to histopathological changes of small intestine and liver of Japanese quail following feeding with diets containing different concentrations of boiled oil (BO). Male Japanese quails (n=120), arranged into four groups each of three replicates, were supplemented with BO at 1%, 2% and 4% at the expense of 4% cotton seed oil (CSO). At the end of the experiment (10 weeks), three birds from each replicate were slaughtered and serum, small intestine and liver were collected for the determination of total testosterone, total protein, albumin and globulin fractions and fat studying the histology of small intestine and liver. The data revealed that feeding with BO led to decrease of total proteins and β-globulins in addition to cellular damages of small intestine and liver. This effect was increased with increasing the BO concentration in the diet

  18. Fractionation, solid-phase immobilization and chemical degradation of long pectin oligogalacturonides. Initial steps towards sequencing of oligosaccharides

    DEFF Research Database (Denmark)

    Guillaumie, Fanny; Justesen, Sune Frederik Lamdahl; Mutenda, K.E.

    2006-01-01

    This work presents the optimized separation of pectin oligomers, their analysis by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), their subsequent immobilization to supports, and our initial steps towards solid-support assisted sequencing. The ambient...... were produced in excellent purity (> 95%). Elution of OGAs followed by direct analysis of the peak fractions by MALDI-TOF MS. Purified OGAs (DP 5-7) were chemoselectively immobilized onto aminooxy-terminated polyethylene glycol polyacrylamide (PEGA) supports. Solid-phase anchoring took place...... at the reducing end of the oligosaccharide and resulted in the formation of an oxime linkage. The very high coupling yields confirmed the general suitability of aminooxy-PEGA resins for the immobilization of OGAs of different lengths. The OGA-functionalized PEGA supports were subsequently treated with aq TFA...

  19. Investigating ER-Associated Degradation with RNAi Screening - and Searching for Model Proteins to Do It with

    DEFF Research Database (Denmark)

    Jensen, Njal Winther

    Abstract In eukaryotes, secretory proteins are translocated into the endoplasmic reticulum (ER) for folding assistance, acquisition of posttranslational modifications and sorting. Proteins that do not obtain their native conformation are eliminated by ER-associated degradation (ERAD). ERAD...... is a sophisticated pathway that recognizes misfolded proteins and targets them for degradation by the 26S proteasome residing in the cytosol. More than 60 diseases including Alzheimer’s disease, Huntington’s disease and Parkinson’s disease have been linked to the ERAD pathway underscoring its crucial role...... for cellular homeostasis. The aim of this thesis has been to gain insight into ERAD. The experimental approach was RNAi screening, which is a fast and efficient method for initial evaluation of a large pool of genes. Since relatively few proteins routinely are used as ERAD substrates, the first goal...

  20. Comparison of protein degradation, protein oxidation, and μ-calpain activation between pale, soft, and exudative and red, firm, and nonexudative pork during postmortem aging.

    Science.gov (United States)

    Yin, Y; Zhang, W G; Zhou, G H; Guo, B

    2014-08-01

    The objective of this study was to investigate the differences in protein modifications between pale, soft, and exudative (PSE) and red, firm, and nonexudative (RFN) pork during postmortem (PM) aging. Longissimus dorsi (LD) including 8 PSE and 8 RFN muscles were individually removed from 16 carcasses. These 16 LD muscles were vacuum packaged at 24 h after slaughter and stored at 4°C for 1, 3, and 5 d. The centrifugation loss, drip loss, color, protein solubility, protein oxidation, protein degradation including desmin, troponin T, and integrin, and μ-calpain activation were determined. The pH of PSE samples was significantly lower than that of RFN samples at both 1 and 24 h PM (P 0.05). In addition, PSE pork presented a lower solubility of sarcoplasmic protein, myofibrillar protein, and total protein than RFN pork except the solubility of myofibrillar protein at d 1 (P firm, and nonexudative pork presented lower intensity of intact 80 kDa calpain and greater intensity of autolyzed 76 kDa product compared to PSE pork (P < 0.01). The results indicate that the degree of μ-calpain activation, the extent of protein degradation including desmin and integrin, and the level of protein solubility in PSE pork could contribute to its low water holding capacity during PM storage.

  1. Effect of Rapid Chilling on Beef Quality and Cytoskeletal Protein Degradation in of Chinese Yellow Crossbred Bulls

    Directory of Open Access Journals (Sweden)

    Yanwei Mao

    2012-08-01

    Full Text Available The objective of this study was to investigate the effect of rapid chilling (RC on beef quality and the degradation of cytoskeletal proteins. Twenty Chinese Yellow crossbred bulls were selected and randomly divided into two groups. RC and conventional chilling (CC were applied to left and right sides of the carcasses respectively after slaughtering. To determine whether electrical stimulation (ES treatment can alleviate the potential hazard of RC on meat quality, ES was applied to one group. The effects of RC and ES were determined by meat color, shear force and cytoskeletal protein degradation postmortem (PM. The results showed that RC decreased beef tenderness at 1 d and 3 d postmortem, but had no detrimental effect on meat color. Western blotting showed that RC decreased the degradation rate of desmin and troponin-T, but the effects weakened gradually as postmortem aging extended. Degradation rates of both desmin and troponin-T were accelerated by ES. The combination of RC and ES could improve beef color, accelerate degradation rate of cytoskeletal protein and improve beef tenderness.

  2. Plasma urea nitrogen and progesterone concentrations and follicular dynamics in ewes fed proteins of different degradability

    Directory of Open Access Journals (Sweden)

    Gustavo Bianchi Lazarin

    2012-07-01

    Full Text Available The effects of overfeeding with protein of different degradability on body condition, plasma urea nitrogen and progesterone concentrations, ovulation number and follicular dynamics were assessed in Santa Ines ewes. Twelve ewes were assigned to a randomized block design according to body weight and received overfeeding with soybean meal or with corn gluten meal or maintenance diet for 28 days before ovulation and during the next estrous cycle. Blood samples were taken on days 7, 14, 21, and 28 after the beginning of treatments for analysis of plasma urea nitrogen and on days 3, 6, 9, 12, and 15 into the estrous cycle for analysis of plasma urea nitrogen and progesterone. Follicular dynamics was monitored daily by ultrasound during one estrous cycle. Dry matter and crude protein intake, weight gain, plasma urea nitrogen concentration before ovulation, number of ovulations, diameter of the largest follicle of the 1st and of the 2nd waves and the growth rate of the largest follicle of the 1st wave were higher in the ewes that received overfeeding. The growth rate of the largest follicle of the 3rd wave was higher in the ewes fed maintenance diet. The back fat thickness, plasma urea nitrogen before ovulation and progesterone concentrations, diameter of the largest follicle of the 2nd wave and growth rate of the largest follicle of the 3rd wave were higher in ewes that received overfeeding with soybean meal. The growth rate of the largest follicle of the 1st wave was higher in ewes that received overfeeding with corn gluten meal. Overfeeding with protein-rich feeds may increase the ovulation number and with soybean meal, it may be effective in increasing plasma progesterone concentration in ewes.

  3. Trafficking and degradation pathways in pathogenic conversion of prions and prion-like proteins in neurodegenerative diseases.

    Science.gov (United States)

    Victoria, Guiliana Soraya; Zurzolo, Chiara

    2015-09-02

    Several neurodegenerative diseases such as transmissible spongiform encephalopathies, Alzheimer's and Parkinson's diseases are caused by the conversion of cellular proteins to a pathogenic conformer. Despite differences in the primary structure and subcellular localization of these proteins, which include the prion protein, α-synuclein and amyloid precursor protein (APP), striking similarity has been observed in their ability to seed and convert naïve protein molecules as well as transfer between cells. This review aims to cover what is known about the intracellular trafficking of these proteins as well as their degradation mechanisms and highlight similarities in their movement through the endocytic pathway that could contribute to the pathogenic conversion and seeding of these proteins which underlies the basis of these diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Antimutagenic and antioxidant activity of a protein fraction from aerial parts of Urtica dioica.

    Science.gov (United States)

    Di Sotto, Antonella; Mazzanti, Gabriela; Savickiene, Nijole; Staršelskytė, Rasa; Baksenskaite, Vaida; Di Giacomo, Silvia; Vitalone, Annabella

    2015-06-01

    Urtica dioica L. (Urticaceae), stinging nettle, has been employed as a folklore remedy for a wide spectrum of ailments, including urinary disorders, prostatic hyperplasia, and liver diseases. It has been also used traditionally for cancer treatment. To evaluate the potential chemopreventive properties of a protein fraction from the aerial part of Urtica dioica (namely UDHL30). UDHL30 has been tested for the antimutagenic activity in bacteria (50-800 μg/plate; Ames test by the preincubation method) and for the cytotoxicity on human hepatoma HepG2 cells (0.06-2 mg/mL; 24 and 48 h incubation). Moreover, the antioxidant activity of UDHL30 (0.1-1200 μg/mL; ABTS and superoxide-radical scavenger assays) was evaluated as potential protective mechanisms. UDHL30 was not cytotoxic on HepG2 cells up to 2 mg/mL; conversely, it exhibited a strong antimutagenic activity against the mutagen 2-aminoanthracene (2AA) in all strains tested (maximum inhibition of 56, 78, and 61% in TA98, TA100, and WP2uvrA strains, respectively, at 800 μg/plate). In addition, a remarkable scavenging activity against ABTS radical and superoxide anion (IC50 values of 19.9 ± 1.0 μg/mL and 75.3 ± 0.9 μg/mL, respectively) was produced. UDHL30 possesses antimutagenic and radical scavenging properties. Being 2AA a pro-carcinogenic agent, we hypothesize that the antimutagenicity of UDHL30 can be due to the inhibition of CYP450-isoenzymes, involved in the mutagen bioactivation. The radical scavenger ability could contribute to 2AA-antimutagenicity. These data encourage further studies in order to better define the potential usefulness of UDHL30 in chemoprevention.

  5. Precision-feeding dairy heifers a high rumen-degradable protein diet with different proportions of dietary fiber and forage-to-concentrate ratios.

    Science.gov (United States)

    Lascano, G J; Koch, L E; Heinrichs, A J

    2016-09-01

    The objective of this experiment was to determine the effects of feeding a high-rumen-degradable protein (RDP) diet when dietary fiber content is manipulated within differing forage-to-concentrate ratio (F:C) on nutrient utilization of precision-fed dairy heifers. Six cannulated Holstein heifers (486.98±15.07kg of body weight) were randomly assigned to 2 F:C, low- (45% forage; LF) and high-forage (90% forage; HF) diets and to a fiber proportion sequence [33% grass hay and wheat straw (HS), 67% corn silage (CS; low fiber); 50% HS, 50% CS (medium fiber); and 67% HS, 33% CS (high fiber)] within forage proportion administered according to a split-plot, 3×3 Latin square design (16-d periods). Heifers fed LF had greater apparent total-tract organic matter digestibility coefficients (dC), neutral detergent fiber, and cellulose than those fed LC diets. Substituting CS with HS resulted in a linear reduction in dry matter, organic matter, and cellulose dC. Nitrogen dC was not different between F:C or with increasing proportions of HS in diets, but N retention tended to decrease linearly as HS was increased in the diets. Predicted microbial protein flow to the duodenum decreased linearly with HS addition and protozoa numbers HS interacted linearly, exhibiting a decrease as HS increased for LF, whereas no effects were observed for HF. Blood urea N increased linearly as HS was incorporated. The LF-fed heifers had a greater ruminal volatile fatty acids concentration. We noted a tendency for a greater dry matter, and a significantly higher liquid fraction turnover rate for HF diets. There was a linear numerical increase in the liquid and solid fraction turnover rate as fiber was added to the diets. Rumen fermentation parameters and fractional passages (solid and liquid) rates support the reduction in dC, N retention, and microbial protein synthesis observed as more dietary fiber is added to the rations of dairy heifers precision-fed a constant proportion of rumen-degradable

  6. Molecular characterization and bio-functional property determination using SDS-PAGE and RP-HPLC of protein fractions from two Nigella species.

    Science.gov (United States)

    Alu'datt, Muhammad H; Rababah, Taha; Alhamad, Mohammad N; Alodat, Moh'd; Al-Mahasneh, Majdi A; Gammoh, Sana; Ereifej, Khalil; Almajwal, Ali; Kubow, Stan

    2017-09-01

    This study aimed to investigate the molecular and bio-functional properties of protein fractions from Nigella damascena and Nigella arvensis, including the albumin, globulin, glutein-1, glutein-2 and prolamin fractions. Protein subunits were not observed in globulin and prolamin fractions. No peaks appeared in RP-HPLC chromatograms of globulin for either species. Two predominant peaks were observed in the RP-HPLC profiles of all protein fractions. Proteins separated by RP-HPLC have potential inhibitory and antioxidant activities in all fractions. Optimum ACE-inhibitory and antioxidant activities of proteins separated by RP-HPLC were observed in glutein-2 and albumin, respectively, for both species. For pepsin and combined pepsin-trypsin hydrolyses, the highest degree of hydrolysis (DH) was obtained in glutein-2 fraction of Nigella arvensis. Highest ACE-inhibitory activity of hydrolyzed protein fractions was found at 4h via pepsin hydrolysis in globulin fraction of Nigella damascena. Highest antioxidant activities of hydrolyzed protein fractions were found in glutelin-2 for both species. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Protein degradation and post-deboning tenderization in broiler breast meat with different degrees of muscle shortening

    Science.gov (United States)

    Deboning broiler breast fillets prior to rigor mortis negatively influences tenderness due to sarcomere shortening. The effects of sarcomere shortening on muscle protein degradation and breast meat tenderization during post-deboning aging are not well understood. The objective of this study was to m...

  8. Fractionation of milk proteins on pilot scale with particular focus on β-casein

    NARCIS (Netherlands)

    Thienel, Katharina J.F.; Holder, Aline; Schubert, Thomas; Boom, Remko M.; Hinrichs, Jörg; Atamer, Zeynep

    2018-01-01

    The aim of this study was to increase the yield and purity of casein fractions at pilot scale and determine the main process parameters influencing the isolation of β-casein, such as cold extraction time, separation speed. The fractions were obtained from micellar casein by means of selective

  9. Determination of total antioxidant capacity of milk by CUPRAC and ABTS methods with separate characterisation of milk protein fractions.

    Science.gov (United States)

    Çekiç, Sema Demirci; Demir, Aslı; Başkan, Kevser Sözgen; Tütem, Esma; Apak, Reşat

    2015-05-01

    Most milk-applied antioxidant assays in literature are based on the isolation and quantification of individual antioxidative compounds, whereas total antioxidant capacity (TAC) gives a more holistic picture due to cooperative action of antioxidants. Recently, the cupric reducing antioxidant capacity (CUPRAC) method has been modified to measure the antioxidant capacities of thiol-containing proteins, where the classical ammonium acetate buffer - that may otherwise precipitate proteins- was replaced with concentrated urea buffer (able to expose embedded thiol groups of proteins to oxidative attack) adjusted to pH 7.0. Thus, antioxidant capacity of milk was investigated with two competing TAC assays, namely CUPRAC and ABTS (2,2'-azinobis(3-ethylbenzothiazoline-6-sulphonic acid))/persulphate, because only these assays were capable of evaluating protein contribution to the observed TAC value. As milk fat caused turbidity, experiments were carried out with skim milk or defatted milk samples. To determine TAC, modified CUPRAC method was applied to whole milk, separated and redissolved protein fractions, and the remaining liquid phase after necessary operations. Both TAC methods were investigated for their dilution sensitivity and antioxidant power assessment of separate milk fractions such as casein and whey. Proteins like β-lactoglobulin and casein (but not simple thiols) exhibited enhanced CUPRAC reactivity with surfactant (SDS) addition. Addition of milk protein fractions to whole skim milk produced significant 'negative-biased' deviations (up to -26% relative standard error) from TAC absorbance additivity in the application of the ABTS method, as opposed to that of the CUPRAC method less affected by chemical deviations from Beer's law thereby producing much smaller deviations from additivity (i.e. the property of additivity is valid when the measured TAC of a mixture is equal to the sum of individual antioxidant capacities of its constituents).

