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Sample records for sarcoplasmic protein fraction

  1. Kinetic characterization of Channa striatus muscle sarcoplasmic and myofibrillar protein hydrolysates.

    Science.gov (United States)

    Ghassem, Masomeh; Fern, See Siau; Said, Mamot; Ali, Zainon Mohd; Ibrahim, Saadiah; Babji, Abdul Salam

    2014-03-01

    This study was conducted to evaluate the kinetic characteristics of proteolytic activity of proteases on Channa striatus protein fractions. Degree of hydrolysis (DH), amino acid composition and kinetic parameters of sarcoplasmic and myofibrillar proteins were investigated when incubated with proteinase K and thermolysin, separately. After 30 min incubation with proteases, a decrease in DH of sarcoplasmic protein was observed whereas, hydrolysis of myofibrillar protein with proteases took 2 h with an increase in DH. The major amino acids were glutamic acid (16.6%) in thermolysin- myofibrillar hydrolysate followed by aspartic acid (11.1%) in sarcoplasmic protein fraction with no enzyme treatment and lysine (10%) in thermolysin-myofibrillar hydrolysate. The apparent Michaelis constant of proteinase K was lower than thermolysin for both sarcoplasmic and myofibrillar proteins. However, rate of turnover and enzyme efficiency suggested that sarcoplasmic and myofibrillar proteins are suitable substrates for proteinase K and thermolysin hydrolytic reaction, respectively.

  2. Kinetic characterization of Channa striatus muscle sarcoplasmic and myofibrillar protein hydrolysates

    OpenAIRE

    Ghassem, Masomeh; Fern, See Siau; Said, Mamot; Ali, Zainon Mohd; Ibrahim, Saadiah; Babji, Abdul Salam

    2011-01-01

    This study was conducted to evaluate the kinetic characteristics of proteolytic activity of proteases on Channa striatus protein fractions. Degree of hydrolysis (DH), amino acid composition and kinetic parameters of sarcoplasmic and myofibrillar proteins were investigated when incubated with proteinase K and thermolysin, separately. After 30 min incubation with proteases, a decrease in DH of sarcoplasmic protein was observed whereas, hydrolysis of myofibrillar protein with proteases took 2 h ...

  3. Bioactive electrospun fish sarcoplasmic proteins as a drug delivery system

    DEFF Research Database (Denmark)

    Stephansen, Karen; Chronakis, Ioannis S.; Jessen, Flemming

    2014-01-01

    Nano-microfibers were made from cod (Gadus morhua) sarcoplasmic proteins (FSP) (Mwelectrospinning technique. The FSP fibers were studied by scanning electron microscopy, and thefiber morphology was found to be strongly dependent on FSP concentration. Interestingly, the FSP...

  4. Affi-gel blue treatment simplifies the protein composition of sarcoplasmic reticulum vesicles.

    Science.gov (United States)

    Papp, S; Dux, L; Martonosi, A

    1986-04-01

    Sarcoplasmic reticulum vesicles isolated by conventional techniques usually contain, in addition to the recognized sarcoplasmic reticulum components, several other proteins (phosphorylase, myosin, glyceraldehyde-3-phosphate dehydrogenase, etc.) in variable amounts; these proteins complicate the interpretation of chemical modification data. Incubation of sarcoplasmic reticulum vesicles with Affi-Gel blue particles for 1-4 h at 2 degrees C, followed by sedimentation of the Affi-Gel in a clinical centrifuge, simplifies the protein composition by selective adsorption of the accessory proteins, and improves the consistency of the preparations. The Affi-Gel blue treatment is recommended as part of the standard procedure for the isolation of sarcoplasmic reticulum vesicles.

  5. Cooked sausage batter cohesiveness as affected by sarcoplasmic proteins.

    Science.gov (United States)

    Farouk, M M; Wieliczko, K; Lim, R; Turnwald, S; Macdonald, G A

    2002-05-01

    In the first trial, m. semitendinosus and m. biceps femoris were held at 0, 10 and 35 °C until they entered rigor, and in the second trial, minced m. semitendinosus was washed in water for 15, 30, 45 or 60 min. The samples from both the trials were then used to make a finely comminuted sausage batter. Soluble sarcoplasmic protein (SSP) levels decreased with increasing rigor temperature (P < 0.05) or washing (P < 0.01). Cooked batter shear stress was not affected by SSP level, but batter shear strain decreased with the decreasing SSP level associated with an increasing rigor temperature (P < 0.05) or washing (P < 0.01). Reducing the SSP content lowered the cook yield (P < 0.05) and emulsion stability (P < 0.01) of the batter from the washed samples compared to that of controls. The results suggest that sarcoplasmic proteins are important in determining the strain values (cohesiveness) of cooked sausage batter.

  6. Effect of losartan on sarcoplasmic reticulum Ca2+ handing proteins in heart failure rabbit

    Institute of Scientific and Technical Information of China (English)

    姚艳

    2006-01-01

    Objective To investigate the effects of losartan on mRNA expression of myocardial sarcoplasmic reticulum calcium handling proteins (SERCA2, RyR2 and PLB) and the role of which in prevention of chronic heart failure in rabbit. Methods After chronic heart failure was

  7. Time Course of the Response of Myofibrillar and Sarcoplasmic Protein Metabolism to Unweighting of the Soleus Muscle

    Science.gov (United States)

    Munoz, Kathryn A.; Satarug, Soisungwan; Tischler, Marc E.

    1993-01-01

    Contributions of altered in vivo protein synthesis and degradation to unweighting atrophy of the soleus muscle in tail-suspended young female rats were analyzed daily for up to 6 days. Specific changes in myofibrillar and sarcoplasmic proteins were also evaluated to assess their contributions to the loss of total protein. Synthesis of myofibrillar and sarcoplasmic proteins was estimated by intramuscular (IM) injection and total protein by intraperitoneal (IP) injection of flooding doses of H-3-phenylaianine. Total protein loss was greatest during the first 3 days following suspension and was a consequence of the loss of myofibrillar rather than sarcoplasmic proteins. However, synthesis of total myofibrillar and sarcoplasmic proteins diminished in parallel beginning in the first 24 hours. Therefore sarcoplasmic proteins must be spared due to a decrease in their degradation. In contrast, myofibrillar protein degradation increased, thus explaining the elevated degradation of the total pool. Following 72 hours of suspension, protein synthesis remained low, but the rate of myofibrillar protein loss diminished, suggesting a slowing of degradation. These various results show acute loss of protein during unweighting atrophy is a consequence of decreased synthesis and increased degradation of myofibrillar proteins, and sarcoplasmic proteins are spared due to slower degradation, likely explaining the sparing of plasma membrane receptors. Based on other published data, we propose that the slowing of atrophy after the initial response may be attributed to an increased effect of insulin.

  8. Shrimp allergy beyond Tropomyosin in Italy: clinical relevance of Arginine Kinase, Sarcoplasmic calcium binding protein and Hemocyanin

    National Research Council Canada - National Science Library

    Giuffrida, M G; Villalta, D; Mistrello, G; Amato, S; Asero, R

    2014-01-01

    .... We detected the prevalence of arginine kinase and sarcoplasmic calcium binding protein sensitization, and identified a high molecular weight allergen that is frequently recognized by Italian shrimp-allergic patients...

  9. Sarcoplasmic phospholamban protein is involved in the mechanisms of postresuscitation myocardial dysfunction and the cardioprotective effect of nitrite during resuscitation.

    Directory of Open Access Journals (Sweden)

    Yu Huang

    Full Text Available OBJECTIVES: Sarcoplasmic reticulum (SR Ca(2+-handling proteins play an important role in myocardial dysfunction after acute ischemia/reperfusion injury. We hypothesized that nitrite would improve postresuscitation myocardial dysfunction by increasing nitric oxide (NO generation and that the mechanism of this protection is related to the modulation of SR Ca(2+-handling proteins. METHODS: We conducted a randomized prospective animal study using male Sprague-Dawley rats. Cardiac arrest was induced by intravenous bolus of potassium chloride (40 µg/g. Nitrite (1.2 nmol/g or placebo was administered when chest compression was started. No cardiac arrest was induced in the sham group. Hemodynamic parameters were monitored invasively for 90 minutes after the return of spontaneous circulation (ROSC. Echocardiogram was performed to evaluate cardiac function. Myocardial samples were harvested 5 minutes and 1 hour after ROSC. RESULTS: Myocardial function was significantly impaired in the nitrite and placebo groups after resuscitation, whereas cardiac function (i.e., ejection fraction and fractional shortening was significantly greater in the nitrite group than in the placebo group. Nitrite administration increased the level of nitric oxide in the myocardium 5 min after resuscitation compared to the other two groups. The levels of phosphorylated phospholamban (PLB were decreased after resuscitation, and nitrite increased the phosphorylation of phospholamban compared to the placebo. No significant differences were found in the expression of sarcoplasmic reticulum Ca(2+ ATPase (SERCA2a and ryanodine receptors (RyRs. CONCLUSIONS: postresuscitation myocardial dysfunction is associated with the impairment of PLB phosphorylation. Nitrite administered during resuscitation improves postresuscitation myocardial dysfunction by preserving phosphorylated PLB protein during resuscitation.

  10. Effects of tetrandrine on calcium transport, protein fluorescences and membrane fluidity of sarcoplasmic reticulum.

    Science.gov (United States)

    Chen, L Y; Chen, X; Tian, X L; Yu, X H

    2000-10-01

    To understand whether the molecular mechanism of Tetrandrine (Tet)'s pharmacological effects is concerned with sarcoplasmic reticulum calcium transport so as to be involved in myocardial contractility, we observed the effects of Tet on calcium transport and membrane structure of rabbit skeletal muscle sarcoplasmic reticulum vesicles (SR) and rat cardiac sarcoplasmic reticulum vesicles (CSR). Calcium uptake was monitored with a dual-wavelength spectrophotometer. Protein conformation and fluorescence polarization were measured by fluospectrophotometric method and membrane lipids labelled with fluorescence probes for SR, respectively. 128 micromol l(-1) Tet reduced the initial rate of calcium uptake to 59% of control 6 min after reaction. Tet un-competitively inhibited SR Ca(2+), Mg(2+)-ATPase activity, causing the stoichiometric ratio of SR Ca(2+)/ATP to decrease to 1.43 from 2.0 of control. Inhibitory rates on SR Ca(2+),Mg(2+)-ATPase by Tet were reduced from 60% in the absence of phosphate to 50% in the presence of phosphate and reduced from 92% in 1 mmol l(-1) ATP to 60% in 5 mmol l(-1) ATP. Tet markedly reduced SR intrinsic protein fluorescence, while it slightly decreased the thiol(SH)-modified protein fluorescence of SR labelled with N-(3-pyrene)-maleimide. Tet slightly increased fluorescence polarization in the middle and deep layers of SR membrane lipids labelled with 7- or 12-(9-anthroyloxy) stearic acid (AS) probes, whereas it did not change that of SR labelled with 1, 6-diphenyl-1,3,5-hexatrine (DPH). These results revealed that prevention of SR calcium uptake by Tet was due to inhibition of the SR calcium pump Ca(2+),Mg(2+)-ATPase, changes in spatial conformation of the pumps protein molecules and a decrease in the extent of motion of membrane lipid molecules, thus altering the regulation of [Ca(2+)](i) and myocardial contractility.

  11. Design and characterization of self-assembled fish sarcoplasmic protein-alginate nanocomplexes

    DEFF Research Database (Denmark)

    Boutrup Stephansen, Karen; Mattebjerg, Maria Ahlm; Wattjes, Jasper;

    2015-01-01

    Macrostructures based on natural polymers are subject to large attention, as the application range is wide within the food and pharmaceutical industries. In this study we present nanocomplexes (NCXs) made from electrostatic self-assembly between negatively charged alginate and positively charged...... fish sarcoplasmic proteins (FSP), prepared by bulk mixing. A concentration screening revealed that there was a range of alginate and FSP concentrations where stable NCXs with similar properties were formed, rather than two exact concentrations. The size of the NCXs was 293 +/- 3 nm, and the zeta...

  12. Chemical and Functional Characterization of Sarcoplasmic Proteins from Giant Squid (Dosidicus gigas Mantle

    Directory of Open Access Journals (Sweden)

    Rosa Linda Lopez-Enriquez

    2015-01-01

    Full Text Available Modification of pH and NaCl concentration changed the physicochemical properties of sarcoplasmic proteins (SP from jumbo squid mantle and consequently their functional properties. Better results of emulsifying activity index (EAI and foam capacity (FC were exhibited at pH 11 in NaCl absence due to higher solubility. But better emulsifying stability index (ESI was obtained at pH 11 in 0.5 M NaCl, while, foaming stability (FS was better at pH near to isoelectric point (pI. These results suggest that SP from jumbo squid may be a promising ingredient, whose functional properties can be manipulated by changing pH and NaCl concentration.

  13. Preliminary investigation of sequence-independent DNA binding proteins in rat skeletal muscle sarcoplasmic reticulum and their function

    Institute of Scientific and Technical Information of China (English)

    赵文; 姜志胜; 倪菊华; 陈光慧; 刘乃奎; 汤健; 贾弘褆; 唐朝枢

    2000-01-01

    To observe the binding of plasmid DNA to non-nuclear DNA binding proteins in sar-coplasmic reticulum (SR) and the effects of this binding on SR function, sarcoplasmic reticulum proteins in rat skeletal muscle were isolated by differential centrifuge and sucrose density-gradient centrifuge. The results showed that there are two sequence-independent DNA binding proteins in SR proteins, the molecular weights of which are 83 and 58 ku, respectively. Ca2+ uptake and release of SR were remarkably promoted by the binding of plasmid DNA to DNA binding proteins in SR, the mechanism is probably through increasing of Ca2+-ATPase activity in SR and changing of character of Ca2+ release channel ryanodine receptors induced by the binding. These results suggest that there exist DNA binding proteins in SR and its binding to DNA may affect Ca2+ transport of SR.

  14. Preliminary investigation of sequence-independent DNA binding proteins in rat skeletal muscle sarcoplasmic reticulum and their function

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    To observe the binding of plasmid DNA to non-nuclear DNA binding proteins in sarcoplasmic reticulum (SR) and the effects of this binding on SR function, sarcoplasmic reticulum proteins in rat skeletal muscle were isolated by differential centrifuge and sucrose density-gradient centrifuge. The results showed that there are two sequence-independent DNA binding proteins in SR proteins, the molecular weights of which are 83 and 58 ku, respectively. Ca2+ uptake and release of SR were remarkably promoted by the binding of plasmid DNA to DNA binding proteins in SR, the mechanism is probably through increasing of Ca2+-ATPase activity in SR and changing of character of Ca2+ release channel ryanodine receptors induced by the binding. These results suggest that there exist DNA binding proteins in SR and its binding to DNA may affect Ca2+ transport of SR.

  15. Phosphorylation of anchoring protein by calmodulin protein kinase associated to the sarcoplasmic reticulum of rabbit fast-twitch muscle.

    Science.gov (United States)

    Damiani, E; Sacchetto, R; Margreth, A

    2000-12-09

    Regulatory phosphorylation of phospholamban and of SR Ca(2+)-ATPase SERCA2a isoform by endogenous CaM-K II in slow-twitch skeletal and cardiac sarcoplasmic reticulum (SR) is well documented, but much less is known of the exact functional role of CaM K II in fast-twitch muscle SR. Recently, it was shown that RNA splicing of brain-specific alpha CaM K II, gives rise to a truncated protein (alpha KAP), consisting mainly of the association domain, serving to anchor CaM K II to SR membrane in rat skeletal muscle [Bayer, K.-U., et al. (1998) EMBO J. 19, 5598-5605]. In the present study, we searched for the presence of alpha KAP in sucrose-density purified SR membrane fractions from representative fast-twitch and slow-twitch limb muscles, both of the rabbit and the rat, using immunoblot techniques and antibody directed against the association domain of alpha CaM K II. Putative alpha KAP was immunodetected as a 23-kDa electrophoretic component on SDS-PAGE of the isolated SR from fast-twitch but not from slow-twitch muscle, and was further identified as a specific substrate of endogenous CaM K II, in the rabbit. Immunodetected, (32)P-labeled, non-calmodulin binding protein, behaved as a single 23-kDa protein species under several electrophoretic conditions. The 23-kDa protein, with defined properties, was isolated as a complex with 60-kDa delta CaM K II isoform, by sucrose-density sedimentation analysis. Moreover, we show here that putative alphaKAP, in spite of its inability to bind CaM in ligand blot overlay, co-eluted with delta CaM K II from CaM-affinity columns. That raises the question of whether CaM K II-mediated phosphorylation of alpha KAP and triadin together might be involved in a molecular signaling pathway important for SR Ca(2+)-release in fast-twitch muscle SR.

  16. Identification and characterization of alpha-I-proteinase inhibitor from common carp sarcoplasmic proteins.

    Science.gov (United States)

    Siriangkanakun, Siriphon; Li-Chan, Eunice C Y; Yongsawadigul, Jirawat

    2016-02-01

    Purification of proteinase inhibitor from common carp (Cyprinus carpio) sarcoplasmic proteins resulted in 2.8% yield with purification fold of 111. Two inhibitors, namely inhibitor I and II, exhibited molecular mass of 47 and 52 kDa, respectively, based on non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both inhibitors I and II were identified to be alpha-1-proteinase inhibitor (α1-PI) based on LC-MS/MS. They were glycoproteins and molecular mass after peptide-N-glycosidase F treatment was 38 and 45 kDa, respectively. The N-glycosylation sites of both inhibitors were determined to be at N214 and N226. The inhibitors specifically inhibited trypsin. The common carp α1-PI showed high thermal stability with denaturation temperatures of 65.43 and 73.31 °C, which were slightly less than those of ovomucoid. High stability toward NaCl was also evident up to 3M. The common carp α1-PI effectively reduced autolytic degradation of bigeye snapper surimi at the concentration as low as 0.025%.

  17. Design and characterization of self-assembled fish sarcoplasmic protein-alginate nanocomplexes.

    Science.gov (United States)

    Stephansen, Karen; Mattebjerg, Maria; Wattjes, Jasper; Milisavljevic, Ana; Jessen, Flemming; Qvortrup, Klaus; Goycoolea, Francisco M; Chronakis, Ioannis S

    2015-05-01

    Macrostructures based on natural polymers are subject to large attention, as the application range is wide within the food and pharmaceutical industries. In this study we present nanocomplexes (NCXs) made from electrostatic self-assembly between negatively charged alginate and positively charged fish sarcoplasmic proteins (FSP), prepared by bulk mixing. A concentration screening revealed that there was a range of alginate and FSP concentrations where stable NCXs with similar properties were formed, rather than two exact concentrations. The size of the NCXs was 293 ± 3 nm, and the zeta potential was -42 ± 0.3 mV. The NCXs were stable in water, gastric buffer, intestinal buffer and HEPES buffered glycose, and at all pH values from 2 to 9 except pH 3, where they aggregated. When proteolytic enzymes were present in the buffer, the NCXs were degraded. Only at high concentrations the NCXs caused a decreased viability in HeLa and U2OS cell lines. The simple processing procedure and the high stability of the NCXs, makes them excellent candidates for use in the food and pharmaceutical industry. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Protein kinase C modulation of the regulation of sarcoplasmic reticular function by protein kinase A-mediated phospholamban phosphorylation in diabetic rats.