  10. Sonicated Protein Fractions of Mycoplasma hyopneumoniae Induce Inflammatory Responses and Differential Gene Expression in a Murine Alveolar Macrophage Cell Line.

    Science.gov (United States)

    Damte, Dereje; Lee, Seung-Jin; Birhanu, Biruk Tesfaye; Suh, Joo-Won; Park, Seung-Chun

    2015-12-28

    Mycoplasma hyopneumoniae is known to cause porcine enzootic pneumonia (EP), an important disease in swine production. The objective of this study was to examine the effects of sonicated protein fractions of M. hyopneumoniae on inflammatory response and gene expression in the murine alveolar macrophage MH-S cell line. The effects of sonicated protein fractions and intact M. hyopneumoniae on the gene expression of cytokines and iNOS were assessed using RT-PCR. The Annealing Control Primer (ACP)-based PCR method was used to screen differentially expressed genes. Increased transcription of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, COX-2, and iNOS mRNA was observed after exposure to the supernatant (SPT), precipitant (PPT), and intact M. hyopneumoniae protein. A time-dependent analysis of the mRNA expression revealed an upregulation after 4 h for IL-6 and iNOS and after 12 h for IL-1β and TNF-α, for both SPT and PPT; the fold change in COX-2 expression was less. A dose- and time-dependent correlation was observed in nitrite (NO) production for both protein fractions; however, there was no significant difference between the effects of the two protein fractions. In a differential gene analysis, PCR revealed differential expression for nine gene bands after 3 h of stimulation - only one gene was downregulated, while the remaining eight were upregulated. The results of this study provide insights that help improve our understanding of the mechanisms underlying the pathogenesis of and macrophage defenses against M. hyopneumoniae assault, and suggest targets for future studies on therapeutic interventions for M. hyopneumoniae infections.

  11. Wogonin inhibits tumor angiogenesis via degradation of HIF-1α protein

    International Nuclear Information System (INIS)

    Song, Xiuming; Yao, Jing; Wang, Fei; Zhou, Mi; Zhou, Yuxin; Wang, Hu; Wei, Libin; Zhao, Li; Li, Zhiyu; Lu, Na; Guo, Qinglong

    2013-01-01

    Wogonin, a plant-derived flavone, has been shown recently to have antitumor effects. However, the mechanisms that wogonin inhibits tumor angiogenesis are not well known. In this study, we investigated the effects of wogonin on expression of hypoxia-inducible factor-1α (HIF-1α) and secretion of vascular endothelial growth factor (VEGF) in tumor cells. We found that wogonin decreased the expression of HIF-1α by affecting its stability and reduced the secretion of VEGF, which suppressed angiogenesis in cancer. Wogonin promoted the degradation of HIF-1α by increasing its prolyl hydroxylation, which depended on prolyl hydroxylase (PHD) and the von Hippel–Lindau tumor suppressor (VHL). Intriguingly, wogonin impeded the binding between heat-shock protein 90 (Hsp90) and HIF-1α. In addition, wogonin down-regulated the Hsp90 client proteins EGFR, Cdk4 and survivin, but did not affect the level of Hsp90. Wogonin also increased ubiquitination of HIF-1α and promoted its degradation in proteasome. We also found that wogonin could inhibit nuclear translocation of HIF-1α. Electrophoresis mobility shift assay (EMSA) showed that wogonin decreased the binding activity of exogenous consensus DNA oligonucleotide with HIF-1α in nuclear extracts from MCF-7 cells. Chromatin immunoprecipitation (ChIP) assay also revealed that HIF-1α directly binded to endogenous hypoxia-responsive element (HRE) and this binding was significantly decreased in MCF-7 cells treated with wogonin. Preliminary results indicated in vivo activity of wogonin against xenograft-induced angiogenesis in nude mice. Taken together, the results suggested that wogonin was a potent inhibitor of HIF-1α and provided a new insight into the mechanisms of wogonin against cancers. - Highlights: • Wogonin is an all around inhibitor of VEGF signaling. • We firstly demonstrate that wogonin inhibits secretion of VEGF by decreasing HIF-1α. • Wogonin enhances PDH and VHL expression and inhibits Hsp90 function.

  12. Wogonin inhibits tumor angiogenesis via degradation of HIF-1α protein

    Energy Technology Data Exchange (ETDEWEB)

    Song, Xiuming; Yao, Jing; Wang, Fei; Zhou, Mi; Zhou, Yuxin; Wang, Hu; Wei, Libin; Zhao, Li; Li, Zhiyu; Lu, Na, E-mail: luna555@163.com; Guo, Qinglong, E-mail: anticancer_drug@yahoo.com.cn

    2013-09-01

    Wogonin, a plant-derived flavone, has been shown recently to have antitumor effects. However, the mechanisms that wogonin inhibits tumor angiogenesis are not well known. In this study, we investigated the effects of wogonin on expression of hypoxia-inducible factor-1α (HIF-1α) and secretion of vascular endothelial growth factor (VEGF) in tumor cells. We found that wogonin decreased the expression of HIF-1α by affecting its stability and reduced the secretion of VEGF, which suppressed angiogenesis in cancer. Wogonin promoted the degradation of HIF-1α by increasing its prolyl hydroxylation, which depended on prolyl hydroxylase (PHD) and the von Hippel–Lindau tumor suppressor (VHL). Intriguingly, wogonin impeded the binding between heat-shock protein 90 (Hsp90) and HIF-1α. In addition, wogonin down-regulated the Hsp90 client proteins EGFR, Cdk4 and survivin, but did not affect the level of Hsp90. Wogonin also increased ubiquitination of HIF-1α and promoted its degradation in proteasome. We also found that wogonin could inhibit nuclear translocation of HIF-1α. Electrophoresis mobility shift assay (EMSA) showed that wogonin decreased the binding activity of exogenous consensus DNA oligonucleotide with HIF-1α in nuclear extracts from MCF-7 cells. Chromatin immunoprecipitation (ChIP) assay also revealed that HIF-1α directly binded to endogenous hypoxia-responsive element (HRE) and this binding was significantly decreased in MCF-7 cells treated with wogonin. Preliminary results indicated in vivo activity of wogonin against xenograft-induced angiogenesis in nude mice. Taken together, the results suggested that wogonin was a potent inhibitor of HIF-1α and provided a new insight into the mechanisms of wogonin against cancers. - Highlights: • Wogonin is an all around inhibitor of VEGF signaling. • We firstly demonstrate that wogonin inhibits secretion of VEGF by decreasing HIF-1α. • Wogonin enhances PDH and VHL expression and inhibits Hsp90 function.

  13. Degradation kinetics of fisetin and quercetin in solutions affected by medium pH, temperature and co-existed proteins

    Directory of Open Access Journals (Sweden)

    Wang Jing

    2016-01-01

    Full Text Available Impacts of medium pH, temperature and coexisted proteins on the degradation of two flavonoids fisetin and quercetin were assessed by spectroscopic method in the present study. Based on the measured degradation rate constants (k, fisetin was more stable than quercetin in all cases. Increasing medium pH from 6.0 to 7.5 at 37°C enhanced respective k values of fisetin and quercetin from 8.30x10−3 and 2.81x10−2 to 0.202 and 0.375 h-1 (P<0.05. In comparison with their degradation at 37°C, fisetin and quercetin showed larger k values at higher temperature (0.124 and 0.245 h−1 at 50°C, or 0.490 and 1.42 h−1 at 65°C. Four protein products in medium could stabilize the two flavonoids (P<0.05, as these proteins at 0.10 g L-1 decreased respective k values of fisetin and quercetin to 2.28x10−2-2.98x10−2 and 4.37´10−2-5.97x10−2 h−1. Hydrophobic interaction between the proteins and the two flavonoids was evidenced responsible for the stabilization, as sodium dodecyl sulfate could destroy the stabilization significantly (P<0.05. Casein and soybean protein provided greater stabilization than whey protein isolate. It is thus concluded that higher temperature and alkaline pH can enhance flavonoid loss, whereas coexisted proteins as flavonoid stabilizers can inhibit flavonoid degradation.

  14. Fractional-order active fault-tolerant force-position controller design for the legged robots using saturated actuator with unknown bias and gain degradation

    Science.gov (United States)

    Farid, Yousef; Majd, Vahid Johari; Ehsani-Seresht, Abbas

    2018-05-01

    In this paper, a novel fault accommodation strategy is proposed for the legged robots subject to the actuator faults including actuation bias and effective gain degradation as well as the actuator saturation. First, the combined dynamics of two coupled subsystems consisting of the dynamics of the legs subsystem and the body subsystem are developed. Then, the interaction of the robot with the environment is formulated as the contact force optimization problem with equality and inequality constraints. The desired force is obtained by a dynamic model. A robust super twisting fault estimator is proposed to precisely estimate the defective torque amplitude of the faulty actuator in finite time. Defining a novel fractional sliding surface, a fractional nonsingular terminal sliding mode control law is developed. Moreover, by introducing a suitable auxiliary system and using its state vector in the designed controller, the proposed fault-tolerant control (FTC) scheme guarantees the finite-time stability of the closed-loop control system. The robustness and finite-time convergence of the proposed control law is established using the Lyapunov stability theory. Finally, numerical simulations are performed on a quadruped robot to demonstrate the stable walking of the robot with and without actuator faults, and actuator saturation constraints, and the results are compared to results with an integer order fault-tolerant controller.

  15. Misfolding, degradation, and aggregation of variant proteins. The molecular pathogenesis of short chain acyl-CoA dehydrogenase (SCAD) deficiency

    DEFF Research Database (Denmark)

    Pedersen, Christina Bak; Bross, P.; Winter, V.S.

    2003-01-01

    and aggregation of variant SCAD proteins. In this study we investigated the processing of a set of disease-causing variant SCAD proteins (R22W, G68C, W153R, R359C, and Q341H) and two common variant proteins (R147W and G185S) that lead to reduced SCAD activity. All SCAD proteins, including the wild type, associate...... proteolytic degradation by mitochondrial proteases or, especially at elevated temperature, aggregation of non-native conformers. The latter finding may indicate that accumulation of aggregated SCAD proteins may play a role in the pathogenesis of SCAD deficiency.......Short chain acyl-CoA dehydrogenase (SCAD) deficiency is an inborn error of the mitochondrial fatty acid metabolism caused by rare variations as well as common susceptibility variations in the SCAD gene. Earlier studies have shown that a common variant SCAD protein (R147W) was impaired in folding...

  16. Selective fermentation of carbohydrate and protein fractions of Scenedesmus, and biohydrogenation of its lipid fraction for enhanced recovery of saturated fatty acids.

    Science.gov (United States)

    Lai, YenJung Sean; Parameswaran, Prathap; Li, Ang; Aguinaga, Alyssa; Rittmann, Bruce E

    2016-02-01

    Biofuels derived from microalgae have promise as carbon-neutral replacements for petroleum. However, difficulty extracting microalgae-derived lipids and the co-extraction of non-lipid components add major costs that detract from the benefits of microalgae-based biofuel. Selective fermentation could alleviate these problems by managing microbial degradation so that carbohydrates and proteins are hydrolyzed and fermented, but lipids remain intact. We evaluated selective fermentation of Scenedesmus biomass in batch experiments buffered at pH 5.5, 7, or 9. Carbohydrates were fermented up to 45% within the first 6 days, protein fermentation followed after about 20 days, and lipids (measured as fatty acid methyl esters, FAME) were conserved. Fermentation of the non-lipid components generated volatile fatty acids, with acetate, butyrate, and propionate being the dominant products. Selective fermentation of Scenedesmus biomass increased the amount of extractable FAME and the ratio of FAME to crude lipids. It also led to biohydrogenation of unsaturated FAME to more desirable saturated FAME (especially to C16:0 and C18:0), and the degree of saturation was inversely related to the accumulation of hydrogen gas after fermentation. Moreover, the microbial communities after selective fermentation were enriched in bacteria from families known to perform biohydrogenation, i.e., Porphyromonadaceae and Ruminococcaceae. Thus, this study provides proof-of-concept that selective fermentation can improve the quantity and quality of lipids that can be extracted from Scenedesmus. © 2015 Wiley Periodicals, Inc.

  17. The highly antigenic 53/25 kDa Taenia solium protein fraction with cathepsin-L like activity is present in the oncosphere/cysticercus and induces non-protective IgG antibodies in pigs

    Science.gov (United States)

    Zimic, Mirko; Pajuelo, Mónica; Gilman, Robert H.; Gutiérrez, Andrés H.; Rueda, Luis D.; Flores, Myra; Chile, Nancy; Verástegui, Manuela; Gonzalez, Armando; García, Héctor H.; Sheen, Patricia

    2011-01-01

    Cathepsin L-like proteases are secreted by several parasites including Taenia solium. The mechanism used by T. solium oncospheres to degrade and penetrate the intestine and infect the host is incompletely understood. It is assumed that intestinal degradation is driven by the proteolytic activity of enzymes secreted by the oncosphere. Blocking the proteolytic activity by an antibody response would prevent the oncosphere penetration and further infection. Serine and cysteine proteases including chymotrypsin, trypsin, elastase, and cathepsin L, are secreted by T. solium and Taenia saginata oncospheres when cultured in vitro, being potential vaccine candidates. However, the purification of a sufficient quantity of proteases secreted by oncospheres to conduct a vaccine trial is costly and lengthy. A 53/25 kDa cathepsin L-like fraction partially purified from T. solium cyst fluid was described previously as an important antigen for immunodiagnostics. In this study we found that this antigen is present in the T. solium oncosphere and is also secreted by the cysticercus. This protein fraction was tested for its ability to protect pigs against an oral challenge with T. solium oncospheres in a vaccine trial. IgG antibodies against the 53/25 kDa cathepsin L-like protein fraction were elicited in the vaccinated animals but did not confer protection. PMID:22119017

  18. Magnitude Differences in Bioactive Compounds, Chemical Functional Groups, Fatty Acid Profiles, Nutrient Degradation and Digestion, Molecular Structure, and Metabolic Characteristics of Protein in Newly Developed Yellow-Seeded and Black-Seeded Canola Lines.

    Science.gov (United States)

    Theodoridou, Katerina; Zhang, Xuewei; Vail, Sally; Yu, Peiqiang

    2015-06-10

    Recently, new lines of yellow-seeded (CS-Y) and black-seeded canola (CS-B) have been developed with chemical and structural alteration through modern breeding technology. However, no systematic study was found on the bioactive compounds, chemical functional groups, fatty acid profiles, inherent structure, nutrient degradation and absorption, or metabolic characteristics between the newly developed yellow- and black-seeded canola lines. This study aimed to systematically characterize chemical, structural, and nutritional features in these canola lines. The parameters accessed include bioactive compounds and antinutrition factors, chemical functional groups, detailed chemical and nutrient profiles, energy value, nutrient fractions, protein structure, degradation kinetics, intestinal digestion, true intestinal protein supply, and feed milk value. The results showed that the CS-Y line was lower (P ≤ 0.05) in neutral detergent fiber (122 vs 154 g/kg DM), acid detergent fiber (61 vs 99 g/kg DM), lignin (58 vs 77 g/kg DM), nonprotein nitrogen (56 vs 68 g/kg DM), and acid detergent insoluble protein (11 vs 35 g/kg DM) than the CS-B line. There was no difference in fatty acid profiles except C20:1 eicosenoic acid content (omega-9) which was in lower in the CS-Y line (P structure spectral profile, there were no significant differences in functional groups of amides I and II, α helix, and β-sheet structure as well as their ratio between the two new lines, indicating no difference in protein structure makeup and conformation between the two lines. In terms of energy values, there were significant differences in total digestible nutrient (TDN; 149 vs 133 g/kg DM), metabolizable energy (ME; 58 vs 52 MJ/kg DM), and net energy for lactation (NEL; 42 vs 37 MJ/kg DM) between CS-Y and CS-B lines. For in situ rumen degradation kinetics, the two lines differed in soluble fraction (S; 284 vs 341 g/kg CP), potential degradation fraction (D; 672 vs 590 g/kg CP), and effective degraded

  19. Effect of the Ethyl Acetate Fraction of Eugenia uniflora on Proteins Global Expression during Morphogenesis in Candida albicans.