    Science.gov (United States)

    Watanuki, Satoko; Matsuda, Naoyuki; Sakuraya, Fumika; Jesmin, Subrina; Hattori, Yuichi

    2004-01-01

    1. The goal of this study was to elucidate the possible mechanisms by which protein kinase A (PKA)-mediated regulation of the sarcoplasmic reticulum (SR) via phospholambin protein phosphorylation is functionally impaired in streptozotocin-induced diabetic rats. 2. Phospholamban (PLB) protein and mRNA levels were 1.3-fold higher in diabetic than in control hearts, while protein expression of cardiac SR Ca(2+)-ATPase (SERCA2a) was unchanged. 3. Basal and isoprenaline-stimulated phosphorylation of PLB at Ser(16) or Thr(17) was unchanged in diabetic hearts. However, stronger immunoreactivity was observed at the basal level in diabetic hearts when antiphosphoserine antibody was used. 4. Basal (32)P incorporation into PLB was significantly higher in diabetic than in control SR vesicles, but the extent of the PKA-mediated increase in PLB phosphorylation was the same in the two groups of vesicles. 5. Stimulation of Ca(2+) uptake by PKA-catalyzed PLB phosphorylation was weaker in diabetic than in control SR vesicles. The PKA-induced increase in Ca(2+) uptake was attenuated when control SR vesicles were preincubated with protein kinase C (PKC). 6. PKC activities were increased by more than two-fold in the membranous fractions from diabetic hearts in comparison with control values, regardless of whether Ca(2+) was present. This was associated with increases in the protein content of PKCdelta, PKCeta, PKCiota, and PKClambda in diabetic membranous fractions. 7. The changes observed in diabetic rats were reversed by insulin therapy. 8. These results suggest that PKA-dependent phosphorylation may incompletely counteract the function of PLB as an inhibitor of SERCA2a activity in diabetes in which PKC expression and activity are enhanced.

  19. Shrimp allergy beyond Tropomyosin in Italy: clinical relevance of Arginine Kinase, Sarcoplasmic calcium binding protein and Hemocyanin.

    Science.gov (United States)

    Giuffrida, M G; Villalta, D; Mistrello, G; Amato, S; Asero, R

    2014-09-01

    Little is known about the prevalence and clinical relevance of sensitization to shrimp allergens other than tropomyosin. We detected the prevalence of arginine kinase and sarcoplasmic calcium binding protein sensitization, and identified a high molecular weight allergen that is frequently recognized by Italian shrimp-allergic patients. Sera from 40 shrimp-allergic patients underwent the detection of IgE specific for arginine kinase (rPen m 2) and sarcoplasmic calcium-binding protein (rPen m 4) by ISAC 112 Microarray platform and immunoblot analysis. A high molecular weight shrimp allergen was identified by N-terminal amino acid sequencing. IgE to rPen m 2 and rPen m 4 were found in 4/40 (10%) and 6/40 (15%) sera, respectively; two sera reacted to both allergens. Clinically, 6/8 Pen m 2 and/or Pen m 4 reactors experienced severe allergies to shrimp. On immunoblot, 4/6 rPen m 4-positive sera showed IgE reactivity at about 20 kDa, whereas no rPen m 2-positive serum reacted at about 40 kDa. Nineteen (47%) sera showed IgE reactivity at molecular weights > 60 kDa. Such profile was not associated with IgE reactivity to rPen m 2 or rPen m 4. N-terminal amino acid sequencing of the high molecular weight allergen led to the identification of hemocyanin. Shrimp arginine kinase and sarcoplasmic calcium-binding protein are minor allergens sensitizing only 10%-15% of Italian shrimp-allergic patients, but are clinically relevant. Hemocyanin is a clinically relevant high molecular weight shrimp allergen possibly cross-reacting to house dust mite.

  20. The use of label-free mass spectrometry for relative quantification of sarcoplasmic proteins during the processing of dry-cured ham.

    Science.gov (United States)

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

    2016-04-01

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

  1. Proteomic study of muscle sarcoplasmic proteins using AUT-PAGE/SDS-PAGE as two-dimensional gel electrophoresis.

    Science.gov (United States)

    Picariello, Gianluca; De Martino, Alessandra; Mamone, Gianfranco; Ferranti, Pasquale; Addeo, Francesco; Faccia, Michele; Spagnamusso, Salvatore; Di Luccia, Aldo

    2006-03-20

    In the present study, an alternative procedure for two-dimensional (2D) electrophoretic analysis in proteomic investigation of the most represented basic muscle water-soluble proteins is suggested. Our method consists of Acetic acid-Urea-Triton polyacrylamide gel (AUT-PAGE) analysis in the first dimension and standard sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) in the second dimension. Although standard two-dimensional Immobilized pH Gradient-Sodium Dodecyl-Sulphate (2D IPG-SDS) gel electrophoresis has been successfully used to study these proteins, most of the water-soluble proteins are spread on the alkaline part of the 2D map and are poorly focused. Furthermore, the similarity in their molecular weights impairs resolution of the classical approach. The addition of Triton X-100, a non-ionic detergent, into the gel induces a differential electrophoretic mobility of proteins as a result of the formation of mixed micelles between the detergent and the hydrophobic moieties of polypeptides, separating basic proteins with a criterion similar to reversed phase chromatography based on their hydrophobicity. The acid pH induces positive net charges, increasing with the isoelectric point of proteins, thus allowing enhanced resolution in the separation. By using 2D AUT-PAGE/SDS electrophoresis approach to separate water-soluble proteins from fresh pork and from dry-cured products, we could spread proteins over a greater area, achieving a greater resolution than that obtained by IPG in the pH range 3-10 and 6-11. Sarcoplasmic proteins undergoing proteolysis during the ripening of products were identified by Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-ToF) mass spectrometry peptide mass fingerprinting in a easier and more effective way. Two-dimensional AUT-PAGE/SDS electrophoresis has allowed to simplify separation of sarcoplasmic protein mixtures making this technique suitable in the defining of quality of dry-cured pork products by immediate

  2. Effects of simvastatin on cardiac performance and expression of sarcoplasmic reticular calcium regulatory proteins in rat heart

    Institute of Scientific and Technical Information of China (English)

    Xia ZHENG; Shen-jiang HU

    2005-01-01

    Aim: To investigate the effect of simvastatin on the cardiac contractile function and the alteration of gene and protein expression of the sarcoplasmic calcium regulatory proteins, including sarcoplasmic reticulum Ca2+-ATPase (SERCA),phospholamban (PLB), and ryanodine receptor 2 (RyR2) in rat hearts. Methods:Langendorff-perfused rat hearts were subjected to 60-min perfusion with different concentrations of simvastatin (1, 3, 10, 30, or 100 μmol/L), and the parameters of cardiac function such as left ventricular developed pressure (LVDP), +dp/dtmax,and -dp/dtmax were determined. The cultured neonatal rat ventricular cardiomyocytes were incubated with simvastatin (1, 3, 10, 30, and 100 μmol/L) for 1 h or 24 h.The levels of SERCA, PLB, and RyR2 expression were measured by reverse transcription-polymerase chain reaction and Western blot. Cytotoxic effect of simvastatin on ventricular cardiomyocytes was assessed by the MTT colorimetric assay.Results: LVDP, +dp/dtmax, and -dp/dtmax of hearts were increased significantly after treatment with simvastatin 3, 10, and 30 μmol/L. In simvastatin-treated isolated hearts, the levels of mRNA expression of SERCA and RyR2 were elevated compared with the control (P<0.05), while the mRNA expression of PLB did not change. After the cultured neonatal rat ventricular cardiomyocytes were incubated with 3, 10, 30, and 100 μmol/L simvastatin for 1 h, SERCA and RyR2 mRNA expressions of cardiomyocytes rose, but there was no alteration in protein expressions. However, with the elongation of simvastatin treatment to 24 h, the protein expression of SERCA and RyR2 were also elevated. Additionally,simvastatin (1-30 μmol/L) had no influence on cell viability of cultured cardiac myocytes, but simvastatin 100 μmol/L inhibited the cell viability. Conclusion:Simvastatin improved cardiac performance accompanied by the elevation of SERCA and RyR2 gene and protein expression.

  3. 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 < 0.01), intramuscular fat (2.15 vs. 0.98%; P < 0.01), and collagen (1.36 vs. 1.22%; P < 0.01), and lower content of protein (18.7 vs. 22.8%; P < 0.01) and ash (1.14 vs. 1.34%; P < 0.01), in comparison with normal fillets. There was a great decline in myofibrillar (14.0 vs. 8.7%; P < 0.01) and sarcoplasmic (3.2 vs. 2.6%; P < 0.01) content and solubility as well as an increase in cooking loss (33.7 vs. 27.4%; P < 0.05) due to white striping defects. Moreover, gel electrophoresis showed that the concentration of 3 myofibrillar proteins corresponding to actin (42 kDa); LC1, slow-twitch light chain myosin (27.5 kDa); and LC3, fast-twitch light chain myosin (16 kDa), and almost all sarcoplasmic proteins were lower than normal. In conclusion, the findings of this study revealed that 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

  4. Effects of pH-treated Fish Sarcoplasmic Proteins on the Functional Properties of Chicken Myofibrillar Protein Gel Mediated by Microbial Transglutaminase.

    Science.gov (United States)

    Hemung, Bung-Orn; Chin, Koo Bok

    2014-01-01

    pH adjustment would be of advantage in improving the water holding capacity of muscle proteins. The objective of this study was to evaluate the addition of fish sarcoplasmic protein (SP) solution, which was adjusted to pH 3.0 or 12.0, neutralized to pH 7.0, and lyophilized to obtain the acid- and alkaline-treated SP samples, on the functional properties of the chicken myofibrillar protein induced by microbial transglutaminase (MTG). The solubility of alkaline-treated SP was higher than that of the acid counterpart; however, those values of the two pH-treated samples were lower than that of normal SP (pproteins (MP) extracted from chicken breast, and incubated with MTG. The shear stresses of MP with acid- and alkaline-treated SP were higher than that of normal SP. The thermal stability of MP mixture reduced upon adding SP, regardless of the pH treatment. The breaking force of MP gels with acid-treated SP increased more than those of alkaline-treated SP, while normal SP showed the highest value. The MP gel lightness increased, but cooking loss reduced, with the addition of SP. Smooth microstructure of the gel surface was observed. These results indicated that adjusting the pH of SP improved the water holding capacity of chicken myofibrillar proteins induced by MTG.

  5. 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

  6. Safety of protein hydrolysates, fractions thereof and

    NARCIS (Netherlands)

    Schaafsma, Gertjan

    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

  7. Gel-based phosphoproteomics analysis of sarcoplasmic proteins in postmortem porcine muscle with pH decline rate and time differences

    DEFF Research Database (Denmark)

    Huang, Honggang; Larsen, Martin Røssel; Karlsson, Anders H

    2011-01-01

    Meat quality development is highly influenced by the pH decline caused by the postmortem (PM) glycolysis. Protein phosphorylation is an important mechanism in regulating the activity of glycometabolic enzymes. Here, a gel-based phosphoproteomic study was performed to analyze the protein...... phosphorylation in sarcoplasmic proteins from three groups of pigs with different pH decline rates from PM 1 to 24¿h. Globally, the fast pH decline group had the highest phosphorylation level at PM 1¿h, but lowest at 24¿h, whereas the slow pH decline group showed the reverse case. The same pattern was also...... observed in most individual bands in 1-DE. The protein phosphorylation levels of 12 bands were significantly affected by the synergy effects of pH and time (p...

  8. Protein-contg. fraction from mussel feet

    NARCIS (Netherlands)

    Van der Leeden, M.C.; Groen, B.W.

    1997-01-01

    Abstract of NL 1000732 (C1) A protein-contg. fraction suitable for use as an adhesive that can provide an adhesive bond with a strength of more than 15 N/cm2 is claimed. The fraction comprises one or more proteins contg. 3,4-dihydroxyphenylalanine (Dopa) and is obtd. by a process comprising trea

  9. Response of Dairy Cows to Protein Fractions

    OpenAIRE

    GÜNEY, Mehtap; KARSLI, M. Akif

    2014-01-01

     Proteins are the most important factors affecting microbial growth and milk production at ruminant animals. The continuation of reproduction, growth and milk production in dairy cattle are possible with existence of amino acids that is the monomers of protein in diet. The importance of protein fraction to determine the protein requirements of ruminant animals, the utilization of nitrogenous sources consumed by dairy cattle with feedstuffs and their effects on milk production has been explain...

  10. Decavanadate interactions with sarcoplasmic reticulum calcium pump

    OpenAIRE

    2007-01-01

    Although not stable, once formed, decameric vanadate (V10) disintegration is in general slow enough to allow the study of its effects even in the micromolar range. Besides, it may become inaccessible to decomposition due to their specific interaction upon target proteins such as the Ca2+-ATPase from sarcoplasmic reticulum (SR). Characterization of the vanadate solutions and interactions with compounds containing phosphate as well as with the SR Ca2+-ATPase was analysed by 51V NMR spectr...

  11. Skeletal muscle myofibrillar and sarcoplasmic protein synthesis rates are affected differently by altitude-induced hypoxia in native lowlanders

    DEFF Research Database (Denmark)

    Holm, Lars; Haslund, Mads Lyhne; Robach, Paul

    2010-01-01

    As a consequence to hypobaric hypoxic exposure skeletal muscle atrophy is often reported. The underlying mechanism has been suggested to involve a decrease in protein synthesis in order to conserve O(2). With the aim to challenge this hypothesis, we applied a primed, constant infusion of 1-(13)C-...

  12. Ameliorated stress related proteins are associated with improved cardiac function by sarcoplasmic reticulum calcium ATPase gene transfer in heart failure

    Institute of Scientific and Technical Information of China (English)

    Zhi-Qing Fu; Xiao-Ying Li; Xiao-Chun Lu; Ya-Fei Mi; Tao Liu; Wei-Hua Ye

    2012-01-01

    Background Previous studies showed that overexpression of sarco-endoplasmic reticulum calcium ATPase (SERCA2a) in a variety of heart failure (HF) models was associated with greatly enhanced cardiac performance. However, it still undefined the effect of SERCA2a overexpression on the systemic inflammatory response and neuro-hormonal factors. Methods A rapid right ventricular pacing model of experimental HF was used in beagles. Then the animals underwent recombinant adeno-associated virus 1 (rAAV1) mediated gene transfection by direct intra-myocardium injection. HF animals were randomized to receive the SERCA2a gene, enhanced green fluorescent protein (control) gene, or equivalent phosphate buffered saline. Thirty days after gene delivery, the cardiac function was evaluated by echocardiographic testing. The protein level of SERCA2a was measured by western blotting. The proteomic analysis of left ventricular (LV) sample was determined using two-dimensional (2-D) gel electrophoresis and MALDI-TOF-MS. The serum levels of the systemic inflammatory and neuro-hormonal factors were assayed using radioimmunoassay kits. Results The cardiac function improved after SERCA- 2a gene transfer due to the significantly increased SERCA2a protein level. Beagles treated with SERCA2a had significantly decreased serum levels of the inflammatory markers (interleukin-6 and tumor necrosis factor-α) and neuro-hormonal factors (brain natriuretic peptide, endothelin-1 and angiotensin Ⅱ) compared with HF animals. The myocardial proteomic analysis showed that haptoglobin heavy chain, heat shock protein (alpha-crystallin-related, B6) were down-regulated, and galectin-1 was up-regulated in SERCA2a group compared with HF group, companied by up-regulated contractile proteins and NADH dehydrogenase. Conclusions These findings demonstrate that regional intramyocardial injections of rAAV1-SERCA2a vectors may improve global LV function, correlating with reverse activation of the systemic inflammatory

  13. Low molecular weight peptides derived from sarcoplasmic proteins produced by an autochthonous starter culture in a beaker sausage model

    Directory of Open Access Journals (Sweden)

    Constanza M. López

    2015-06-01

    Significance: The selection of a specific autochthonous starter culture guarantees the hygiene and typicity of fermented sausages. The identification of new peptides as well as new target proteins by means of peptidomics represents a significant step toward the elucidation of the role of microorganisms in meat proteolysis. Moreover, these peptides may be further used as biomarkers capable to certify the use of the applied autochthonous starter culture described here.

  14. Skeletal muscle myofibrillar and sarcoplasmic protein synthesis rates are affected differently by altitude-induced hypoxia in native lowlanders

    DEFF Research Database (Denmark)

    Holm, Lars; Haslund, Mads Lyhne; Robach, Paul;

    2010-01-01

    and expired breath samples were collected hourly during the 4 hour trial and vastus lateralis muscle biopsies obtained at 1 and 4 hours after tracer priming in the overnight fasted state. Myofibrillar protein synthesis rate was doubled; 0.041±0.018 at sea-level to 0.080±0.018%·hr(-1) (p0.05). Trends...

  15. 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 functiona

  16. Synaptosomal protein synthesis in P2 and Ficoll purified fractions.

    Science.gov (United States)

    Eyman, Maria; Cefaliello, Carolina; Bruno, Anna Paola; Bruno, Annapaola; Crispino, Marianna; Giuditta, Antonio

    2012-01-30

    Cytoplasmic protein synthesis of brain synaptosomes has generally been determined in the Ficoll purified fraction which contains fewer contaminating mitochondria, microsomes and myelin fragments than the parent P2 fraction. Using a highly selective assay of this activity we have compared the total translation activity and the specific activity of the proteins synthesized by either fraction in control rats and in rats trained for a two-way active avoidance task. In control rats the specific activity remained essentially the same in both fractions but in trained rats the value of the Ficoll fraction was markedly lower (38.5%) than in the P2 fraction. Furthermore, the total translation activity of the Ficoll fraction was 30% lower than in the P2 fraction in control rats and 62% lower in trained rats. These decrements indicate that a large proportion of active synaptosomes present in the P2 fraction is not recovered in the Ficoll fraction, notably in rats undergoing plastic brain changes. We conclude that cytoplasmic protein synthesis of brain synaptosomes is better preserved in the P2 fraction.

  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. 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 antioxidative activity obtained from a cod protein hydrolysate. The free amino acids in all fractions were dominated by Ala, Gly, Glu and Ser. The total amino acid composition had high proportions of Lys, Ala...... to the antioxidative activity of the peptide fractions, and Tyr seemed to play a major role in the antioxidant activity....

  19. Protein microarrays using liquid phase fractionation of cell lysates.

    Science.gov (United States)

    Yan, Fang; Sreekumar, Arun; Laxman, Bharathi; Chinnaiyan, Arul M; Lubman, David M; Barder, Timothy J

    2003-07-01

    We describe an approach in which protein microarrays are produced using a two-dimensional (2-D) liquid phase fractionation of cell lysates. The method involves a pI-based fractionation using chromatofocusing in the first dimension followed by nonporous reversed-phase high-performance liquid chromatography (HPLC) of each pI fraction in the second dimension. This allows fractionation of cellular proteins in the liquid phase that could then be arrayed on nitrocellulose slides and used to study humoral response in cancer. Protein microarrays have been used to identify potential serum biomarkers for prostate cancer. It is shown that specific fractions are immunoreactive against prostate cancer serum but not against serum from healthy individuals. These proteins could serve as sero-diagnostic markers for prostate cancer. Importantly, this method allows for use of post-translationally modified proteins as baits for detection of humoral response. Proteins eliciting an immune response are identified using the molecular mass and peptide sequence data obtained using mass spectrometric analysis of the liquid fractions. The fractionation of proteins in the liquid phase make this method amenable to automation.