    Science.gov (United States)

    Silva-Rocha, Walicyranison P; de Azevedo, Matheus F; Ferreira, Magda R A; da Silva, Julhiany de Fátima; Svidzinski, Terezinha I E; Milan, Eveline P; Soares, Luiz A L; Rocha, Keyla B F; Uchôa, Adriana F; Mendes-Giannini, Maria J S; Fusco Almeida, Ana M; Chaves, Guilherme M

    2017-01-01

    Candida albicans is able to switch from yeast to hyphal growth and this is an essential step for tissue invasion and establishment of infection. Due to the limited drug arsenal used to treat fungal infections and the constant emergence of resistant strains, it is important to search for new therapeutic candidates. Therefore, this study aimed to investigate by proteomic analysis the role of a natural product ( Eugenia uniflora ) in impairing hypha formation in C. albicans . We also tested the potential action of E. uniflora to prevent and treat oral candidiasis induced in a murine model of oral infection and the ability of polymorphonuclear neutrophils to phagocytize C. albicans cells treated with the ethyl acetate fraction of the extract. We found that this fraction greatly reduced hypha formation after morphogenesis induction in the presence of serum. Besides, several proteins were differentially expressed in cells treated with the fraction. Surprisingly, the ethyl acetate fraction significantly reduced phagocytosis in C. albicans (Mean 120.36 ± 36.71 yeasts/100 PMNs vs. 44.68 ± 19.84 yeasts/100 PMNs). Oral candidiasis was attenuated when C. albicans cells were either pre-incubated in the presence of E. uniflora or when the fraction was applied to the surface of the oral cavity after infection. These results were consistent with the reduction in CFU counts (2.36 vs. 1.85 Log10 CFU/ml) and attenuation of tissue damage observed with histopathological analysis of animals belonging to treated group. We also observed shorter true hyphae by direct examination and histopathological analysis, when cells were treated with the referred natural product. The E. uniflora ethyl acetate fraction was non-toxic to human cells. E. uniflora may act on essential proteins mainly related to cellular structure, reducing the capacity of filamentation and attenuating infection in a murine model, without causing any toxic effect on human cells, suggesting that it may be a future

  20. Thermolysin damages animal life through degradation of plasma proteins enhanced by rapid cleavage of serpins and activation of proteases.

    Science.gov (United States)

    Kong, Lulu; Lu, Anrui; Guan, Jingmin; Yang, Bing; Li, Muwang; Hillyer, Julián F; Ramarao, Nalini; Söderhäll, Kenneth; Liu, Chaoliang; Ling, Erjun

    2015-01-01

    Thermolysin, a metallopeptidase secreted by pathogenic microbes, is concluded as an important virulence factor due to cleaving purified host proteins in vitro. Using the silkworm Bombyx mori as a model system, we found that thermolysin injection into larvae induces the destruction of the coagulation response and the activation of hemolymph melanization, which results in larval death. Thermolysin triggers the rapid degradation of insect and mammalian plasma proteins at a level that is considerably greater than expected in vitro and/or in vivo. To more specifically explore the mechanism, thermolysin-induced changes to key proteins belonging to the insect melanization pathway were assessed as a window for observing plasma protein cleavage. The application of thermolysin induced the rapid cleavage of the melanization negative regulator serpin-3, but did not directly activate the melanization rate-limiting enzyme prophenoloxidase (PPO) or the terminal serine proteases responsible for PPO activation. Terminal serine proteases of melanization are activated indirectly after thermolysin exposure. We hypothesize that thermolysin induces the rapid degradation of serpins and the activation of proteases directly or indirectly, boosting uncontrolled plasma protein degradation in insects and mammalians. © 2014 Wiley Periodicals, Inc.

  1. Different redox sensitivity of endoplasmic reticulum associated degradation clients suggests a novel role for disulphide bonds in secretory proteins.

    Science.gov (United States)

    Medraño-Fernandez, Iria; Fagioli, Claudio; Mezghrani, Alexandre; Otsu, Mieko; Sitia, Roberto

    2014-04-01

    To maintain proteostasis in the endoplasmic reticulum (ER), terminally misfolded secretory proteins must be recognized, partially unfolded, and dislocated to the cytosol for proteasomal destruction, in a complex process called ER-associated degradation (ERAD). Dislocation implies reduction of inter-chain disulphide bonds. When in its reduced form, protein disulphide isomerase (PDI) can act not only as a reductase but also as an unfoldase, preparing substrates for dislocation. PDI oxidation by Ero1 favours substrate release and transport across the ER membrane. Here we addressed the redox dependency of ERAD and found that DTT stimulates the dislocation of proteins with DTT-resistant disulphide bonds (i.e., orphan Ig-μ chains) but stabilizes a ribophorin mutant (Ri332) devoid of them. DTT promotes the association of Ri332, but not of Ig-µ, with PDI. This discrepancy may suggest that disulphide bonds in cargo proteins can be utilized to oxidize PDI, hence facilitating substrate detachment and degradation also in the absence of Ero1. Accordingly, Ero1 silencing retards Ri332 degradation, but has little if any effect on Ig-µ. Thus, some disulphides can increase the stability and simultaneously favour quality control of secretory proteins.

  2. SCF(SAP) controls organ size by targeting PPD proteins for degradation in Arabidopsis thaliana.

    Science.gov (United States)

    Wang, Zhibiao; Li, Na; Jiang, Shan; Gonzalez, Nathalie; Huang, Xiahe; Wang, Yingchun; Inzé, Dirk; Li, Yunhai

    2016-04-06

    Control of organ size by cell proliferation and growth is a fundamental process, but the mechanisms that determine the final size of organs are largely elusive in plants. We have previously revealed that the ubiquitin receptor DA1 regulates organ size by repressing cell proliferation in Arabidopsis. Here we report that a mutant allele of STERILE APETALA (SAP) suppresses the da1-1 mutant phenotype. We show that SAP is an F-box protein that forms part of a SKP1/Cullin/F-box E3 ubiquitin ligase complex and controls organ size by promoting the proliferation of meristemoid cells. Genetic analyses suggest that SAP may act in the same pathway with PEAPOD1 and PEAPOD2, which are negative regulators of meristemoid proliferation, to control organ size, but does so independently of DA1. Further results reveal that SAP physically associates with PEAPOD1 and PEAPOD2, and targets them for degradation. These findings define a molecular mechanism by which SAP and PEAPOD control organ size.

  3. In vivo imaging of brain ischemia using an oxygen-dependent degradative fusion protein probe.

    Directory of Open Access Journals (Sweden)

    Youshi Fujita

    Full Text Available Within the ischemic penumbra, blood flow is sufficiently reduced that it results in hypoxia severe enough to arrest physiological function. Nevertheless, it has been shown that cells present within this region can be rescued and resuscitated by restoring perfusion and through other protective therapies. Thus, the early detection of the ischemic penumbra can be exploited to improve outcomes after focal ischemia. Hypoxia-inducible factor (HIF-1 is a transcription factor induced by a reduction in molecular oxygen levels. Although the role of HIF-1 in the ischemic penumbra remains unknown, there is a strong correlation between areas with HIF-1 activity and the ischemic penumbra. We recently developed a near-infrared fluorescently labeled-fusion protein, POH-N, with an oxygen-dependent degradation property identical to the alpha subunit of HIF-1. Here, we conduct in vivo imaging of HIF-active regions using POH-N in ischemic brains after transient focal cerebral ischemia induced using the intraluminal middle cerebral artery occlusion technique in mice. The results demonstrate that POH-N enables the in vivo monitoring and ex vivo detection of HIF-1-active regions after ischemic brain injury and suggest its potential in imaging and drug delivery to HIF-1-active areas in ischemic brains.

  4. Variable-order fractional MSD function to describe the evolution of protein lateral diffusion ability in cell membranes

    Science.gov (United States)

    Yin, Deshun; Qu, Pengfei

    2018-02-01

    Protein lateral diffusion is considered anomalous in the plasma membrane. And this diffusion is related to membrane microstructure. In order to better describe the property of protein lateral diffusion and find out the inner relationship between protein lateral diffusion and membrane microstructure, this article applies variable-order fractional mean square displacement (f-MSD) function for characterizing the anomalous diffusion. It is found that the variable order can reflect the evolution of diffusion ability. The results of numerical simulation demonstrate variable-order f-MSD function can predict the tendency of anomalous diffusion during the process of confined diffusion. It is also noted that protein lateral diffusion ability during the processes of confined and hop diffusion can be split into three parts. In addition, the comparative analyses reveal that the variable order is related to the confinement-domain size and microstructure of compartment boundary too.

  5. Analyzing pepsin degradation assay conditions used for allergenicity assessments to ensure that pepsin susceptible and pepsin resistant dietary proteins are distinguishable.

    Directory of Open Access Journals (Sweden)

    Rong Wang

    Full Text Available The susceptibility of a dietary protein to proteolytic degradation by digestive enzymes, such as gastric pepsin, provides information on the likelihood of systemic exposure to a structurally intact and biologically active macromolecule, thus informing on the safety of proteins for human and animal consumption. Therefore, the purpose of standardized in vitro degradation studies that are performed during protein safety assessments is to distinguish whether proteins of interest are susceptible or resistant to pepsin degradation via a study design that enables study-to-study comparison. Attempting to assess pepsin degradation under a wide-range of possible physiological conditions poses a problem because of the lack of robust and consistent data collected under a large-range of sub-optimal conditions, which undermines the needs to harmonize in vitro degradation conditions. This report systematically compares the effects of pH, incubation time, and pepsin-to-substrate protein ratio on the relative degradation of five dietary proteins: three pepsin susceptible proteins [ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco, horseradish peroxidase (HRP, hemoglobin (Hb], and two pepsin resistant proteins [lipid transfer protein (LTP and soybean trypsin inhibitor (STI]. The results indicate that proteins susceptible to pepsin degradation are readily distinguishable from pepsin-resistant proteins when the reaction conditions are within the well-characterized optima for pepsin. The current standardized in vitro pepsin resistant assay with low pH and high pepsin-to-substrate ratio fits this purpose. Using non-optimal pH and/or pepsin-to-substrate protein ratios resulted in susceptible proteins no longer being reliably degraded by this stomach enzyme, which compromises the ability of this in vitro assay to distinguish between resistant and susceptible proteins and, therefore, no longer providing useful data to an overall weight-of-evidence approach to

  6. Rumen Degradability and Post-ruminal Digestion of Dry Matter, Nitrogen and Amino Acids of Three Protein Supplements

    Directory of Open Access Journals (Sweden)

    Wei Gao

    2015-04-01

    Full Text Available This study evaluated the in situ ruminal degradability, and subsequent small intestinal digestibility (SID of dry matter, crude protein (CP, and amino acids (AA of cottonseed meal (CSM, sunflower seed meal (SFSM and distillers dried grains with solubles (DDGS by using the modified three-step in vitro procedure. The ruminal degradability and subsequent SID of AA in rumen-undegradable protein (RUP-AA varied among three protein supplements. The result show that the effective degradability of DM for SFSM, CSM, and DDGS was 60.8%, 56.4%, and 41.0% and their ruminal fermentable organic matter was 60.0%, 55.9%, and 39.9%, respectively. The ruminal degradable protein (RDP content in CP for SFSM, CSM, and DDGS was 68.3%, 39.0%, and 32.9%, respectively, at the ruminal solid passage rate of 1.84%/h. The SFSM is a good source of RDP for rumen micro-organisms; however, the SID of RUP of SFSM was lower. The DDGS and CSM are good sources of RUP for lambs to digest in the small intestine to complement ruminal microbial AA of growing lambs. Individual RUP-AA from each protein source was selectively removed by the rumen micro-organisms, especially for Trp, Arg, His, and Lys (p<0.01. The SID of individual RUP-AA was different within specific RUP origin (p<0.01. Limiting amino acid was Leu for RUP of CSM and Lys for both RUP of SFSM and DDGS, respectively. Therefore, different protein supplements with specific limitations should be selected and combined carefully in growing lambs ration to optimize AA balance.

  7. Rumen Degradability and Post-ruminal Digestion of Dry Matter, Nitrogen and Amino Acids of Three Protein Supplements.

    Science.gov (United States)

    Gao, Wei; Chen, Aodong; Zhang, Bowen; Kong, Ping; Liu, Chenli; Zhao, Jie

    2015-04-01

    This study evaluated the in situ ruminal degradability, and subsequent small intestinal digestibility (SID) of dry matter, crude protein (CP), and amino acids (AA) of cottonseed meal (CSM), sunflower seed meal (SFSM) and distillers dried grains with solubles (DDGS) by using the modified three-step in vitro procedure. The ruminal degradability and subsequent SID of AA in rumen-undegradable protein (RUP-AA) varied among three protein supplements. The result show that the effective degradability of DM for SFSM, CSM, and DDGS was 60.8%, 56.4%, and 41.0% and their ruminal fermentable organic matter was 60.0%, 55.9%, and 39.9%, respectively. The ruminal degradable protein (RDP) content in CP for SFSM, CSM, and DDGS was 68.3%, 39.0%, and 32.9%, respectively, at the ruminal solid passage rate of 1.84%/h. The SFSM is a good source of RDP for rumen micro-organisms; however, the SID of RUP of SFSM was lower. The DDGS and CSM are good sources of RUP for lambs to digest in the small intestine to complement ruminal microbial AA of growing lambs. Individual RUP-AA from each protein source was selectively removed by the rumen micro-organisms, especially for Trp, Arg, His, and Lys (p<0.01). The SID of individual RUP-AA was different within specific RUP origin (p<0.01). Limiting amino acid was Leu for RUP of CSM and Lys for both RUP of SFSM and DDGS, respectively. Therefore, different protein supplements with specific limitations should be selected and combined carefully in growing lambs ration to optimize AA balance.

  8. F-Box Protein FBXO22 Mediates Polyubiquitination and Degradation of CD147 to Reverse Cisplatin Resistance of Tumor Cells.

    Science.gov (United States)

    Wu, Bo; Liu, Zhen-Yu; Cui, Jian; Yang, Xiang-Min; Jing, Lin; Zhou, Yang; Chen, Zhi-Nan; Jiang, Jian-Li

    2017-01-20

    Drug resistance remains a major clinical obstacle to successful treatment of cancer. As posttranslational modification is becoming widely recognized to affect the function of oncoproteins, targeting specific posttranslational protein modification provides an attractive strategy for anticancer drug development. CD147 is a transmembrane glycoprotein contributing to chemo-resistance of cancer cells in a variety of human malignancies. Ubiquitination is an important posttranslational modification mediating protein degradation. Degradation of oncoproteins, CD147 included, emerges as an attractive alternative for tumor inhibition. However, the ubiquitination of CD147 remains elusive. Here in this study, we found that deletion of the CD147 intracellular domain (CD147-ICD) prolonged the half-life of CD147 in HEK293T cells, and we identified that CD147-ICD interacts with FBXO22 using mass spectrometry and Western blot. Then, we demonstrated that FBXO22 mediates the polyubiquitination and degradation of CD147 by recognizing CD147-ICD. While knocking down of FBXO22 prolonged the half-life of CD147 in HEK293T cells, we found that FBXO22 regulates CD147 protein turnover in SMMC-7721, Huh-7 and A549 cells. Moreover, we found that the low level of FBXO22 contributes to the accumulation of CD147 and thereafter the cisplatin resistance of A549/DDP cells. To conclude, our study demonstrated that FBXO22 mediated the polyubiquitination and degradation of CD147 by interacting with CD147-ICD, and CD147 polyubiquitination by FBXO22 reversed cisplatin resistance of tumor cells.

  9. Rift Valley fever virus NSs inhibits host transcription independently of the degradation of dsRNA-dependent protein kinase PKR.

    Science.gov (United States)

    Kalveram, Birte; Lihoradova, Olga; Indran, Sabarish V; Lokugamage, Nandadeva; Head, Jennifer A; Ikegami, Tetsuro

    2013-01-20

    Rift Valley fever virus (RVFV) encodes one major virulence factor, the NSs protein. NSs suppresses host general transcription, including interferon (IFN)-β mRNA synthesis, and promotes degradation of the dsRNA-dependent protein kinase (PKR). We generated a novel RVFV mutant (rMP12-NSsR173A) specifically lacking the function to promote PKR degradation. rMP12-NSsR173A infection induces early phosphorylation of eIF2α through PKR activation, while retaining the function to inhibit host general transcription including IFN-β gene inhibition. MP-12 NSs but not R173A NSs binds to wt PKR. R173A NSs formed filamentous structure in nucleus in a mosaic pattern, which was distinct from MP-12 NSs filament pattern. Due to early phosphorylation of eIF2α, rMP12-NSsR173A could not efficiently accumulate viral proteins. Our results suggest that NSs-mediated host general transcription suppression occurs independently of PKR degradation, while the PKR degradation is important to inhibit the phosphorylation of eIF2α in infected cells undergoing host general transcription suppression. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. Distribution of Animal Drugs among Curd, Whey, and Milk Protein Fractions in Spiked Skim Milk and Whey.

    Science.gov (United States)

    Shappell, Nancy W; Shelver, Weilin L; Lupton, Sara J; Fanaselle, Wendy; Van Doren, Jane M; Hakk, Heldur

    2017-02-01

    It is important to understand the partitioning of drugs in processed milk and milk products, when drugs are present in raw milk, in order to estimate the potential consumer exposure. Radioisotopically labeled erythromycin, ivermectin, ketoprofen, oxytetracycline, penicillin G, sulfadimethoxine, and thiabendazole were used to evaluate the distribution of animal drugs among rennet curd, whey, and protein fractions from skim cow milk. Our previous work reported the distribution of these same drugs between skim and fat fractions of milk. Drug distribution between curd and whey was significantly correlated (R 2 = 0.70) to the drug's lipophilicity (log P), with improved correlation using log D (R 2 = 0.95). Distribution of drugs was concentration independent over the range tested (20-2000 nM). With the exception of thiabendazole and ivermectin, more drug was associated with whey protein than casein on a nmol/g protein basis (oxytetracycline experiment not performed). These results provide insights into the distribution of animal drug residues, if present in cow milk, among milk fractions, with possible extrapolation to milk products.

  11. Novel Phosphorylation and Ubiquitination Sites Regulate Reactive Oxygen Species-dependent Degradation of Anti-apoptotic c-FLIP Protein*

    Science.gov (United States)

    Wilkie-Grantham, Rachel P.; Matsuzawa, Shu-Ichi; Reed, John C.

    2013-01-01

    The cytosolic protein c-FLIP (cellular Fas-associated death domain-like interleukin 1β-converting enzyme inhibitory protein) is an inhibitor of death receptor-mediated apoptosis that is up-regulated in a variety of cancers, contributing to apoptosis resistance. Several compounds found to restore sensitivity of cancer cells to TRAIL, a TNF family death ligand with promising therapeutic potential, act by targeting c-FLIP ubiquitination and degradation by the proteasome. The generation of reactive oxygen species (ROS) has been implicated in c-FLIP protein degradation. However, the mechanism by which ROS post-transcriptionally regulate c-FLIP protein levels is not well understood. We show here that treatment of prostate cancer PPC-1 cells with the superoxide generators menadione, paraquat, or buthionine sulfoximine down-regulates c-FLIP long (c-FLIPL) protein levels, which is prevented by the proteasome inhibitor MG132. Furthermore, pretreatment of PPC-1 cells with a ROS scavenger prevented ubiquitination and loss of c-FLIPL protein induced by menadione or paraquat. We identified lysine 167 as a novel ubiquitination site of c-FLIPL important for ROS-dependent degradation. We also identified threonine 166 as a novel phosphorylation site and demonstrate that Thr-166 phosphorylation is required for ROS-induced Lys-167 ubiquitination. The mutation of either Thr-166 or Lys-167 was sufficient to stabilize c-FLIP protein levels in PPC-1, HEK293T, and HeLa cancer cells treated with menadione or paraquat. Accordingly, expression of c-FLIP T166A or K167R mutants protected cells from ROS-mediated sensitization to TRAIL-induced cell death. Our findings reveal novel ROS-dependent post-translational modifications of the c-FLIP protein that regulate its stability, thus impacting sensitivity of cancer cells to TRAIL. PMID:23519470

  12. Genome comparison implies the role of Wsm2 in membrane trafficking and protein degradation

    Directory of Open Access Journals (Sweden)

    Guorong Zhang

    2018-04-01

    Wsm2 may combat WSMV disease through a molecular mechanism involving protein degradation and/or membrane trafficking. The 93 putative Wsm2 ancestor loci discovered in this study could serve as good candidates for future genetic isolation of the true Wsm2 locus.

  13. Prolyl hydroxylation regulates protein degradation, synthesis, and splicing in human induced pluripotent stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Stoehr, Andrea; Yang, Yanqin; Patel, Sajni; Evangelista, Alicia M; Aponte, Angel; Wang, Guanghui; Liu, Poching; Boylston, Jennifer; Kloner, Philip H; Lin, Yongshun; Gucek, Marjan; Zhu, Jun; Murphy, Elizabeth

    2016-06-01

    Protein hydroxylases are oxygen- and α-ketoglutarate-dependent enzymes that catalyse hydroxylation of amino acids such as proline, thus linking oxygen and metabolism to enzymatic activity. Prolyl hydroxylation is a dynamic post-translational modification that regulates protein stability and protein-protein interactions; however, the extent of this modification is largely uncharacterized. The goals of this study are to investigate the biological consequences of prolyl hydroxylation and to identify new targets that undergo prolyl hydroxylation in human cardiomyocytes. We used human induced pluripotent stem cell-derived cardiomyocytes in combination with pulse-chase amino acid labelling and proteomics to analyse the effects of prolyl hydroxylation on protein degradation and synthesis. We identified 167 proteins that exhibit differences in degradation with inhibition of prolyl hydroxylation by dimethyloxalylglycine (DMOG); 164 were stabilized. Proteins involved in RNA splicing such as serine/arginine-rich splicing factor 2 (SRSF2) and splicing factor and proline- and glutamine-rich (SFPQ) were stabilized with DMOG. DMOG also decreased protein translation of cytoskeletal and sarcomeric proteins such as α-cardiac actin. We searched the mass spectrometry data for proline hydroxylation and identified 134 high confidence peptides mapping to 78 unique proteins. We identified SRSF2, SFPQ, α-cardiac actin, and cardiac titin as prolyl hydroxylated. We identified 29 prolyl hydroxylated proteins that showed a significant difference in either protein degradation or synthesis. Additionally, we performed next-generation RNA sequencing and showed that the observed decrease in protein synthesis was not due to changes in mRNA levels. Because RNA splicing factors were prolyl hydroxylated, we investigated splicing ± inhibition of prolyl hydroxylation and detected 369 alternative splicing events, with a preponderance of exon skipping. This study provides the first extensive

  14. Degradation of p53 by human Alphapapillomavirus E6 proteins shows a stronger correlation with phylogeny than oncogenicity.

    Directory of Open Access Journals (Sweden)

    Leiping Fu

    2010-09-01

    Full Text Available Human Papillomavirus (HPV E6 induced p53 degradation is thought to be an essential activity by which high-risk human Alphapapillomaviruses (alpha-HPVs contribute to cervical cancer development. However, most of our understanding is derived from the comparison of HPV16 and HPV11. These two viruses are relatively distinct viruses, making the extrapolation of these results difficult. In the present study, we expand the tested strains (types to include members of all known HPV species groups within the Alphapapillomavirus genus.We report the biochemical activity of E6 proteins from 27 HPV types representing all alpha-HPV species groups to degrade p53 in human cells. Expression of E6 from all HPV types epidemiologically classified as group 1 carcinogens significantly reduced p53 levels. However, several types not associated with cancer (e.g., HPV53, HPV70 and HPV71 were equally active in degrading p53. HPV types within species groups alpha 5, 6, 7, 9 and 11 share a most recent common ancestor (MRCA and all contain E6 ORFs that degrade p53. A unique exception, HPV71 E6 ORF that degraded p53 was outside this clade and is one of the most prevalent HPV types infecting the cervix in a population-based study of 10,000 women. Alignment of E6 ORFs identified an amino acid site that was highly correlated with the biochemical ability to degrade p53. Alteration of this amino acid in HPV71 E6 abrogated its ability to degrade p53, while alteration of this site in HPV71-related HPV90 and HPV106 E6s enhanced their capacity to degrade p53.These data suggest that the alpha-HPV E6 proteins' ability to degrade p53 is an evolved phenotype inherited from a most recent common ancestor of the high-risk species that does not always segregate with carcinogenicity. In addition, we identified an amino-acid residue strongly correlated with viral p53 degrading potential.

  15. Distribution of protein fraction in the milk of West African dwarf goat ...

    African Journals Online (AJOL)

    Milks from lactating West African dwarf goat and white Fulani cow were analysed for total protein, casein, whey protein, non protein nitrogen, Globulin and albumin and proteose peptone contents. Milk samples were from healthy goats and cows (n=24) in mid-lactation. The data were grouped and analysed as treatment 1 ...

  16. Degradation of vitamin C in a product made from mango (Mangifera indica L. and whey protein

    Directory of Open Access Journals (Sweden)

    Fernando Alonso Mendoza-Corvis

    2017-01-01

    Full Text Available This study aimed to determine the kinetics of vitamin C degradation in a product made from mango pulp (Mangifera indica L. and whey protein powder, in order to determine the effect of temperature on its conservation and further evaluate the behavior of the L*, a*, b* and the total color difference (ΔE in the powder product. Vitamin C was determined by the aoac 967.21/90 method using 2,6-dichlorophenol indophenol, and the color was quantified with a HunterLab Color Flex EZ colorimeter. Vitamin C showed greater stability in the powder product ResumoEsta pesquisa procurou determinar a cinética de degradação da vitamina C em um produto em pó elaborado a base de polpa de manga (Mangifera indica L. var. Hilacha e lactisoro, com o fim de conhecer o efeito da temperatura na sua conservação e ademais avaliar o comportamento dos parâmetros L*, a*, b* e a diferença total da cor (ΔE no produto. A vitamina C determinou-se mediante o método aoac 967.21/90, com 2,6-diclorofenol indofe-nol; enquanto que a cor se quantificou com um colorímetro cor Flex EZ marca HunterLab. A vitamina C exibiu maior estabilidade no produto em pó armazenado a uma temperatura de 4 °C, stored at 4 °C with a concentration at the end of the eighth sampling week of 13.94 ± 1.2 mg/10 0 g-1 sample and showing a first order degradation kinetics with k1 values of 0.014 and 0.041 mg/100 g/week at temperatures of 4 °C and 28 °C, respec-tively. The greatest variations in color occurred in samples stored at 28 °C, indicating the influence of the temperature change on the product compo-nents. In addition, L*, a* and b* parameters were less affected under storage temperature of 4 °C, and their values correspond to a second degree polynomial.

  17. Variation in ruminal in situ degradation of crude protein and starch from maize grains compared to in vitro gas production kinetics and physical and chemical characteristics.

    Science.gov (United States)

    Seifried, Natascha; Steingaß, Herbert; Schipprack, Wolfgang; Rodehutscord, Markus

    2016-10-01

    The objectives of this study were (1) to evaluate in situ ruminal dry matter (DM), crude protein (CP) and starch degradation characteristics and in vitro gas production (GP) kinetics using a set of 20 different maize grain genotypes and (2) to predict the effective degradation (ED) of CP and starch from chemical and physical characteristics alone or in combination with in vitro GP measurements. Maize grains were characterised by different chemical and physical characteristics. Ruminal in situ degradation was measured in three lactating Jersey cows. Ground grains (sieve size: 2 mm) were incubated in bags for 1, 2, 4, 8, 16, 24, 48 and 72 h. Bag residues were analysed for CP and starch content. Degradation kinetics was determined and the ED of DM, CP and starch calculated using a ruminal passage rate of 5%/h and 8%/h. The GP of the grains (sieve size: 1 mm) was recorded after 2, 4, 6, 8, 12, 24, 48 and 72 h incubation in buffered rumen fluid and fitted to an exponential equation to determine GP kinetics. Correlations and stepwise multiple linear regressions were evaluated for the prediction of ED calculated for a passage rate of 5%/h (ED5) for CP (EDCP5) and starch (EDST5). The in situ parameters and ED5 varied widely between genotypes with average values (±SD) of 64% ± 4.2, 62% ± 4.1 and 65% ± 5.2 for ED5 of DM, EDCP5 and EDST5 and were on average 10 percentage points lower for a passage rate of 8%/h. Degradation rates varied between 4.8%/h and 7.4%/h, 4.1%/h and 6.5%/h and 5.3%/h and 8.9%/h for DM, CP and starch, respectively. These rates were in the same range as GP rates (6.0-8.3%/h). The EDCP5 and EDST5 were related to CP concentration and could be evaluated in detail using CP fractions and specific amino acids. In vitro GP measurements and GP rates correlated well with EDCP5 and EDST5 and predicted EDCP5 and EDST5 in combination with the chemical characteristics of the samples. Equations can be used to obtain quick and cost effective information

  18. Changes in the protein fraction of Merluccius bilinearis muscle under lactic acid bacterial fermentation using a Lactobacillus Acidophilus starter culture (ESP

    Directory of Open Access Journals (Sweden)

    Luis J. Elizondo

    2016-03-01

    Full Text Available The effect of lactic acid bacterial fermentation on the protein fraction of Merluccius bilinearis muscle was evaluated. The non-protein fraction increased progressively with corresponding decreases in the percentage protein (dry weight indicating proteolytic activity during fermentation. Significant increases in the percentages of the amino acids cystine, isoleucine, phenylalanine and tyrosine were observed after two months of fermentation. Percentages of arginine decreased significantly after one week and again after two months of fermentation.

  19. Changes in the protein fraction of Merluccius bilinearis muscle under lactic acid bacterial fermentation using a Lactobacillus Acidophilus starter culture (ESP)

    OpenAIRE

    Elizondo, Luis J.

    2016-01-01

    The effect of lactic acid bacterial fermentation on the protein fraction of Merluccius bilinearis muscle was evaluated. The non-protein fraction increased progressively with corresponding decreases in the percentage protein (dry weight) indicating proteolytic activity during fermentation. Significant increases in the percentages of the amino acids cystine, isoleucine, phenylalanine and tyrosine were observed after two months of fermentation. Percentages of arginine decreased significantly aft...

  20. Comprehensive analysis of proteins of pH fractionated samples using monolithic LC/MS/MS, intact MW measurement and MALDI-QIT-TOF MS

    Science.gov (United States)

    Yoo, Chul; Patwa, Tasneem H.; Kreunin, Paweena; Miller, Fred R.; Huber, Christian G.; Nesvizhskii, Alexey I.; Lubman, David M.

    2012-01-01

    A comprehensive platform that integrates information from the protein and peptide levels by combining various MS techniques has been employed for the analysis of proteins in fully malignant human breast cancer cells. The cell lysates were subjected to chromatofocusing fractionation, followed by tryptic digestion of pH fractions for on-line monolithic RP-HPLC interfaced with linear ion trap MS analysis for rapid protein identification. This unique approach of direct analysis of pH fractions resulted in the identification of large numbers of proteins from several selected pH fractions, in which approximately 1.5 μg of each of the pH fraction digests was consumed for an analysis time of ca 50 min. In order to combine valuable information retained at the protein level with the protein identifications obtained from the peptide level information, the same pH fraction was analyzed using nonporous (NPS)-RP-HPLC/ESI-TOF MS to obtain intact protein MW measurements. In order to further validate the protein identification procedures from the fraction digest analysis, NPS-RP-HPLC separation was performed for off-line protein collection to closely examine each protein using MALDI-TOF MS and MALDI-quadrupole ion trap (QIT)-TOF MS, and excellent agreement of protein identifications was consistently observed. It was also observed that the comparison to intact MW and other MS information was particularly useful for analyzing proteins whose identifications were suggested by one sequenced peptide from fraction digest analysis. PMID:17206599

  1. Monitoring protein turnover during phosphate starvation-dependent autophagic degradation using a photoconvertible fluorescent protein aggregate in tobacco BY-2 cells.