  20. Aqueous fractionation yields chemically stable lupin protein isolates

    NARCIS (Netherlands)

    Berghout, J.A.M.; Marmolejo-Garcia, C.; Berton-Carabin, C.C.; Nikiforidis, C.V.; Boom, R.M.; Goot, van der A.J.

    2015-01-01

    The chemical stability of lupin protein isolates (LPIs) obtained through aqueous fractionation (AF, i.e. fractionation without the use of an organic solvent) at 4 °C or 20 °C was assessed. AF of lupin seeds results in LPIs containing 2 wt.% oil. This oil is composed of mono- and poly-unsaturated fat

  1. Structure characterization of protein fractions from lotus ( Nelumbo nucifera) seed

    Science.gov (United States)

    Zeng, Hong-Yan; Cai, Lian-Hui; Cai, Xi-Ling; Wang, Ya-Ju; Li, Yu-Qin

    2011-08-01

    Protein fractionation of lotus seed was carried out and the structures of the protein fractions were studied. Fourier transform infrared spectroscopy (FTIR) as well as ultraviolet visible spectroscopy (UV-vis) was used to investigate changes in molecular structures of the protein fractions. FTIR and UV-vis spectra showed the protein fractions had different protein molecular structures. FTIR spectra showed β-sheets and β-turns as the major secondary structures in the individual protein fractions, while the amounts of α-helix and random coil structures among the different fractions did not significantly change. The amounts of β-sheet structures of albumin and globulin were significantly higher than ones of prolamin and glutelin, implying albumin and globulin had high stabilities because of the high content in β-sheet structures. The observed similarity in the amounts of α-helix, random coil, β-sheet and β-turn structures shared by albumin and globulin indicated that their interior conformations were similar.

  2. [Protein fraction distribution in milling and screened physical fractions of grain amaranth].

    Science.gov (United States)

    Búcaro Segura, María Ester; Bressani, Ricardo

    2002-06-01

    The purpose of the study was to establish the protein distribution based on solubility in physical fractions of amaranth flour, in particular between the flour from the germ and that from the perisperm. The protein distribution was obtained applying a series of solvents sequentially utilized in the classical methodology of Osborne & Mendel. The sample of A. cruentus weighing 2000 g was divided into 4 subsamples of 500 g each. One was left as the control while the other 3 were ground individually with a mill. Each flour was screened through 18, 20, 30 and 40 mesh screens, so that 5 fractions were obtained from each of the whole grain flours. Samples of each screened fractions were observed by stereoscopy and analyzed for moisture, fat and protein. This characterization suggested that the fraction above the 30 mesh screen and the flour which passed the 40 mesh screen probably were the perisperm and germ respectively. The 30 mesh sample contained 2.34 fat and 9.05% protein while the 40 mesh contained 16.18% fat and 26.46% protein. The extraction and partitioning of the proteins indicated that the most important fractions in germ and perisperm were the water soluble and glutelins measured by Kjeldahl. The relationship of the water soluble + globulin to glutelins ratio was 2.1 to 1 in the whole grain, 1.9 to 1 in the perisperm and 1.7 to 1 in the germ. The distribution of proteins was very much alike between germ and perisperm. The levels of prolamines were quite low. The protein extraction of the perisperm proteins retained on the 30 mesh screen was low (71.1%) measured by Kjeldahl and 47.4% with the Bradford method to measure protein.

  3. 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

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

    Science.gov (United States)

    Sen, M; Bhattacharyya, D K

    2000-01-01

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

  5. PROTEIN FRACTIONATION AND IN VITRO DIGESTIBILITY OF AZOLLA IN RUMINANTS

    Directory of Open Access Journals (Sweden)

    S. PARASHURAMULU

    2013-05-01

    Full Text Available A study was undertaken to evaluate the nutritive value and digestibility of Azolla in ruminants by in vitro techniques. The crude protein, crude fibre and ether extract contents were at a level of 21.37%, 12.5% and 2.3%, respectively. The neutral and acid detergent fibre levels were about 35.4 and 23.9%, respectively. The average in vitro dry matter digestibility, in vitro organic matter digestibility and metabolozable energy contents were 79.5%, 63.8 mg/200mg and 7.36 MJ/kg DM (1759 kcal/kg, respectively. The various protein fractions A, B1, B2, B3 and C estimated by Cornell net crude protein solubility system were 18.22, 42.56, 15.15, 7.47 and 16.61% of total protein, respectively. The Azolla contained significantly higher B1 fraction followed by A, B2 and C and lowest fraction of C. Thus in view of above, present study indicated Azolla to be a good source protein supplement with 21.37% crude protein with highest B protein fractions, moderate source of energy (1759 kcal ME/kg, high dry matter and organic matter digestibilities and rich in trace minerals thus could be used as an alternate protein supplement or as supplementary protein supplement to ruminants.

  6. Physicochemical and Functional Characteristics Changes of Catfish Sarcoplasmic Proteins Subjected to pH-Shift Method%pH变化对鲶鱼肌浆蛋白理化和功能特性的影响

    Institute of Scientific and Technical Information of China (English)

    李鹏; 李沛然; 郭耀华; 岳兰昕; 张乃琳; 刘彩虹; 马俪珍

    2014-01-01

    This paper used the sarcoplasmic protein of washing water from catfish (Clarias gariepinus), and subjected to pH-shift method (pH=3.0→7.0, pH=5.0→7.0, pH=7.1→7.0, pH=9.0→7.0, pH=11.0→7.0) as the research object. Each treatment group was analyzed by protein solubility, surface hydrophobicity, total sulfhydryl content, SDS-PAGE, rheological property and the change of thermal denaturation. Results showed, as the environment of pH to the direction of the acidic or alkaline, sarcoplasm protein solubility, surface hydrophobicity and total sulfydryl content had decreased, protein oxiution increased, meanwhile, rheological properties (viscosity) and thermal denaturation temperature improved.%提取鲶鱼肉漂洗液中的肌浆蛋白,用酸或碱以5种方式(①pH=3.0→7.0;②pH=5.0→7.0;③pH=7.1→7.0;④pH=9.0→7.0;⑤pH=11.0→7.0)调节其pH后,研究经过这种pH变化后的肌浆蛋白的蛋白溶解度、表面疏水性、总巯基含量、SDS-PAGE、流变性和热变性等指标的变化,以期为漂洗液中肌浆蛋白的综合利用奠定理论基础。研究结果表明:随着pH向酸性或者碱性的方向逐渐变化,肌浆蛋白溶解度、表面疏水性和总巯基含量不断下降,蛋白氧化加剧,流变学特性(黏度值)和热变性温度不断增加。

  7. 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.

  8. Composition and molecular weight distribution of carob germ protein fractions.

    Science.gov (United States)

    Smith, Brennan M; Bean, Scott R; Schober, Tilman J; Tilley, Michael; Herald, Thomas J; Aramouni, Fadi

    2010-07-14

    Biochemical properties of carob germ proteins were analyzed using a combination of selective extraction, reversed-phase high-performance liquid chromatography (RP-HPLC), size exclusion chromatography (SEC) coupled with multiangle laser light scattering (SEC-MALS), and electrophoretic analysis. Using a modified Osborne extraction procedure, carob germ flour proteins were found to contain approximately 32% albumin and globulin and approximately 68% glutelin with no prolamins detected. The albumin and globulin fraction was found to contain low amounts of disulfide-bonded polymers with relatively low M(w) ranging up to 5 x 10(6) Da. The glutelin fraction, however, was found to contain large amounts of high molecular weight disulfide-bonded polymers with M(w) up to 8 x 10(7) Da. When extracted under nonreducing conditions and divided into soluble and insoluble proteins as typically done for wheat gluten, carob germ proteins were found to be almost entirely ( approximately 95%) in the soluble fraction with only ( approximately 5%) in the insoluble fraction. As in wheat, SEC-MALS analysis showed that the insoluble proteins had a greater M(w) than the soluble proteins and ranged up to 8 x 10(7) Da. The lower M(w) distribution of the polymeric proteins of carob germ flour may account for differences in functionality between wheat and carob germ flour.

  9. [Electrophoretic studies of serum protein fractions in horses with laminitis].

    Science.gov (United States)

    Edinger, H; Miller, I; Stanek, C; Gemeiner, M

    1992-10-01

    The spectrum of serum proteins was evaluated in 46 horses affected with spontaneous laminitis and correlations between the severity of the disease and changes of the protein pattern were analyzed. The investigation was made in two groups; group A consisted of 21 horses of various breeds (warmblood, thoroughbred, standardbred) and group B of 25 ponys. Each group was subdivided according to the severity of the disease, using the OBEL-grade (OG) classification system. Serum proteins were separated by different one- and two-dimensional electrophoretic methods. Sera analysed by cellulose acetate electrophoresis showed a significant difference in the alpha 1-globulin fraction between OG II and OG IV affected horses. An increasing severity of the disease was correlated with a decrease of the alpha 1-globulins. The other protein fractions didn't show a uniform tendency. In group B there was a significant difference in the alpha 1-globulin fractions of OG II and OG III and in the beta 2-globulin fractions of OG I and OG II affected ponys. The acute phase proteins C3c, C4, Hp and fibronectin could be determined in a preliminary study in horse serum using the cross-reactivity of antibodies against the homologous human proteins.

  10. [Fractional and amino acid composition of krill proteins and the potential for obtaining protein preparations].