    Science.gov (United States)

    Tasaki, Maiko; Asatsuma, Satoru; Matsuoka, Ken

    2014-01-01

    We have developed a system for quantitative monitoring of autophagic degradation in transformed tobacco BY-2 cells using an aggregate-prone protein comprised of cytochrome b5 (Cyt b5) and a tetrameric red fluorescent protein (RFP). Unfortunately, this system is of limited use for monitoring the kinetics of autophagic degradation because the proteins synthesized before and after induction of autophagy cannot be distinguished. To overcome this problem, we developed a system using kikume green-red (KikGR), a photoconvertible and tetrameric fluorescent protein that changes its fluorescence from green to red upon irradiation with purple light. Using the fusion protein of Cyt b5 and KikGR together with a method for the bulk conversion of KikGR, which we had previously used to convert the Golgi-localized monomeric KikGR fusion protein, we were able to monitor both the growth and de novo formation of aggregates. Using this system, we found that tobacco cells do not cease protein synthesis under conditions of phosphate (Pi)-starvation. Induction of autophagy under Pi-starvation, but not under sugar- or nitrogen-starvation, was specifically inhibited by phosphite, which is an analog of Pi with a different oxidation number. Therefore, the mechanism by which BY-2 cells can sense Pi-starvation and induce autophagy does not involve sensing a general decrease in energy supply and a specific Pi sensor might be involved in the induction of autophagy under Pi-starvation.

  2. Isolation and characterization of a tomato non-specific lipid transfer protein involved in polygalacturonase-mediated pectin degradation.

    Science.gov (United States)

    Tomassen, Monic M M; Barrett, Diane M; van der Valk, Henry C P M; Woltering, Ernst J

    2007-01-01

    An important aspect of the ripening process of tomato fruit is softening. Softening is accompanied by hydrolysis of the pectin in the cell wall by pectinases, causing loss of cell adhesion in the middle lamella. One of the most significant pectin-degrading enzymes is polygalacturonase (PG). Previous reports have shown that PG in tomato may exist in different forms (PG1, PG2a, PG2b, and PGx) commonly referred to as PG isoenzymes. The gene product PG2 is differentially glycosylated and is thought to associate with other proteins to form PG1 and PGx. This association is thought to modulate its pectin-degrading activity in planta. An 8 kDa protein that is part of the tomato PG1 multiprotein complex has been isolated, purified, and functionally characterized. This protein, designated 'activator' (ACT), belongs to the class of non-specific lipid transfer proteins (nsLTPs). ACT is capable of 'converting' the gene product PG2 into a more active and heat-stable form, which increases PG-mediated pectin degradation in vitro and stimulates PG-mediated tissue breakdown in planta. This finding suggests a new, not previously identified, function for nsLTPs in the modification of hydrolytic enzyme activity. It is proposed that ACT plays a role in the modulation of PG activity during tomato fruit softening.

  3. GSK3 is required for rapalogs to induce degradation of some oncogenic proteins and to suppress cancer cell growth.

    Science.gov (United States)

    Koo, Junghui; Wang, Xuerong; Owonikoko, Taofeek K; Ramalingam, Suresh S; Khuri, Fadlo R; Sun, Shi-Yong

    2015-04-20

    The single-agent activity of rapalogs (rapamycin and its analogues) in most tumor types has been modest at best. The underlying mechanisms are largely unclear. In this report, we have uncovered a critical role of GSK3 in regulating degradation of some oncogenic proteins induced by rapalogs and cell sensitivity to rapalogs. The basal level of GSK3 activity was positively correlated with cell sensitivity of lung cancer cell lines to rapalogs. GSK3 inhibition antagonized rapamycin's growth inhibitory effects both in vitro and in vivo, while enforced activation of GSK3β sensitized cells to rapamycin. GSK3 inhibition rescued rapamcyin-induced reduction of several oncogenic proteins such as cyclin D1, Mcl-1 and c-Myc, without interfering with the ability of rapamycin to suppress mTORC1 signaling and cap binding. Interestingly, rapamycin induces proteasomal degradation of these oncogenic proteins, as evidenced by their decreased stabilities induced by rapamcyin and rescue of their reduction by proteasomal inhibition. Moreover, acute or short-time rapamycin treatment dissociated not only raptor, but also rictor from mTOR in several tested cell lines, suggesting inhibition of both mTORC1 and mTORC2. Thus, induction of GSK3-dependent degradation of these oncogenic proteins is likely secondary to mTORC2 inhibition; this effect should be critical for rapamycin to exert its anticancer activity.

  4. Cyclin A degradation by primate cytomegalovirus protein pUL21a counters its innate restriction of virus replication.

    Directory of Open Access Journals (Sweden)

    Nicolas Caffarelli

    Full Text Available Cyclin A is critical for cellular DNA synthesis and S phase progression of the cell cycle. Human cytomegalovirus (HCMV can reduce cyclin A levels and block cellular DNA synthesis, and cyclin A overexpression can repress HCMV replication. This interaction has only been previously observed in HCMV as murine CMV does not downregulate cyclin A, and the responsible viral factor has not been identified. We previously reported that the HCMV protein pUL21a disrupted the anaphase-promoting complex (APC, but a point mutant abrogating this activity did not phenocopy a UL21a-deficient virus, suggesting that pUL21a has an additional function. Here we identified a conserved arginine-x-leucine (RxL cyclin-binding domain within pUL21a, which allowed pUL21a to interact with cyclin A and target it for proteasome degradation. Homologous pUL21a proteins from both chimpanzee and rhesus CMVs also contained the RxL domain and similarly degraded cyclin A, indicating that this function is conserved in primate CMVs. The RxL point mutation disabled the virus' ability to block cellular DNA synthesis and resulted in a growth defect similar to pUL21a-deficient virus. Importantly, knockdown of cyclin A rescued growth of UL21a-deficient virus. Together, these data show that during evolution, the pUL21a family proteins of primate CMVs have acquired a cyclin-binding domain that targets cyclin A for degradation, thus neutralizing its restriction on virus replication. Finally, the combined proteasome-dependent degradation of pUL21a and its cellular targets suggests that pUL21a may act as a novel suicide protein, targeting its protein cargos for destruction.

  5. Intracellular protein breakdown. 8

    International Nuclear Information System (INIS)

    Bohley, P.; Kirschke, H.; Langner, J.; Wiederanders, B.; Ansorge, S.

    1976-01-01

    Double-labelled proteins from rat liver cytosol ( 14 C in long-lived, 3 H in short-lived proteins after in-vivo-labelling) are used as substrates for unlabelled proteinases in vitro. Differences in the degradation rates of short-lived and long-lived proteins in vitro by different proteinases and after addition of different effectors allow conclusions concerning their importance for the in-vivo-turnover of substrate proteins. The main activity (>90%) of soluble lysosomal proteinases at pH 6.1 and pH 6.9 is caused by thiolproteinases, which degrade preferentially short-lived cytosol proteins. These proteinases are inhibited by leupeptin. Autolysis of double-labelled cell fractions shows a remarkably faster breakdown of short-lived substrate proteins only in the soluble part of lysosomes. Microsomal fractions degrade in vitro preferentially long-lived substrate proteins. (author)

  6. Characterization of Enzymatic Activity of MlrB and MlrC Proteins Involved in Bacterial Degradation of Cyanotoxins Microcystins.

    Science.gov (United States)

    Dziga, Dariusz; Zielinska, Gabriela; Wladyka, Benedykt; Bochenska, Oliwia; Maksylewicz, Anna; Strzalka, Wojciech; Meriluoto, Jussi

    2016-03-16

    Bacterial degradation of toxic microcystins produced by cyanobacteria is a common phenomenon. However, our understanding of the mechanisms of these processes is rudimentary. In this paper several novel discoveries regarding the action of the enzymes of the mlr cluster responsible for microcystin biodegradation are presented using recombinant proteins. In particular, the predicted active sites of the recombinant MlrB and MlrC were analyzed using functional enzymes and their inactive muteins. A new degradation intermediate, a hexapeptide derived from linearized microcystins by MlrC, was discovered. Furthermore, the involvement of MlrA and MlrB in further degradation of the hexapeptides was confirmed and a corrected biochemical pathway of microcystin biodegradation has been proposed.

  7. Characterization of Enzymatic Activity of MlrB and MlrC Proteins Involved in Bacterial Degradation of Cyanotoxins Microcystins

    Directory of Open Access Journals (Sweden)

    Dariusz Dziga

    2016-03-01

    Full Text Available Bacterial degradation of toxic microcystins produced by cyanobacteria is a common phenomenon. However, our understanding of the mechanisms of these processes is rudimentary. In this paper several novel discoveries regarding the action of the enzymes of the mlr cluster responsible for microcystin biodegradation are presented using recombinant proteins. In particular, the predicted active sites of the recombinant MlrB and MlrC were analyzed using functional enzymes and their inactive muteins. A new degradation intermediate, a hexapeptide derived from linearized microcystins by MlrC, was discovered. Furthermore, the involvement of MlrA and MlrB in further degradation of the hexapeptides was confirmed and a corrected biochemical pathway of microcystin biodegradation has been proposed.

  8. Protective protein/cathepsin A down-regulates osteoclastogenesis by associating with and degrading NF-kappaB p50/p65.

    Science.gov (United States)

    Masuhara, Masaaki; Sato, Takuya; Hada, Naoto; Hakeda, Yoshiyuki

    2009-01-01

    Disruption of the cooperative function balance between osteoblasts and osteoclasts causes various bone disorders, some of which are attributed to abnormal osteoclast recruitment. Osteoclast differentiation is dependent on the receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) as well as the macrophage colony-stimulating factor. The osteoclast formation induced by cytokines requires activation of NF-kappaB, AP-1 and nuclear factor of activated T cells c1. However, osteoclasts are not the only cell types that express these transcription factors, suggesting that some unknown molecules specific for osteoclasts may associate with the transcription factors. Here, we explored the possibility of molecules binding directly to NF-kappaB and cloned protective protein/cathepsin A (PPCA) by yeast two-hybrid screening using a cDNA library of osteoclast precursors. Forced expression of PPCA with p50/p65 in HEK293 cells decreased both the level of p50/p65 proteins and the transcriptional activity. Abundant PPCA was detected in the lysosomes of the transfected HEK293 cells, but a small amount of this enzyme was also present in the cytosolic fraction. In addition, over-expression of PPCA caused the disappearance of p50/p65 in both the lysosomal and cytosolic fractions. PPCA was expressed throughout osteoclastogenesis, and the expression was slightly up-regulated by RANKL signaling. Knockdown of PPCA in osteoclast precursors with PPCA siRNA stimulated binding of nuclear proteins to oligonucleotides containing an NF-kappaB binding motif and increased osteoclastogenesis. Our present results indicate a novel role for PPCA in osteoclastogenesis via down-regulation of NF-kappaB activity and suggest a new function for PPCA as an NF-kappaB-degrading enzyme in addition to its known multifunctional properties.

  9. Polymorphism in ovine ANXA9 gene and physic-chemical properties and the fraction of protein in milk.

    Science.gov (United States)

    Pecka-Kiełb, Ewa; Czerniawska-Piątkowska, Ewa; Kowalewska-Łuczak, Inga; Vasil, Milan

    2018-04-16

    Annexin A9 (ANXA9) is a specific fatty acid transport protein. ANXA9 gene is expressed in various tissues, including secretory tissue and mammary glands. The association between three SNPs of the ANXA9 gene and sheep's milk compositions was assessed. Genotype analysis was performed with the use of PCR-RFLP method. The studied ANXA9 polymorphisms had the following MAF (Major Allele Frequency): SNP1: allele G 0,66; SNP2: allele G 0,54; SNP3: allele C 0,57. The study found the most desired profile of protein fractions, namely an increased kappa-casein fractions and a decreased level of whey protein in sheep's milk for SNP1 and SNP3 polymorphisms. Sheep with the SNP1 GA genotype had the highest (P <0.05) content of fat and dry matter in milk. AXNA9 gene polymorphism did not influence the levels of protein, lactose or urea in sheep's milk. The information contained in this study may be useful for determining the impact of the ANXA9 gene on sheep's milk. The ANXA9 SNP1 and SNP3 polymorphisms results could be included in the breeding programs to select the sheep with the genotypes ensuring the highest kappa-casein levels in milk. However, it is worth conducting further research on ANXA9 and milk composition in larger herds of animals and various breeds of sheep. This article is protected by copyright. All rights reserved.

  10. Influence of different phenolic fractions on red wine astringency based on polyphenol/protein binding

    OpenAIRE

    Ren, M.; Wang, X.; Du, G.; Tian, C.; Zhang, J.; Song, X.; Zhu, D.

    2017-01-01

    The presence of phenolic compounds can make a great contribution to the perception of astringency in red wines based on their interactions with proteins. Human salivary protein and bovine serum albumin were used in this study to investigate the relationship between astringency and polyphenol composition. The interactions between polyphenols and proteins were analysed by means of electrophoresis and fluorescence spectra, and they were further confirmed by sensory analysis. The results indicate...

  11. HIV-1 tat protein recruits CIS to the cytoplasmic tail of CD127 to induce receptor ubiquitination and proteasomal degradation

    Energy Technology Data Exchange (ETDEWEB)

    Sugden, Scott, E-mail: scott.sugden@ircm.qc.ca [The Ottawa Hospital Research Institute, 501 Smyth Road, Ottawa, Ontario, Canada K1H 8L6 (Canada); Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5 (Canada); Ghazawi, Feras [The Ottawa Hospital Research Institute, 501 Smyth Road, Ottawa, Ontario, Canada K1H 8L6 (Canada); Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5 (Canada); MacPherson, Paul, E-mail: pmacpherson@toh.on.ca [The Ottawa Hospital Research Institute, 501 Smyth Road, Ottawa, Ontario, Canada K1H 8L6 (Canada); Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5 (Canada); Division of Infectious Diseases, The Ottawa Hospital General Campus, 501 Smyth Road, Ottawa, Ontario, Canada K1H 8L6 (Canada)

    2016-11-15

    HIV-1 Tat protein down regulates expression of the IL-7 receptor alpha-chain (CD127) from the surface of CD8 T cells resulting in impaired T cell proliferation and cytolytic capacity. We have previously shown that soluble Tat protein is taken up by CD8 T cells and interacts with the cytoplasmic tail of CD127 to induce receptor degradation. The N-terminal domain of Tat interacts with CD127 while the basic domain directs CD127 to the proteasome. We have also shown that upon IL-7 binding to its receptor, CD127 is phosphorylated resulting in CIS-mediated proteasomal degradation. Here, we show that Tat mimics this process by recruiting CIS to CD127 in the absence of IL-7 and receptor phosphorylation, leading to CD127 ubiquitination and degradation. Tat therefore acts as an adapter to induce cellular responses under conditions where they may not otherwise occur. Thusly, Tat reduces IL-7 signaling and impairs CD8 T cell survival and function. -- Highlights: •Soluble HIV-1 Tat decreases CD127 expression on CD8 T cells, causing dysfunction. •Tat induces CD127 ubiquitination without activating IL-7 signaling. •Tat binds CD127 and recruits the E3 ubiquitin ligase CIS via its basic domain. •Tat hijacks a normal cellular mechanism to degrade CD127 without IL-7 signaling.