    Science.gov (United States)

    Orlova, T A; Churina, E E; Kuranova, L K

    1985-01-01

    Studies of the fractional composition of krill proteins demonstrated that the content of protein fractions changes depending on the time of krill catch. The highest amount of water-soluble proteins is contained by krill caught in December (64%), of salt-soluble by krill caught in June (12%), base-soluble by krill caught in May, September and February (34%). Krill protein contains from 50 to 60% of water- and salt-soluble fractions. Analysis of the amino acid composition of krill proteins showed that it does not differ essentially from that of adequate food proteins.

  11. Native antigen fractionation protein microarrays for biomarker discovery.

    Science.gov (United States)

    Caiazzo, Robert J; O'Rourke, Dennis J; Barder, Timothy J; Nelson, Bryce P; Liu, Brian C-S

    2011-01-01

    In this protocol, we used the T24 human bladder cancer cell line as a source of native antigens to construct fractionated lysate microarrays. Subsequently, these microarrays were used to compare the autoantibody responses of individuals with interstitial cystitis/painful bladder syndrome (IC/PBS) to those of normal female controls. To accomplish this, T24 cells were lysed under nondenaturing conditions to obtain native antigens. These native antigens were then fractionated in 2D using a PF-2D liquid chromatography; the first dimension separated the proteins by their isoelectric points, and the second separated them according to hydrophobicity. The resulting protein fractions were printed onto nitrocellulose-coated glass slides (PATH slides) to create a set of fractionated lysate microarrays. To compare the autoantibody responses of IC/PBS patients with normal controls, the fractionated lysate arrays were competitively hybridized with fluorescently labeled IgG samples purified from both IC/PBS and control sera. This protocol presents a detailed description of the creation and use of native antigen fractionated lysate microarrays for autoantibody profiling.

  12. Fractionation and Characterization of Brewers' Spent Grain Protein Hydrolysates

    OpenAIRE

    Celus, Inge; BRIJS, Kristof; Delcour, Jan

    2009-01-01

    Protein hydrolysates with a low and high degree of hydrolysis were enzymatically produced from brewers' spent grain (BSG), the insoluble residue of barley malt resulting from the manufacture of wort in the production of beer. To that end, BSG protein concentrate (BPC), prepared by alkaline extraction of BSG and subsequent acid precipitation, was enzymatically hydrolyzed with Alcalase during both 1.7 and 120 min. Because these hydrolysates contained many different peptides, fractionation of th...

  13. Biochemical and morphological characterization of light and heavy sarcoplasmic reticulum vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, K.P.

    1978-01-01

    Light and heavy sarcoplasmic reticulum vesicles isolated from rabbit leg muscle have been used in a study of chloride-induced calcium release. The biochemical and morphological data indicate that light sarcoplasmic reticulum vesicles are derived from the longitudinal reticulum and heavy sarcoplasmic reticulum vesicles are derived from the terminal cisternae of the sarcoplasmic reticulum. The light and heavy sarcoplasmic reticulum vesicles were both able to accumulate calcium in the presence of ATP to amounts greater than 100 nmoles Ca/sup + +/ per mg of protein in less than one minute. Light and heavy sarcoplasmic reticulum vesicles each had a biphasic time course of calcium uptake. The initial uptake was followed by a rapid release after approximately one minute, of 30 to 40% of the accumulated calcium, which was then followed by a slower phase of calcium accumulation. Results indicate that the chloride induced release of calcium may be acting by two mechanisms, osmotic swelling and depolarization. The release of calcium from the light SR vesicles is probably due to osmotic swelling and the release of calcium from the heavy SR vesicles is probably due to depolarization.

  14. Protein fractionation byproduct from canola meal for dairy cattle.

    Science.gov (United States)

    Heendeniya, R G; Christensen, D A; Maenz, D D; McKinnon, J J; Yu, P

    2012-08-01

    Fiber-protein is a byproduct arising from a process for fractionating high-quality protein from canola meal. The objective of this study was to evaluate the fiber-protein fraction by examining the chemical profiles, rumen degradation, and intestinal digestive characteristics and determining the nutritive value of the fiber-protein fraction as dietary components for dairy cattle in comparison with commercial canola meal and soybean meal. Available energy values were estimated based on National Research Council guidelines, whereas total true protein content potentially absorbable in the small intestine (DVE) were predicted using the predicted DVE/degraded protein balance (OEB) model. The results show that fiber-protein was a highly fibrous material [neutral detergent fiber (NDF): 556; acid detergent fiber (ADF): 463; acid detergent lignin: 241 g/kg of dry matter (DM)] compared with canola meal (NDF: 254; ADF: 212; acid detergent lignin: 90 g/kg of DM) due to the presence of a higher level of seed hulls in fiber-protein. Compared with canola meal, fiber-protein contained 90 g/kg of DM less crude protein (CP), 25% of which consisted of undegradable acid detergent-insoluble CP. Most of the ruminally undegradable nutrient components present in canola meal appeared to be concentrated into fiber-protein during the manufacturing process and, as a result, fiber-protein showed a consistently lower effective degradability of DM, organic matter, CP, NDF, and ADF compared with both canola meal and soybean meal. Available energy content in fiber-protein contained two-thirds of that of canola meal. The DVE was one-third that of soybean meal and one-fifth that of canola meal [DVE value: 58 vs. 180 (soybean) and 291 g/kg of DM (canola meal)]. The OEB value of fiber protein was positive and about half of that of soybean and canola meal [OEB value: 74 vs. 162 (soybean) and 137 g/kg of DM (canola meal)]. Fiber-protein can be considered as a secondary source of protein in ruminant feed.

  15. Sarcoplasmatic and myofibrillar protein changes caused by acute heat stress in broiler chicken Alterações nas proteínas sarcoplasmáticas e miofibrilares em frangos de corte causadas por estresse térmico agudo

    Directory of Open Access Journals (Sweden)

    Carolina de Castro Santos

    2008-01-01

    Full Text Available Acute heat stress (AHS modifies the structure of myofibrils affecting functional properties of meat, mainly the water holding capacity. This experiment aimed to identify changes in proteolysis and migration between the myofibrillar and sarcoplasmatic fractions due to pre-slaughter AHS. Myofibrillar fragmentation index (MFI, SDS-PAGE, western blot of vinculin (WB and shear force (SF were determined. Six hundred broilers (Gallus gallus were slaughtered in three different days (ST. In each ST, groups of ten animals were placed in transport crates and submitted to AHS (35ºC, 75 - 85% RH for 2 hours. Simultaneously, the non-stressed broilers (NS were kept in thermoneutral environment (22ºC, 83 ± 6.6% RH within the crates in the same density. After slaughter, the breast muscles were kept refrigerated until the withdrawal of all samples (0, 1, 2, 6 and 24 hours after slaughter. Sampling within AHS and NS birds was collected according to lightness value (normal L* 51, except for determination of MFI and SF. The lightness was used later to perform SDS-PAGE and WB analyses. MFI kinetics showed that the fragmentation rate was superior in animals NS, indicating that AHS can harm proteolysis and rate of myofibrillar fragmentation. However, the extent of fragmentation did not change, as well as SF values. SDS-PAGE for Troponin fragments indicated a differentiated pattern between AHS and NS. The WB did not show alterations in vinculin fragmentation. Modifications in sarcoplasmatic fraction are observed in meat with high L*values, independent of environmental condition.O estresse térmico agudo (ET causa alterações na estrutura das miofibrilas, afetando propriedades funcionais da carne, principalmente a capacidade de retenção de água. Identificaram-se mudanças na proteólise e migração entre as frações miofibrilar e sarcoplasmática, decorrentes do ET pré-abate, através do índice de fragmentação miofibrilar (MFI, SDS-PAGE para troponina (SDS

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

    Science.gov (United States)

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

    2013-12-15

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

  17. Biochemical and morphological characterization of light and heavy sarcoplasmic reticulum vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, Kevin Peter [Univ. of Rochester, NY (United States)

    1978-01-01

    Light (30 to 32.5% sucrose) and heavy (38.5 to 42% sucrose) sarcoplasmic reticulum vesicles (LSR,HSR) were isolated from rabbit leg muscle using a combination of differential centrifugation and isopycnic zonal ultracentrifugation. Thin-section electron microscopy of LSR vesicles reveals empty vesicles of various sizes and shapes whereas the HSR vesicles appear as rounded vesicles of uniform size filled with electron dense material, similar to that seen in the terminal cisternae of the sarcoplasmic reticulum. The sucrose HSR vesicles have an additional morphological feature which appears as membrane projections that resemble the SR feet. The freeze-fracture morphology of either type of SR reveals an asymmetric distribution of intramembraneous particles in the same orientation and distribution as the sarcoplasmic reticulum in vivo. Biochemical studies were made on the content of Ca, Mg, ATPase, and protein of the vesicles and phosphorylation of the vesicles. The biochemical and morphological data indicate that the LSR is derived from the longitudinal sarcoplasmic reticulum and the HSR is derived from the terminal cisternae of the sarcoplasmic reticulum, contains junctional SR membrane and has three unique proteins (calsequestrin, an intrinsic 30,000 dalton protein and a 9000 dalton proteolipid).

  18. HUMAN AND MARE'S MILK - PROTEIN FRACTION AND LIPID COMPOSITION

    Directory of Open Access Journals (Sweden)

    Vesna Gantner

    2014-12-01

    Full Text Available In human population if the infants are not breast-fed, a substitute for breast milk is nee¬ded. Use of cow's milk can induce allergies during the first 3 years of life. Alternative could be mare's milk. The objectives of this review were to compare human and mare's milk protein fraction and lipid composition as well as to determine adequacy of mare's milk as substitute for breast milk. Similarities are found regarding the protein and salt content; whey protein and NPN concentrations; structure of protein micelles and lipid globules; proportion of saturated fatty acids and unsaturated fatty acids. Taking into account determined similarities of human and mare's milk, it could be concluded that mare's milk is suitable nourishment for infants.