  12. HIV-1 tat protein recruits CIS to the cytoplasmic tail of CD127 to induce receptor ubiquitination and proteasomal degradation

    International Nuclear Information System (INIS)

    Sugden, Scott; Ghazawi, Feras; MacPherson, Paul

    2016-01-01

    HIV-1 Tat protein down regulates expression of the IL-7 receptor alpha-chain (CD127) from the surface of CD8 T cells resulting in impaired T cell proliferation and cytolytic capacity. We have previously shown that soluble Tat protein is taken up by CD8 T cells and interacts with the cytoplasmic tail of CD127 to induce receptor degradation. The N-terminal domain of Tat interacts with CD127 while the basic domain directs CD127 to the proteasome. We have also shown that upon IL-7 binding to its receptor, CD127 is phosphorylated resulting in CIS-mediated proteasomal degradation. Here, we show that Tat mimics this process by recruiting CIS to CD127 in the absence of IL-7 and receptor phosphorylation, leading to CD127 ubiquitination and degradation. Tat therefore acts as an adapter to induce cellular responses under conditions where they may not otherwise occur. Thusly, Tat reduces IL-7 signaling and impairs CD8 T cell survival and function. -- Highlights: •Soluble HIV-1 Tat decreases CD127 expression on CD8 T cells, causing dysfunction. •Tat induces CD127 ubiquitination without activating IL-7 signaling. •Tat binds CD127 and recruits the E3 ubiquitin ligase CIS via its basic domain. •Tat hijacks a normal cellular mechanism to degrade CD127 without IL-7 signaling.

  13. Unique nonstructural proteins of Pneumonia Virus of Mice (PVM) promote degradation of interferon (IFN) pathway components and IFN-stimulated gene proteins.

    Science.gov (United States)

    Dhar, Jayeeta; Barik, Sailen

    2016-12-01

    Pneumonia Virus of Mice (PVM) is the only virus that shares the Pneumovirus genus of the Paramyxoviridae family with Respiratory Syncytial Virus (RSV). A deadly mouse pathogen, PVM has the potential to serve as a robust animal model of RSV infection, since human RSV does not fully replicate the human pathology in mice. Like RSV, PVM also encodes two nonstructural proteins that have been implicated to suppress the IFN pathway, but surprisingly, they exhibit no sequence similarity with their RSV equivalents. The molecular mechanism of PVM NS function, therefore, remains unknown. Here, we show that recombinant PVM NS proteins degrade the mouse counterparts of the IFN pathway components. Proteasomal degradation appears to be mediated by ubiquitination promoted by PVM NS proteins. Interestingly, NS proteins of PVM lowered the levels of several ISG (IFN-stimulated gene) proteins as well. These results provide a molecular foundation for the mechanisms by which PVM efficiently subverts the IFN response of the murine cell. They also reveal that in spite of their high sequence dissimilarity, the two pneumoviral NS proteins are functionally and mechanistically similar.

  14. Antioxidant and Antihypertensive Potential of Protein Fractions from Flour and Milk Substitutes from Canary Seeds (Phalaris canariensis L.).

    Science.gov (United States)

    Valverde, María Elena; Orona-Tamayo, Domancar; Nieto-Rendón, Blanca; Paredes-López, Octavio

    2017-03-01

    Canary seed (Phalaris canariensis) is used to feed birds but it has been recently considered a promising cereal with nutraceutical potential for humans. The aim of this work was to analyze the protein fractions from canary seed flour and from milk substitutes (prepared by soaking the seeds in water 12 and 24 h), and to evaluate antioxidant and antihypertensive capacity of peptides obtained after in vitro digestion. Prolamins were the major protein fraction, followed by glutelins. After digestion, albumins and prolamins fractions from milks presented higher levels of peptides than flour, globulins showed more peptides in flour and glutelins were found in similar concentrations in all samples; 24 h milk prolamins had the highest concentration of peptides. Purification by high performance liquid chromatography (HPLC), sequencing of peptides, in vitro antioxidant ABTS (2,2'-azino-bis, 3-ethylbenzothiazoline-6-sulphonic acid) and DPPH (2,2-diphenyl-1-picrylhydrazyl) assays, and antihypertensive capacity (angiotensin converting enzyme (ACE) assay), indicated that peptides from canary seed prolamins were the most efficient compounds with antioxidant and antihypertensive activity. Canary seeds may be considered an accessible and cheap source to prepare milk substitutes with high contents of bioactive peptides with remarkable functional properties to promote better human health and healthy ageing.

  15. Molecular characterization of whey protein hydrolysate fractions with ferrous chelating and enhanced iron solubility capabilities.

    Science.gov (United States)

    O'Loughlin, Ian B; Kelly, Phil M; Murray, Brian A; FitzGerald, Richard J; Brodkorb, Andre

    2015-03-18

    The ferrous (Fe2+) chelating capabilities of WPI hydrolysate fractions produced via cascade membrane filtration were investigated, specifically 1 kDa permeate (P) and 30 kDa retentate (R) fractions. The 1 kDa-P possessed a Fe2+ chelating capability at 1 g L(-1) equivalent to 84.4 μM EDTA (for 30 kDa-R the value was 8.7 μM EDTA). Fourier transformed infrared (FTIR) spectroscopy was utilized to investigate the structural characteristics of hydrolysates and molecular interactions with Fe2+. Solid-phase extraction was employed to enrich for chelating activity; the most potent chelating fraction was enriched in histidine and lysine. The solubility of ferrous sulfate solutions (10 mM) over a range of pH values was significantly (Piron solubility was improved by 72% in the presence of the 1 kDa-P fraction following simulated gastrointestinal digestion (SGID) compared to control FeSO4·7H2O solutions.

  16. NSs Virulence Factor of Rift Valley Fever Virus Engages the F-Box Proteins FBXW11 and β-TRCP1 To Degrade the Antiviral Protein Kinase PKR.

    Science.gov (United States)

    Kainulainen, Markus; Lau, Simone; Samuel, Charles E; Hornung, Veit; Weber, Friedemann

    2016-07-01

    Rift Valley fever virus (RVFV, family Bunyaviridae, genus Phlebovirus) is a relevant pathogen of both humans and livestock in Africa. The nonstructural protein NSs is a major virulence factor known to suppress the type I interferon (IFN) response by inhibiting host cell transcription and by proteasomal degradation of a major antiviral IFN effector, the translation-inhibiting protein kinase PKR. Here, we identified components of the modular SCF (Skp1, Cul1, F-box protein)-type E3 ubiquitin ligases as mediators of PKR destruction by NSs. Small interfering RNAs (siRNAs) against the conserved SCF subunit Skp1 protected PKR from NSs-mediated degradation. Consequently, RVFV replication was severely reduced in Skp1-depleted cells when PKR was present. SCF complexes have a variable F-box protein subunit that determines substrate specificity for ubiquitination. We performed an siRNA screen for all (about 70) human F-box proteins and found FBXW11 to be involved in PKR degradation. The partial stabilization of PKR by FBXW11 depletion upregulated PKR autophosphorylation and phosphorylation of the PKR substrate eIF2α and caused a shutoff of host cell protein synthesis in RVFV-infected cells. To maximally protect PKR from the action of NSs, knockdown of structurally and functionally related FBXW1 (also known as β-TRCP1), in addition to FBXW11 deletion, was necessary. Consequently, NSs was found to interact with both FBXW11 and β-TRCP1. Thus, NSs eliminates the antiviral kinase PKR by recruitment of SCF-type E3 ubiquitin ligases containing FBXW11 and β-TRCP1 as substrate recognition subunits. This antagonism of PKR by NSs is essential for efficient RVFV replication in mammalian cells. Rift Valley fever virus is a pathogen of humans and animals that has the potential to spread from Africa and the Arabian Peninsula to other regions. A major virulence mechanism is the proteasomal degradation of the antiviral kinase PKR by the viral protein NSs. Here, we demonstrate that NSs

  17. Protein identification and in vitro digestion of fractions from Tenebrio molitor

    NARCIS (Netherlands)

    Yi, Liya; Boekel, van M.A.J.S.; Boeren, Sjef; Lakemond, Catriona M.M.

    2016-01-01

    The nutritional value of insect protein is evaluated not only in amino acid composition, but also in protein digestibility. The general amino acid composition of Tenebrio molitor has been reported before, but limited knowledge is available on its digestibility. The objective of this study was to

  18. Semi-continuous protein fractionating using affinity cross-flow filtration

    NARCIS (Netherlands)

    Borneman, Zandrie; Zhang, W.; van den Boomgaard, Anthonie; Smolders, C.A.

    2002-01-01

    Protein purification by means of downstream processing is increasingly important. At the University of Twente a semi-continuous process is developed for the isolation of BSA out of crude protein mixtures. For this purpose an automated Affinity Cross-Flow Filtration, ACFF, process is developed. This

  19. Biorefinery methods for separation of protein and oil fractions from rubber seed kernel

    NARCIS (Netherlands)

    Widyarani, R.; Ratnaningsih, E.; Sanders, J.P.M.; Bruins, M.E.

    2014-01-01

    Biorefinery of rubber seeds can generate additional income for farmers, who already grow rubber trees for latex production. The aim of this study was to find the best method for protein and oil production from rubber seed kernel, with focus on protein recovery. Different pre-treatments and oil

  20. 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 ...

  1. Water binding of proteins in the processing frankfurter-type sausages. Part. 1. Water-binding ability of freeze-dried meat fractions containing myofibrillar and stromal proteins.

    Science.gov (United States)

    Heinevetter, L; Gassmann, B; Kroll, J

    1987-01-01

    As soon as possible and 48 h after slaughter respectively, from both blade-bone muscle groups of cattle and pig carcasses the "thick pieces" were excised, extracted, and fractionated. Residues and precipitates from water and salt extracts resulted were freeze-dried, and an improved Baumann capillary suction apparatus was used to measure their water binding capacity (WBC) with and without addition of 2% sodium chloride and/or heating to 80 degrees C. With one exception the WBC results followed a relative pattern demonstrating the final residues (stromal proteins and leavings of myofibrillar proteins) binding the highest amount of added water, precipitates of dialysis (mainly containing myofibrillar proteins) a remarkable amount and powdered meats the least. As scanning electron micrographs confirmed, there were no fibrous structures in the precipitates resulted from dialysis of salt solutions (1.0 mol/1). Heating decreased the spontaneous water uptake of all fractions. Addition of sodium chloride had only a noticeable capillary-suction and swelling effect on unheated samples. Hence swelling of undissolved protein structures (extraction of myosin and possibly of actomyosin) is therefore not the only way for water binding in frankfurter-type sausages.

  2. Vitamin C degradation products and pathways in the human lens.

    Science.gov (United States)

    Nemet, Ina; Monnier, Vincent M

    2011-10-28

    Vitamin C and its degradation products participate in chemical modifications of proteins in vivo through non-enzymatic glycation (Maillard reaction) and formation of different products called advanced glycation end products. Vitamin C levels are particularly high in selected tissues, such as lens, brain and adrenal gland, and its degradation products can inflict substantial protein damage via formation of advanced glycation end products. However, the pathways of in vivo vitamin C degradation are poorly understood. Here we have determined the levels of vitamin C oxidation and degradation products dehydroascorbic acid, 2,3-diketogulonic acid, 3-deoxythreosone, xylosone, and threosone in the human lens using o-phenylenediamine to trap both free and protein-bound adducts. In the protein-free fraction and water-soluble proteins (WSP), all five listed degradation products were identified. Dehydroascorbic acid, 2,3-diketogulonic acid, and 3-deoxythreosone were the major products in the protein-free fraction, whereas in the WSP, 3-deoxythreosone was the most abundant measured dicarbonyl. In addition, 3-deoxythreosone in WSP showed positive linear correlation with age (p degradation product bound to human lens proteins provides in vivo evidence for the non-oxidative pathway of dehydroascorbate degradation into erythrulose as a major pathway for vitamin C degradation in vivo.

  3. Betulinic acid selectively increases protein degradation and enhances prostate cancer-specific apoptosis: possible role for inhibition of deubiquitinase activity.

    Directory of Open Access Journals (Sweden)

    Teresita Reiner

    Full Text Available Inhibition of the ubiquitin-proteasome system (UPS of protein degradation is a valid anti-cancer strategy and has led to the approval of bortezomib for the treatment of multiple myeloma. However, the alternative approach of enhancing the degradation of oncoproteins that are frequently overexpressed in cancers is less developed. Betulinic acid (BA is a plant-derived small molecule that can increase apoptosis specifically in cancer but not in normal cells, making it an attractive anti-cancer agent. Our results in prostate cancer suggested that BA inhibited multiple deubiquitinases (DUBs, which resulted in the accumulation of poly-ubiquitinated proteins, decreased levels of oncoproteins, and increased apoptotic cell death. In normal fibroblasts, however, BA did not inhibit DUB activity nor increased total poly-ubiquitinated proteins, which was associated with a lack of effect on cell death. In the TRAMP transgenic mouse model of prostate cancer, treatment with BA (10 mg/kg inhibited primary tumors, increased apoptosis, decreased angiogenesis and proliferation, and lowered androgen receptor and cyclin D1 protein. BA treatment also inhibited DUB activity and increased ubiquitinated proteins in TRAMP prostate cancer but had no effect on apoptosis or ubiquitination in normal mouse tissues. Overall, our data suggests that BA-mediated inhibition of DUBs and induction of apoptotic cell death specifically in prostate cancer but not in normal cells and tissues may provide an effective non-toxic and clinically selective agent for chemotherapy.

  4. Protein-Rich Fraction of Cnidoscolus urens (L. Arthur Leaves: Enzymatic Characterization and Procoagulant and Fibrinogenolytic Activities

    Directory of Open Access Journals (Sweden)

    Yamara A. S. de Menezes

    2014-03-01

    Full Text Available Proteolytic enzymes are important macromolecules in the regulation of biochemical processes in living organisms. Additionally, these versatile biomolecules have numerous applications in the industrial segment. In this study we have characterized a protein-rich fraction of Cnidoscolus urens (L. Arthur leaves, rich in proteolytic enzymes, and evaluated its effects on the coagulation cascade. Three protein-rich fractions were obtained from the crude extract of C. urens leaves by precipitation with acetone. Fraction F1.0 showed higher proteolytic activity upon azocasein, and thus, was chosen for subsequent tests. The proteolytic activity of F1.0 on fibrinogen was dose-dependent and time-dependent. The extract demonstrated procoagulant activity on citrated plasma and reduced the APTT, not exerting effects on PT. Despite the fibrin(ogenolytic activity, F1.0 showed no defibrinogenating activity in vivo. The fraction F1.0 did not express hemorrhagic nor hemolytic activities. The proteolytic activity was inhibited by E-64, EDTA and in the presence of metal ions, and increased when pretreated with reducing agents, suggesting that the observed activity was mostly due to cysteine proteases. Several bands with proteolytic activity were detected by zymography with gelatin, albumin and fibrinogen. The optimal enzymatic activity was observed in temperature of 60 °C and pH 5.0, demonstrating the presence of acidic proteases. In conclusion, these results could provide basis for the pharmacological application of C. urens proteases as a new source of bioactive molecules to treat bleeding and thrombotic disorders.

  5. Effects of Supplementation of Branched-Chain Amino Acids to Reduced-Protein Diet on Skeletal Muscle Protein Synthesis and Degradation in the Fed and Fasted States in a Piglet Model

    Directory of Open Access Journals (Sweden)

    Liufeng Zheng

    2016-12-01

    Full Text Available Supplementation of branched-chain amino acids (BCAA has been demonstrated to promote skeletal muscle mass gain, but the mechanisms underlying this observation are still unknown. Since the regulation of muscle mass depends on a dynamic equilibrium (fasted losses–fed gains in protein turnover, the aim of this study was to investigate the effects of BCAA supplementation on muscle protein synthesis and degradation in fed/fasted states and the related mechanisms. Fourteen 26- (Experiment 1 and 28-day-old (Experiment 2 piglets were fed reduced-protein diets without or with supplemental BCAA. After a four-week acclimation period, skeletal muscle mass and components of anabolic and catabolic signaling in muscle samples after overnight fasting were determined in Experiment 1. Pigs in Experiment 2 were implanted with carotid arterial, jugular venous, femoral arterial and venous catheters, and fed once hourly along with the intravenous infusion of NaH13CO3 for 2 h, followed by a 6-h infusion of [1-13C]leucine. Muscle leucine kinetics were measured using arteriovenous difference technique. The mass of most muscles was increased by BCAA supplementation. During feeding, BCAA supplementation increased leucine uptake, protein synthesis, protein degradation and net transamination. The greater increase in protein synthesis than in protein degradation resulted in elevated protein deposition. Protein synthesis was strongly and positively correlated with the intramuscular net production of α-ketoisocaproate (KIC and protein degradation. Moreover, BCAA supplementation enhanced the fasted-state phosphorylation of protein translation initiation factors and inhibited the protein-degradation signaling of ubiquitin-proteasome and autophagy-lysosome systems. In conclusion, supplementation of BCAA to reduced-protein diet increases fed-state protein synthesis and inhibits fasted-state protein degradation, both of which could contribute to the elevation of skeletal muscle

  6. TOTAL AND FRACTIONAL CONTENTS OF PROTEINS IN BEAN SEEDS UNDER THE CONDITIONS OF VARIED FERTILISATION WITH MICROELEMENTS

    Directory of Open Access Journals (Sweden)

    Wojciech KOZERA

    2013-03-01

    Full Text Available Over 2003-2005 at the Experiment Station at Wierzchucinek at the University of Technology and Life Sciences in Bydgoszcz, there was performed a strict one-factor micro-plot experiment in split-splot design. The factor tested was a type of microelements [n=5: Cu, Zn, Mn, Mo, B]. The microelements were foliar sprayed in a chelated form, as the series of Symfonia fertilizers. The study aimed at comparing the effect of five agricultural-engineering basic microelements on the contents and protein composition of the seeds of Aura cultivar. The fertilization applied, boron and manganese in particular, showed an effect on the increase in the contents of total protein in bean seeds. It also modified the fractional composition of the bean seed protein. There was observed a clear increase in the fraction of albumins and globulins in seeds as a result of the microelements applied, except for boron. The fertilization with molybdenum, boron, copper and zinc reduced the content of glutelins, and the sum of glulelins and prolamines in the bean seeds.

  7. Measurement of Muscle Protein Fractional Synthetic Rate by Capillary Gas Chromatography/Combustion Isotope Ratio Mass Spectrometry

    Science.gov (United States)

    Yarasheski, Kevin E.; Smith, Kenneth; Rennie, Michael J.; Bier, Dennis M.

    2014-01-01

    The measurement of skeletal muscle protein fractional synthetic rate using an infusion of (1-13C)leucine and measuring the isotopic abundance of the tracer in skeletal muscle protein by preparative gas chromatography (GC)/ninhydrin isotope ratio mass spectrometry (IRMS) is laborious and subject to errors owing to contamination by 12C. The purpose of this study was to compare muscle (13C)leucine enrichment measured with the conventional preparative GC/ninhydrin IRMS approach to a new, continuous-flow technique using capillary GC/combustion IRMS. Quadriceps muscles were removed from four Sprague–Dawley rats after each was infused at a different rate with (1-13C)leucine for 6–8 h. Muscle leucine enrichment (at.% excess) measured by both methods differed by less than 4%, except at low (13C)leucine enrichments (IRMS was used to assess muscle (13C)leucine enrichment and fractional muscle protein synthesis rate in ten normal young men and women infused with (1,2-13C2)leucine for 12–14 h. This approach reduced the variability of the isotope abundance measure and gave estimates of muscle protein synthesis rate (0.050 ± 0.011% h−1 (mean ± SEM); range = 0.023–0.147% h−1) that agree with published values determined using the standard analytical approach. The measurement of (13C)leucine enrichment from skeletal muscle protein by capillary GC/combustion IRMS provides a simple, acceptable and practical alternative to preparative GC/ninhydrin IRMS. PMID:1420371

  8. Effect of the Ethyl Acetate Fraction of Eugenia uniflora on Proteins Global Expression during Morphogenesis in Candida albicans

    Directory of Open Access Journals (Sweden)

    Walicyranison P. Silva-Rocha

    2017-09-01

    Full Text Available Candida albicans is able to switch from yeast to hyphal growth and this is an essential step for tissue invasion and establishment of infection. Due to the limited drug arsenal used to treat fungal infections and the constant emergence of resistant strains, it is important to search for new therapeutic candidates. Therefore, this study aimed to investigate by proteomic analysis the role of a natural product (Eugenia uniflora in impairing hypha formation in C. albicans. We also tested the potential action of E. uniflora to prevent and treat oral candidiasis induced in a murine model of oral infection and the ability of polymorphonuclear neutrophils to phagocytize C. albicans cells treated with the ethyl acetate fraction of the extract. We found that this fraction greatly reduced hypha formation after morphogenesis induction in the presence of serum. Besides, several proteins were differentially expressed in cells treated with the fraction. Surprisingly, the ethyl acetate fraction significantly reduced phagocytosis in C. albicans (Mean 120.36 ± 36.71 yeasts/100 PMNs vs. 44.68 ± 19.84 yeasts/100 PMNs. Oral candidiasis was attenuated when C. albicans cells were either pre-incubated in the presence of E. uniflora or when the fraction was applied to the surface of the oral cavity after infection. These results were consistent with the reduction in CFU counts (2.36 vs. 1.85 Log10 CFU/ml and attenuation of tissue damage observed with histopathological analysis of animals belonging to treated group. We also observed shorter true hyphae by direct examination and histopathological analysis, when cells were treated with the referred natural product. The E. uniflora ethyl acetate fraction was non-toxic to human cells. E. uniflora may act on essential proteins mainly related to cellular structure, reducing the capacity of filamentation and attenuating infection in a murine model, without causing any toxic effect on human cells, suggesting that it may be a

  9. Whole linted cottonseed meal (Gossypium hirsutum L. protein and fiber degradability in the rumen

    Directory of Open Access Journals (Sweden)

    Deborah Clea Ruy

    1996-12-01

    3 x 3 change-over design to evaluate the following treatments: A = 0% WLC; B = 6.6% WLC; and C = 15.0% WLC. Sorghum silage contributed with 70% in all three treatments. DM degradability at 48h incubation time was statistically different (p < 0.05 (A = 54.4%; B = 54.2% and C = 58.7%, as well as PB degradability at 12h (A = 40.3%; B = 47.7% and C = 53.1% and ADF degradability at 48h (A = 40.3%; B = 41.2% and C = 45.6%. Ruminal volume, turn overtime and ruminal pH weren’t affected by the experimental diets. Substitution of WLC for cottonseed meal up to 15% diet increased degradability of DM, CP and ADF of WLC.

  10. The structural determinants responsible for c-Fos protein proteasomal degradation differ according to the conditions of expression.

    Science.gov (United States)

    Ferrara, Patrizia; Andermarcher, Elisabetta; Bossis, Guillaume; Acquaviva, Claire; Brockly, Frédérique; Jariel-Encontre, Isabelle; Piechaczyk, Marc

    2003-03-13

    c-fos gene is expressed constitutively in a number of tissues as well as in certain tumor cells and is inducible, in general rapidly and transiently, in virtually all other cell types by a variety of stimuli. Its protein product, c-Fos, is a short-lived transcription factor that heterodimerizes with various protein partners within the AP-1 transcription complex via leucine zipper/leucine zipper interactions for binding to specific DNA sequences. It is mostly, if not exclusively, degraded by the proteasome. To localize the determinant(s) responsible for its instability, we have conducted a genetic analysis in which the half-lives of c-Fos mutants and chimeras made with the stable EGFP reporter protein were compared under two experimental conditions taken as example of continous and inducible expression. Those were constitutive expression in asynchronously growing Balb/C 3T3 mouse embryo fibroblasts and transient induction in the same cells undergoing the G0/G1 phase transition upon stimulation by serum. Our work shows that c-Fos is degraded faster in synchronous- than in asynchronous cells. This difference in turnover is primarily accounted for by several mechanisms. First, in asynchronous cells, a unique C-terminal destabilizer is active whereas, in serum-stimulated cells two destabilizers located at both extremities of the protein are functional. Second, heterodimerization and/or binding to DNA accelerates protein degradation only during the G0/G1 phase transition. Adding another level of complexity to turnover control, phosphorylation at serines 362 and 374, which are c-Fos phosphorylation sites largely modified during the G0/G1 phase transition, stabilizes c-Fos much more efficiently in asynchronous than in serum-stimulated cells. In both cases, the reduced degradation rate is due to inhibition of the activity of the C-terminal destabilizer. However, in serum-stimulated cells, this effect is partially masked by the activation of the N-terminal destabilizer and

  11. F-Box Protein FBXO22 Mediates Polyubiquitination and Degradation of CD147 to Reverse Cisplatin Resistance of Tumor Cells

    Directory of Open Access Journals (Sweden)

    Bo Wu

    2017-01-01

    Full Text Available Drug resistance remains a major clinical obstacle to successful treatment of cancer. As posttranslational modification is becoming widely recognized to affect the function of oncoproteins, targeting specific posttranslational protein modification provides an attractive strategy for anticancer drug development. CD147 is a transmembrane glycoprotein contributing to chemo-resistance of cancer cells in a variety of human malignancies. Ubiquitination is an important posttranslational modification mediating protein degradation. Degradation of oncoproteins, CD147 included, emerges as an attractive alternative for tumor inhibition. However, the ubiquitination of CD147 remains elusive. Here in this study, we found that deletion of the CD147 intracellular domain (CD147-ICD prolonged the half-life of CD147 in HEK293T cells, and we identified that CD147-ICD interacts with FBXO22 using mass spectrometry and Western blot. Then, we demonstrated that FBXO22 mediates the polyubiquitination and degradation of CD147 by recognizing CD147-ICD. While knocking down of FBXO22 prolonged the half-life of CD147 in HEK293T cells, we found that FBXO22 regulates CD147 protein turnover in SMMC-7721, Huh-7 and A549 cells. Moreover, we found that the low level of FBXO22 contributes to the accumulation of CD147 and thereafter the cisplatin resistance of A549/DDP cells. To conclude, our study demonstrated that FBXO22 mediated the polyubiquitination and degradation of CD147 by interacting with CD147-ICD, and CD147 polyubiquitination by FBXO22 reversed cisplatin resistance of tumor cells.

  12. Optimization of protein fractionation by skim milk microfiltration: Choice of ceramic membrane pore size and filtration temperature.

    Science.gov (United States)

    Jørgensen, Camilla Elise; Abrahamsen, Roger K; Rukke, Elling-Olav; Johansen, Anne-Grethe; Schüller, Reidar B; Skeie, Siv B

    2016-08-01

    The objective of this study was to investigate how ceramic membrane pore size and filtration temperature influence the protein fractionation of skim milk by cross flow microfiltration (MF). Microfiltration was performed at a uniform transmembrane pressure with constant permeate flux to a volume concentration factor of 2.5. Three different membrane pore sizes, 0.05, 0.10, and 0.20µm, were used at a filtration temperature of 50°C. Furthermore, at pore size 0.10µm, 2 different filtration temperatures were investigated: 50 and 60°C. The transmission of proteins increased with increasing pore size, giving the permeate from MF with the 0.20-µm membrane a significantly higher concentration of native whey proteins compared with the permeates from the 0.05- and 0.10-µm membranes (0.50, 0.24, and 0.39%, respectively). Significant amounts of caseins permeated the 0.20-µm membrane (1.4%), giving a permeate with a whitish appearance and a casein distribution (αS2-CN: αS1-CN: κ-CN: β-CN) similar to that of skim milk. The 0.05- and 0.10-µm membranes were able to retain all caseins (only negligible amounts were detected). A permeate free from casein is beneficial in the production of native whey protein concentrates and in applications where transparency is an important functional characteristic. Microfiltration of skim milk at 50°C with the 0.10-µm membrane resulted in a permeate containing significantly more native whey proteins than the permeate from MF at 60°C. The more rapid increase in transmembrane pressure and the significantly lower concentration of caseins in the retentate at 60°C indicated that a higher concentration of caseins deposited on the membrane, and consequently reduced the native whey protein transmission. Optimal protein fractionation of skim milk into a casein-rich retentate and a permeate with native whey proteins were obtained by 0.10-µm MF at 50°C. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All

  13. Arginine depletion by arginine deiminase does not affect whole protein metabolism or muscle fractional protein synthesis rate in mice

    Science.gov (United States)

    Due to the absolute need for arginine that certain cancer cells have, arginine depletion is a therapy in clinical trials to treat several types of cancers. Arginine is an amino acids utilized not only as a precursor for other important molecules, but also for protein synthesis. Because arginine depl...

  14. Retraction: Myostatin Induces Degradation of Sarcomeric Proteins through a Smad3 Signaling Mechanism During Skeletal Muscle Wasting

    Science.gov (United States)

    Lokireddy, Sudarsanareddy; McFarlane, Craig; Ge, Xiaojia; Zhang, Huoming; Sze, Siu Kwan; Sharma, Mridula

    2011-01-01

    Ubiquitination-mediated proteolysis is a hallmark of skeletal muscle wasting manifested in response to negative growth factors, including myostatin. Thus, the characterization of signaling mechanisms that induce the ubiquitination of intracellular and sarcomeric proteins during skeletal muscle wasting is of great importance. We have recently characterized myostatin as a potent negative regulator of myogenesis and further demonstrated that elevated levels of myostatin in circulation results in the up-regulation of the muscle-specific E3 ligases, Atrogin-1 and muscle ring finger protein 1 (MuRF1). However, the exact signaling mechanisms by which myostatin regulates the expression of Atrogin-1 and MuRF1, as well as the proteins targeted for degradation in response to excess myostatin, remain to be elucidated. In this report, we have demonstrated that myostatin signals through Smad3 (mothers against decapentaplegic homolog 3) to activate forkhead box O1 and Atrogin-1 expression, which further promotes the ubiquitination and subsequent proteasome-mediated degradation of critical sarcomeric proteins. Smad3 signaling was dispensable for myostatin-dependent overexpression of MuRF1. Although down-regulation of Atrogin-1 expression rescued approximately 80% of sarcomeric protein loss induced by myostatin, only about 20% rescue was seen when MuRF1 was silenced, implicating that Atrogin-1 is the predominant E3 ligase through which myostatin manifests skeletal muscle wasting. Furthermore, we have highlighted that Atrogin-1 not only associates with myosin heavy and light chain, but it also ubiquitinates these sarcomeric proteins. Based on presented data we propose a model whereby myostatin induces skeletal muscle wasting through targeting sarcomeric proteins via Smad3-mediated up-regulation of Atrogin-1 and forkhead box O1. PMID:21964591

  15. THE CHANGE OF TOTAL PROTEIN FRACTION OF MUSCLE TISSUE OF PORK WITH BIO- AND PHYSICO-CHEMICAL SPECIFIC IN THE PROCESS OF COOKING AT DIFFERENT TEMPERATURES

    Directory of Open Access Journals (Sweden)

    O. Shalimova

    2012-03-01

    Full Text Available The character of changes in total protein fraction of muscle tissue of pork with PSE defects in the process of cooking at temperatures ranging from 40 to 72 g.C in steps of 2 g.C is investigated. Our studies have revealed differences in the change of state the total fraction of muscle proteins with defects PSE pork during cooking.

  16. Protein degradation in skeletal muscle during experimental hyperthyroidism in rats and the effect of beta-blocking agents.

    Science.gov (United States)

    Angerås, U; Hasselgren, P O

    1987-04-01

    beta-Blocking agents are increasingly used in the management of hyperthyroid patients. The effect of this treatment on increased muscle protein breakdown in the hyperthyroid state is not known. In the present study, experimental hyperthyroidism was induced in rats by daily ip injections of T3 (100 micrograms/100 g BW) during a 10-day period. Control animals received corresponding volumes of solvent. In groups of rats the selective beta-1-blocking agent metoprolol or the nonselective beta-blocker propranolol was infused by miniosmotic pumps implanted sc on the backs of the animals. Protein degradation was measured in incubated intact soleus and extensor digitorum longus muscles by determining tyrosine release into the incubation medium. The protein degradation rate in incubated extensor digitorum longus and soleus muscles was increased by 50-60% during T3 treatment. Metoprolol or propranolol did not influence muscle protein breakdown in either T3-treated or control animals. The results suggest that T3-induced increased muscle proteolysis is not mediated by beta-receptors, and muscle weakness and wasting in hyperthyroidism might not be affected by beta-blockers.