  19. Sarcoplasmic Reticulum Ca2+ Cycling Protein Phosphorylation in a Physiologic Ca2+ Milieu Unleashes a High-Power, Rhythmic Ca2+ Clock in Ventricular Myocytes: Relevance to Arrhythmias and Bio-Pacemaker Design

    Science.gov (United States)

    Sirenko, Syevda; Maltsev, Victor A; Maltseva, Larissa A; Yang, Dongmei; Lukyanenko, Yevgeniya; Vinogradova, Tatiana; Jones, Larry; Lakatta, Edward G.

    2014-01-01

    Basal phosphorylation of sarcoplasmic reticulum (SR) Ca2+ proteins is high in sinoatrial nodal cells (SANC), which generate partially synchronized, spontaneous, rhythmic, diastolic local Ca2+ releases (LCRs), but low in ventricular myocytes (VM), which exhibit rare diastolic, stochastic SR-generated Ca2+ sparks. We tested the hypothesis that in a physiologic Ca2+ milieu, and independent of increased Ca2+ influx, an increase in basal phosphorylation of SR Ca2+ cycling proteins will convert stochastic Ca2+ sparks into periodic, high-power Ca2+ signals of the type that drives SANC normal automaticity. We measured phosphorylation of SR-associated proteins, phospholamban (PLB) and ryanodine receptors (RyR), and spontaneous local Ca2+ release characteristics (LCR) in permeabilized single, rabbit VM in physiologic [Ca2+], prior to and during inhibition of protein phosphatase (PP) and phosphodiesterase (PDE), or addition of exogenous cAMP, or in the presence of an antibody (2D12), that specifically inhibits binding of the PLB to SERCA-2. In the absence of the aforementioned perturbations, VM could only generate stochastic local Ca2+ releases of low power and low amplitude, as assessed by confocal Ca2+ imaging and spectral analysis. When the kinetics of Ca2+ pumping into the SR were increased by an increase in PLB phosphorylation (via PDE and PP inhibition or addition of cAMP) or by 2D12, self-organized, “clock-like” local Ca2+ releases, partially synchronized in space and time (Ca2+ wavelets), emerged, and the ensemble of these rhythmic local Ca2+ wavelets generated a periodic high-amplitude Ca2+ signal. Thus, a Ca2+ clock is not specific to pacemaker cells, but can also be unleashed in VM when SR Ca2+ cycling increases and spontaneous local Ca2+ release becomes partially synchronized. This unleashed Ca2+ clock that emerges in a physiological Ca2+ milieu in VM has two faces, however: it can provoke ventricular arrhythmias; or if harnessed, can be an important feature

  20. Enhanced sarcoplasmic reticulum Ca(2+) release following intermittent sprint training

    DEFF Research Database (Denmark)

    Ørtenblad, Niels; Lunde, Per; Levin, Kasper

    2000-01-01

    To evaluate the effect of intermittent sprint training on sarcoplasmic reticulum (SR) function, nine young men performed a 5 wk high-intensity intermittent bicycle training, and six served as controls. SR function was evaluated from resting vastus lateralis muscle biopsies, before and after...... the training period. Intermittent sprint performance (ten 8-s all-out periods alternating with 32-s recovery) was enhanced 12% (P training. The 5-wk sprint training induced a significantly higher (P ...-977) arbitrary units Ca(2+). g protein(-1). min(-1) (after). The relative SR density of functional ryanodine receptors (RyR) remained unchanged after training; there was, however, a 48% (P

  1. Enhanced sarcoplasmic reticulum Ca(2+) release following intermittent sprint training

    DEFF Research Database (Denmark)

    Ørtenblad, Niels; Lunde, Per; Levin, Kasper

    2000-01-01

    To evaluate the effect of intermittent sprint training on sarcoplasmic reticulum (SR) function, nine young men performed a 5 wk high-intensity intermittent bicycle training, and six served as controls. SR function was evaluated from resting vastus lateralis muscle biopsies, before and after...... the training period. Intermittent sprint performance (ten 8-s all-out periods alternating with 32-s recovery) was enhanced 12% (P training. The 5-wk sprint training induced a significantly higher (P ...-977) arbitrary units Ca(2+). g protein(-1). min(-1) (after). The relative SR density of functional ryanodine receptors (RyR) remained unchanged after training; there was, however, a 48% (P

  2. 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

    in the nonfractionated colostrum (NF) and seven fractions (F1-F7) using six different fractionation techniques. Fractionation contributed with 69 additional proteins in the fluid phase compared with NF. Different fractionation techniques each resulted in detection of unique subsets of proteins. Whey production by high...

  3. 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......-MS/MS) and GC-tandem MS (GC-MS/MS) have made these techniques an option for human muscle FSR measurements. Human muscle biopsies were freeze dried, cleaned, and hydrolyzed, and the amino acids derivatized using either N-acetyl-n-propyl, phenylisothiocyanate, or N.......89 ± 0.01, P muscle FSR, (2) LC-MS/MS comes quite close and is a good alternative when tissue quantities are too small for GC-C-IRMS, and (3) If GC-MS/MS is to be used, then the HFBA derivative should be used instead...

  4. Generation, Fractionation, and Characterization of Iron-Chelating Protein Hydrolysate from Palm Kernel Cake Proteins.

    Science.gov (United States)

    Zarei, Mohammad; Ghanbari, Rahele; Tajabadi, Naser; Abdul-Hamid, Azizah; Bakar, Fatimah Abu; Saari, Nazamid

    2016-02-01

    Palm kernel cake protein was hydrolyzed with different proteases namely papain, bromelain, subtilisin, flavourzyme, trypsin, chymotrypsin, and pepsin to generate different protein hydrolysates. Peptide content and iron-chelating activity of each hydrolysate were evaluated using O-phthaldialdehyde-based spectrophotometric method and ferrozine-based colorimetric assay, respectively. The results revealed a positive correlation between peptide contents and iron-chelating activities of the protein hydrolysates. Protein hydrolysate generated by papain exhibited the highest peptide content of 10.5 mM and highest iron-chelating activity of 64.8% compared with the other hydrolysates. Profiling of the papain-generated hydrolysate by reverse phase high performance liquid chromatography fractionation indicated a direct association between peptide content and iron-chelating activity in most of the fractions. Further fractionation using isoelectric focusing also revealed that protein hydrolysate with basic and neutral isoelectric point (pI) had the highest iron-chelating activity, although a few fractions in the acidic range also exhibited good metal chelating potential. After identification and synthesis of papain-generated peptides, GGIF and YLLLK showed among the highest iron-chelating activities of 56% and 53%, whereas their IC50 were 1.4 and 0.2 μM, respectively.

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

    Science.gov (United States)

    Sabeena Farvin, K H; Andersen, Lisa Lystbæk; Otte, Jeanette; Nielsen, Henrik Hauch; Jessen, Flemming; Jacobsen, Charlotte

    2016-08-01

    This study aimed to characterise peptide fractions (>5kDa, 3-5kDa and fractions were dominated by Ala, Gly, Glu and Ser. The total amino acid composition had high proportions of Lys, Ala and Glu. The 3-5kDa and fractions were further fractionated by size exclusion chromatography. All sub-fractions showed high Fe(2+) chelating activity. The DPPH radical-scavenging activity of the 3-5kDa fraction was exerted mainly by one sub-fraction dominated by peptides with masses below 600Da. 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 to the antioxidative activity of the peptide fractions, and Tyr seemed to play a major role in the antioxidant activity.

  6. 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...... the identification of most major proteins in the PSD fraction with the use of an analytical method based on mass spectrometry coupled with searching of the protein sequence databases. At least one protein in each of 26 prominent protein bands from the PSD fraction has now been identified. We found 7 proteins...... 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...

  7. Fractionation of proteins with two-sided electro-ultrafiltration.

    Science.gov (United States)

    Käppler, Tobias; Posten, Clemens

    2007-03-10

    Downstream processing is a major challenge in bioprocess industry due to the high complexity of bio-suspensions itself, the low concentration of the product and the stress sensitivity of the valuable target molecules. A multitude of unit operations have to be joined together to achieve an acceptable purity and concentration of the product. Since each of the unit operations leads to a certain product loss, one important aim in downstream-research is the combination of different separation principles into one unit operation. In the current work a dead-end membrane process is combined with an electrophoresis operation. In the past this concept has proven successfully for the concentration of biopolymers. The present work shows that using different ultrafiltration membranes in a two-sided electro-filter apparatus with flushed electrodes brought significant enhancement of the protein fractionation process. Due to electrophoretic effects, the filtration velocity could be kept on a very high level for a long time, furthermore, the selectivity of a binary separation process carried out exemplarily for bovine serum albumin (BSA) and lysozyme (LZ) could be greatly increased; in the current case up to a value of more than 800. Thus the new two-sided electro-ultrafiltration technique achieves both high product purity and short separation times.