  17. Performing Isoelectric Focusing and Simultaneous Fractionation of Proteins on A Rotary Valve Followed by Sodium Dodecyl – Polyacrylamide Gel Electrophoresis

    Science.gov (United States)

    Wang, Wei; Lu, Joann J.; Gu, Congying; Zhou, Lei; Liu, Shaorong

    2013-01-01

    In this technical note, we design and fabricate a novel rotary valve and demonstrate its feasibility for performing isoelectric focusing and simultaneous fractionation of proteins, followed by sodium dodecyl – polyacrylamide gel electrophoresis. The valve has two positions. In one position, the valve routes a series of capillary loops together into a single capillary tube where capillary isoelectric focusing (CIEF) is performed. By switching the valve to another position, the CIEF-resolved proteins in all capillary loops are isolated simultaneously, and samples in the loops are removed and collected in vials. After the collected samples are briefly processed, they are separated via sodium dodecyl – polyacrylamide gel electrophoresis (SDS-PAGE, the 2nd-D separation) on either a capillary gel electrophoresis instrument or a slab-gel system. The detailed valve configuration is illustrated, and the experimental conditions and operation protocols are discussed. PMID:23819755

  18. BAG3 down-modulation reduces anaplastic thyroid tumor growth by enhancing proteasome-mediated degradation of BRAF protein.

    Science.gov (United States)

    Chiappetta, Gennaro; Basile, Anna; Arra, Claudio; Califano, Daniela; Pasquinelli, Rosa; Barbieri, Antonio; De Simone, Veronica; Rea, Domenica; Giudice, Aldo; Pezzullo, Luciano; De Laurenzi, Vincenzo; Botti, Gerardo; Losito, Simona; Conforti, Daniela; Turco, Maria Caterina

    2012-01-01

    Anaplastic thyroid tumors (ATC) express high levels of BAG3, a member of the BAG family of cochaperone proteins that is involved in regulating cell apoptosis through multiple mechanisms. The objective of the study was the investigation of the influence of B-cell lymphoma-2-associated athanogene 3 (BAG3) on ATC growth. We investigated the effects of BAG3 down-modulation, obtained by using a specific small interfering RNA, on in vitro and in vivo growth of the human ATC cell line 8505C. Because BRAF protein plays an important role in ATC cell growth, we analyzed the effects of BAG3 down-modulation on BRAF protein levels. Furthermore, by using a proteasome inhibitor, we verified whether BAG3-mediated regulation of BRAF levels involved a proteasome-dependent mechanism. BAG3 down-modulation significantly inhibits ATC growth in vitro and in vivo. BAG3 coimmunoprecipitates with BRAF protein, and its down-modulation results in a significant reduction of BRAF protein levels, which can be reverted by incubation with the proteasome inhibitor MG132. BAG3 protein sustains ATC growth in vitro and in vivo. The underlying molecular mechanism appears to rely on BAG3 binding to BRAF, thus protecting it from proteasome-dependent degradation. These results are in line with the reported ability of BAG3 to interfere with the proteasomal delivery of a number of other client proteins.

  19. Physical degradation of proteins in well-defined fluid flows studied within a four-roll apparatus.

    Science.gov (United States)

    Simon, S; Krause, H J; Weber, C; Peukert, W

    2011-12-01

    In most applications of biotechnology and downstream processing proteins are exposed to fluid stresses in various flow configurations which often lead to the formation of unwanted protein aggregates. In this paper we present physical degradation experiments for proteins under well-defined flow conditions in a four-roll apparatus. The flow field was characterized numerically by computational fluid dynamics (CFD) and experimentally by particle image velocimetry (PIV). The local shear strain rate as well as the local shear and elongation rate was used to characterize the hydrodynamic stress environment acting on the proteins. Lysozyme was used as a model protein and subjected to well-defined fluid stresses in high and low stress environment. By using in situ turbidity measurements during stressing the aggregate formation was monitored directly in the fluid flow. An increase in absorbance at 350 nm was attributed to a higher content of visible particles (>1 µm). In addition to lysozyme, the formation of aggregates was confirmed for two larger proteins (bovine serum albumin and alcohol dehydrogenase). Thus, the presented experimental setup is a helpful tool to monitor flow-induced protein aggregation with high reproducibility. For instance, screening experiments for formulation development of biopharmaceuticals for fill and finish operations can be performed in the lab-scale in a short time-period if the stress distributions in the application are transferred and applied in the four-roll mill. Copyright © 2011 Wiley Periodicals, Inc.

  20. Theobromine suppresses adipogenesis through enhancement of CCAAT-enhancer-binding protein β degradation by adenosine receptor A1.

    Science.gov (United States)

    Mitani, Takakazu; Watanabe, Shun; Yoshioka, Yasukiyo; Katayama, Shigeru; Nakamura, Soichiro; Ashida, Hitoshi

    2017-12-01

    Theobromine, a methylxanthine derived from cacao beans, reportedly has various health-promoting properties but molecular mechanism by which effects of theobromine on adipocyte differentiation and adipogenesis remains unclear. In this study, we aimed to clarify the molecular mechanisms of the anti-adipogenic effect of theobromine in vitro and in vivo. ICR mice (4week-old) were administered with theobromine (0.1g/kg) for 7days. Theobromine administration attenuated gains in body and epididymal adipose tissue weights in mice and suppressed expression of adipogenic-associated genes in mouse adipose tissue. In 3T3-L1 preadipocytes, theobromine caused degradation of C/EBPβ protein by the ubiquitin-proteasome pathway. Pull down assay showed that theobromine selectively interacts with adenosine receptor A1 (AR1), and AR1 knockdown inhibited theobromine-induced C/EBPβ degradation. Theobromine increased sumoylation of C/EBPβ at Lys133. Expression of the small ubiquitin-like modifier (SUMO)-specific protease 2 (SENP2) gene, coding for a desumoylation enzyme, was suppressed by theobromine. In vivo knockdown studies showed that AR1 knockdown in mice attenuated the anti-adipogenic effects of theobromine in younger mice. Theobromine suppresses adipocyte differentiation and induced C/EBPβ degradation by increasing its sumoylation. Furthermore, the inhibition of AR1 signaling is important for theobromine-induced C/EBPβ degradation. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Quantitative profiling of serum samples using TMT protein labelling, fractionation and LC-MS/MS.

    Science.gov (United States)

    Sinclair, John; Timms, John F

    2011-08-01

    Blood-borne biomarkers are urgently required for the early detection, accurate diagnosis and prognosis of disease. Additionally, improved methods of profiling serum and plasma proteins for biomarker discovery efforts are needed. Herein, we report a quantitative method based on amino-group labelling of serum proteins (rather than peptides) with isobaric tandem mass tags (TMT) and incorporating immune-based depletion, gel-based and strong anion exchange separation of proteins prior to differential endoproteinase treatment and liquid chromatography tandem mass spectrometry. We report a generally higher level of quantitative coverage of the serum proteome compared to other peptide-based isobaric tagging approaches and show the potential of the method by applying it to a set of unique samples that pre-date the diagnosis of pancreatic cancer. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. Sterol metabolism regulates neuroserpin polymer degradation in the absence of the unfolded protein response in the dementia FENIB.

    Science.gov (United States)

    Roussel, Benoit D; Newton, Timothy M; Malzer, Elke; Simecek, Nikol; Haq, Imran; Thomas, Sally E; Burr, Marian L; Lehner, Paul J; Crowther, Damian C; Marciniak, Stefan J; Lomas, David A

    2013-11-15

    Mutants of neuroserpin are retained as polymers within the endoplasmic reticulum (ER) of neurones to cause the autosomal dominant dementia familial encephalopathy with neuroserpin inclusion bodies or FENIB. The cellular consequences are unusual in that the ordered polymers activate the ER overload response (EOR) in the absence of the canonical unfolded protein response. We use both cell lines and Drosophila models to show that the G392E mutant of neuroserpin that forms polymers is degraded by UBE2j1 E2 ligase and Hrd1 E3 ligase while truncated neuroserpin, a protein that lacks 132 amino acids, is degraded by UBE2g2 (E2) and gp78 (E3) ligases. The degradation of G392E neuroserpin results from SREBP-dependent activation of the cholesterol biosynthetic pathway in cells that express polymers of neuroserpin (G392E). Inhibition of HMGCoA reductase, the limiting enzyme of the cholesterol biosynthetic pathway, reduced the ubiquitination of G392E neuroserpin in our cell lines and increased the retention of neuroserpin polymers in both HeLa cells and primary neurones. Our data reveal a reciprocal relationship between cholesterol biosynthesis and the clearance of mutant neuroserpin. This represents the first description of a link between sterol metabolism and modulation of the proteotoxicity mediated by the EOR.

  3. Cheliensisin A (Chel A) induces apoptosis in human bladder cancer cells by promoting PHLPP2 protein degradation.

    Science.gov (United States)

    Zhang, Ruowen; Che, Xun; Zhang, Jingjie; Li, Yang; Li, Jingxia; Deng, Xu; Zhu, Junlan; Jin, Honglei; Zhao, Qinshi; Huang, Chuanshu

    2016-10-11

    Cheliensisin A (Chel A), a styryl-lactone compound extracted from Goniothalamus cheliensis, is reported to have significant anti-cancer effects in various cancer cells. Here we demonstrated that Chel A treatment resulted in apoptosis and an inhibition of anchorage-independent growth in human bladder cancer T24, T24T and U5637 cells. Mechanistic studies showed that such effect is mediated by PH domain and Leucine rich repeat Protein Phosphatases (PHLPP2) protein. Chel A treatment led to PHLPP2 degradation and subsequently increased in c-Jun phosphorylation. Moreover PHLPP2 degradation could be attenuated by inhibition of autophagy, which was mediated by Beclin 1. Collectively, we discover that Chel A treatment induces Beclin-dependent autophagy, consequently mediates PHLPP2 degradation and JNK/C-Jun phosphorylation and activation, further in turn contributing to apoptosis in human bladder cancer cells. Current studies provide a significant insight into understanding of anticancer effect of Chel A in treatment of human bladder cancer.

  4. Proteasome-mediated degradation of integral inner nuclear membrane protein emerin in fibroblasts lacking A-type lamins

    International Nuclear Information System (INIS)

    Muchir, Antoine; Massart, Catherine; Engelen, Baziel G. van; Lammens, Martin; Bonne, Gisele; Worman, Howard J.

    2006-01-01

    We previously identified and characterized a homozygous LMNA nonsense mutation leading to the absence of A-type lamins in a premature neonate who died at birth. We show here that the absence of A-type lamins is due to degradation of the aberrant mRNA transcript with a premature termination codon. In cultured fibroblasts from the subject with the homozygous LMNA nonsense mutation, there was a decreased steady-state expression of the integral inner nuclear membrane proteins emerin and nesprin-1α associated with their mislocalization to the bulk endoplasmic reticulum and a hyperphosphorylation of emerin. To determine if decreased emerin expression occurred post-translationally, we treated cells with a selective proteasome inhibitor and observed an increase in expression. Our results show that mislocalization of integral inner nuclear membrane proteins to the endoplasmic reticulum in human cells lacking A-type lamins leads to their degradation and provides the first evidence that their degradation is mediated by the proteasome

  5. Separate photosensitizers mediate degradation of the 32-kDa photosystem II reaction center protein in the visible and UV spectral regions

    International Nuclear Information System (INIS)

    Greenberg, B.M.; Gaba, V.; Canaani, O.; Malkin, S.; Mattoo, A.K.; Edelman, M.

    1989-01-01

    A component of the photosystem II reaction center, the 32-kDa protein, is rapidly turned over in the light. The mechanism of its light-dependent metabolism is largely unknown. We quantified the rate of 32-kDa protein degradation over a broad spectral range (UV, visible, and far red). The quantum yield for degradation was highest in the UVB (280-320 nm) region. Spectral evidence demonstrates two distinctly different photosensitizers for 32-kDa protein degradation. The data implicate the bulk photosynthetic pigments (primarily chlorophyll) in the visible and far red regions, and plastoquinone (in one or more of its redox states) in the UV region. A significant portion of 32-kDa protein degradation in sunlight is attributed to UVB irradiance

  6. Comparison of the adhesive performances of soy meal, water washed meal fractions, and protein isolates

    Science.gov (United States)

    Adhesive bonding of wood plays an increasing role in the forest products industry and is a key factor for efficiently utilizing timber and other lignocellulosic resources. In this work, we obtained five soy meal products through commercial sources or in-house preparations. The protein content was 49...

  7. Proteomic Analysis of PEG-Fractionated UV-C Stress-Response Proteins in Globe Artichoke

    NARCIS (Netherlands)

    Falvo, S.; Acquadro, A.; Albo, A.G.; America, A.H.P.; Lanteri, S.

    2012-01-01

    Plants respond to UV stress by producing antioxidant molecules and by altering their metabolism through the regulation of specific gene family members. Globe artichoke (Cynara cardunculus var. scolymus L.-Compositae family) is an attractive model species for studying the protein networks involved in

  8. Method Development to Increase Protein Enrichment During Dry Fractionation of Starch-Rich Legumes

    NARCIS (Netherlands)

    Pelgrom, P.J.M.; Boom, R.M.; Schutyser, M.A.I.

    2015-01-01

    A facile method was developed to establish milling settings that optimally separate starch granules from protein bodies and cell wall fibres for starch-rich legumes. Optimal separation was obtained for pea, bean, lentil and chickpea when the particle size distribution curve of flour and isolated

  9. Study of the Changes in Protein Fractions and Amino Acids of an ...

    African Journals Online (AJOL)

    Many studies have been conducted on the changes of the proteins in fermented sausages, which is aided by the proteolytic enzymes or lactic acid producing organisms. Information on the changes brought about by the meat enzymes and drying process as such is still unknown. We report results in this respect as observed ...

  10. Role of pH-induced structural change in protein aggregation in foam fractionation of bovine serum albumin

    Directory of Open Access Journals (Sweden)

    Rui Li

    2016-03-01

    Full Text Available For reducing protein aggregation in foam fractionation, the role of pH-induced structural change in the interface-induced protein aggregation was analyzed using bovine serum albumin (BSA as a model protein. The results show that the decrease in pH from 7.0 to 3.0 gradually unfolded the BSA structure to increase the molecular size and the relative content of β-sheet and thus reduced the stability of BSA in the aqueous solution. At the isoelectric point (pH 4.7, BSA suffered the lowest level in protein aggregation induced by the gas–liquid interface. In the pH range from 7.0 to 4.7, most BSA aggregates were formed in the defoaming process while in the pH range from 4.7 to 3.0, the BSA aggregates were formed at the gas–liquid interface due to the unfolded BSA structure and they further aggregated to form insoluble ones in the desorption process.

  11. Carrier ampholyte-free isoelectric focusing on a paper-based analytical device for the fractionation of proteins.

    Science.gov (United States)

    Xie, Song-Fang; Gao, Han; Niu, Li-Li; Xie, Zhen-Sheng; Fang, Fang; Wu, Zhi-Yong; Yang, Fu-Quan

    2018-01-25

    Isoelectric focusing plays a critical role in the analysis of complex protein samples. Conventionally, isoelectric focusing is implemented with carrier ampholytes in capillary or immobilized pH gradient gel. In this study, we successfully exhibited a carrier ampholyte-free isoelectric focusing on paper-based analytical device. Proof of the concept was visually demonstrated with color model proteins. Experimental results showed that not only a pH gradient was well established along the open paper fluidic channel as confirmed by pH indicator strip, the pH gradient range could also be tuned by the catholyte or anolyte. Furthermore, the isoelectric f