  8. 心肺复苏后心功能障碍与心肌内质网Ca2+调控蛋白表达关系的研究%The relationship between sarcoplasmic reticulum Ca2+modulation proteins and postresuscitation myocardial dysfunction

    Institute of Scientific and Technical Information of China (English)

    黄煜; 何庆

    2014-01-01

    Objective To investigate the relationship between sarcoplasmic reticulum Ca2+modulation proteins and postresuscitation myocardial dysfunction. Methods Thirty-eight SPF male Sprague-Dawley (SD) rats were randomly divided into control group(n=12)and cardiac arrest(CA)group(n=26). CA was induced by intravenous bolus of potassium chloride(40μg/g),and cardiopulmonary resuscitation(CPR)was conducted 8 minutes later. No CA was induced in control group except catheter placement for monitoring cardiopulmonary parameters after anesthesia. Invasive hemodynamic parameters were monitored for 1 hour after CPR. Echocardiogram was performed to evaluate cardiac function. Myocardial samples were harvested 5 minutes and 1 hour after restoration of spontaneous circulation (ROSC),and sarcoplasmic reticulum Ca2+ ATPase (SERCA2a),phosphorylated phospholamban (p-PLB) and rynodine receptor(RyR)were determined by Western Blot. Results ROSC rate of CA group was 92.3%(24/26),and mean recovery time was (68 ±39)seconds. Cardiac function was significantly impaired in CA group at 1 hour after resuscitation, and ejection fraction, fraction shortening (FS), the maximal rate of left ventricular pressure increase/decline (±dp/dt max)were significantly decreased compared with those in control group 〔ejection fraction:0.548±0.060 vs. 0.809±0.043,F=71.692,P=0.000;FS:(34.4±4.4)%vs. (46.0±3.5)%,F=55.443,P=0.000;+dp/dt max(mmHg/s):4 718±743 vs. 7 098±394,P0.05). Conclusions The impairment of the p-PLB is closely related to postresuscitation myocardial dysfunction.%目的探讨心肌内质网Ca2+调控蛋白表达与心肺复苏(CPR)后心功能障碍的关系。方法38只SPF级雄性SD大鼠按随机数字表法分为对照组(12只)和心搏骤停组(26只)。静脉弹丸式注射氯化钾40μg/g诱导心搏骤停,8 min后进行CPR;对照组大鼠仅麻醉后置管并监测指标,不诱导心搏骤停。在复苏后进行有创血流动力学监测1 h,采用超声心动

  9. ACUTE PHASE PROTEIN AND ELECTROPHORESIS PROTEIN FRACTION VALUES FOR CAPTIVE AMERICAN FLAMINGOS (PHOENICOPTERUS RUBER).

    Science.gov (United States)

    Delk, Katie W; Wack, Raymund F; Burgdorf-Moisuk, Anne; Kass, Philip H; Cray, Carolyn

    2015-12-01

    Protein electrophoresis has recognized applications in determining the health status of various species. While reference intervals for electrophoresis have been determined for psittacine and raptor species, there are none reported for Phoenicopteriformes species. Reference intervals for haptoglobin and protein fractions obtained by electrophoresis were determined for the American flamingo (Phoenicopterus ruber) based on plasma samples from 39 captive birds. The reference intervals were as follows: haptoglobin, 0.17-0.8 mg/ml; total protein, 3.65-6.38 g/dl; prealbumin, 0.26-1.9 g/dl; albumin, 1.51-3.12 g/dl; α-1 globulin, 0.06-0.38 g/dl; α-2 globulin, 0.17-0.67 g/dl; β globulin, 0.38-1.33 g/dl; γ globulin, 0.26-0.68 g/dl; albumin : globulin ratio, 0.93-2.17. As captive flamingos often suffer from pododermatitis, feet of all flamingos were scored to determine if pododermatitis would be reflected in the acute phase proteins. Spearman rank correlation was performed on each of the protein fractions and pododermatitis scores, and only albumin had a significant correlation. This indicates that albumin, as a negative acute phase protein, may be a marker for this disease process.

  10. Protein bodies of castor bean endosperm: isolation, fractionation, and the characterization of protein components.

    Science.gov (United States)

    Tully, R E; Beevers, H

    1976-12-01

    Protein bodies in the endosperm of castor bean seeds (Ricinus communis L.) contain phytin globoids and protein crystalloids embedded in an amorphous proteinaceous matrix. The protein bodies are apparently surrounded by a single membrane. The protein bodies were isolated by grinding and centrifuging in glycerol. Such isolated protein bodies were almost identical (after cytological fixation) to those observed in situ, except that the globoids were lost. However, membrane-like structures appear to have surrounded the globoids. Histochemical analysis of the isolated protein bodies showed that carbohydrates (glycoproteins) are localized only in the matrix region.Addition of water to protein bodies in glycerol caused dissolution of the matrix, and release of the globoids and crystalloids. When the crystalloids were centrifuged on sucrose density gradients, they were recovered at an equilibrium density of 1.29 to 1.30 g/ml. The crystalloids were only slightly soluble in most aqueous buffers but were very soluble in sodium dodecyl sulfate, urea, or NaOH solutions.Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and chromatography on ion exchange celluloses show that the protein bodies are composed of one major and several minor anodic proteins. The major protein, along with a few of the minor proteins, is localized in the crystalloids.The major protein (molecular weight 65,000) was converted by mercaptoethanol into subunits with molecular weights of 32,000 and 15,800. It is proposed that the protein is made up of two of the smaller subunits and one of the larger, linked by disulfide bridges. None of the crystalloid proteins appear to be glycosylated.The water-soluble matrix fraction is composed mainly of two proteins, with molecular weights of 12,500 and 10,300 on the gels. Neither is a glycoprotein, and neither can be reduced with mercaptoethanol to give subunits. The soluble fraction also contains other lesser components among which are

  11. Impaired relaxation despite upregulated calcium-handling protein atrial myocardium from type 2 diabetic patients with preserved ejection fraction.

    Science.gov (United States)

    Lamberts, Regis R; Lingam, Shivanjali J; Wang, Heng-Yu; Bollen, Ilse A E; Hughes, Gillian; Galvin, Ivor F; Bunton, Richard W; Bahn, Andrew; Katare, Rajesh; Baldi, J Chris; Williams, Michael J A; Saxena, Pankaj; Coffey, Sean; Jones, Peter P

    2014-04-05

    Diastolic dysfunction is a key factor in the development and pathology of cardiac dysfunction in diabetes, however the exact underlying mechanism remains unknown, especially in humans. We aimed to measure contraction, relaxation, expression of calcium-handling proteins and fibrosis in myocardium of diabetic patients with preserved systolic function. Right atrial appendages from patients with type 2 diabetes mellitus (DM, n = 20) and non-diabetic patients (non-DM, n = 36), all with preserved ejection fraction and undergoing coronary artery bypass grafting (CABG), were collected. From appendages, small cardiac muscles, trabeculae, were isolated to measure basal and β-adrenergic stimulated myocardial function. Expression levels of calcium-handling proteins, sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) and phospholamban (PLB), and of β1-adrenoreceptors were determined in tissue samples by Western blot. Collagen deposition was determined by picro-sirius red staining. In trabeculae from diabetic samples, contractile function was preserved, but relaxation was prolonged (Tau: 74 ± 13 ms vs. 93 ± 16 ms, non-DM vs. DM, p = 0.03). The expression of SERCA2a was increased in diabetic myocardial tissue (0.75 ± 0.09 vs. 1.23 ± 0.15, non-DM vs. DM, p = 0.007), whereas its endogenous inhibitor PLB was reduced (2.21 ± 0.45 vs. 0.42 ± 0.11, non-DM vs. DM, p = 0.01). Collagen deposition was increased in diabetic samples. Moreover, trabeculae from diabetic patients were unresponsive to β-adrenergic stimulation, despite no change in β1-adrenoreceptor expression levels. Human type 2 diabetic atrial myocardium showed increased fibrosis without systolic dysfunction but with impaired relaxation, especially during β-adrenergic challenge. Interestingly, changes in calcium-handling protein expression suggests accelerated active calcium re-uptake, thus improved relaxation, indicating a compensatory calcium-handling mechanism in diabetes

  12. Dynamic Changes in Sarcoplasmic Reticulum Structure in Ventricular Myocytes

    Directory of Open Access Journals (Sweden)

    Amanda L. Vega

    2011-01-01

    sarcoplasmic reticulum (SR and the sarcolemma where Ca2+ release is activated. Here, we tested the hypothesis that the SR is a structurally inert organelle in ventricular myocytes. Our data suggest that rather than being static, the SR undergoes frequent dynamic structural changes. SR boutons expressing functional ryanodine receptors moved throughout the cell, approaching or moving away from the sarcolemma of ventricular myocytes. These changes in SR structure occurred in the absence of changes in [Ca2+] during EC coupling. Microtubules and the molecular motors dynein and kinesin 1(Kif5b were important regulators of SR motility. These findings support a model in which the SR is a motile organelle capable of molecular motor protein-driven structural changes.

  13. 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.

  14. Protein precipitating capacity and antioxidant activity of Turkish Tombul hazelnut phenolic extract and its fractions.

    Science.gov (United States)

    Pelvan Pelitli, Ebru; Janiak, Michał Adam; Amarowicz, Ryszard; Alasalvar, Cesarettin

    2017-03-01

    Natural (raw) hazelnut was extracted with 80% (v/v) acetone to obtain crude phenolic extract that was then fractionated for elution of low-molecular weight (LMW) and high-molecular weight (HMW) fractions. LMW fraction was further purified (LWM-FP) to remove sugars and organic acids. The crude extract and its fractions were determined by measuring their protein precipitating capacity (PPC) using two different proteins [bovine serum albumin (BSA) and gelatin], molecular weights, total phenolics, condensed tannins, and various antioxidant activities. Significant differences (p<0.05) existed in the contents of total phenolics, condensed tannins, antioxidant activities, and PPC among the crude extract and fractions, albeit to different extends. BSA and gelatin was effectively precipitated by HMW fraction. HMW fraction had the highest total phenolics, condensed tannins, and antioxidant activities, followed by crude extract, LWM-FP, and LMW, respectively. The present study suggests that HMW fraction could be utilised as a source of polyphenols for the food industry.

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

    Science.gov (United States)

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

    2015-01-01

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

  16. 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% ...

  17. 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.

  18. Protein nutritional quality of cowpea and navy bean residue fractions ...

    African Journals Online (AJOL)

    and navy bean residue-wheat diets was determined using in-vivo and in-vitro protein ... Phytohemagglutinin activity was only detectable in the raw cowpea ... Legume residues after protein extraction could be recommended for human food if

  19. Amino acid composition of Lagenaria siceraria seed flour and protein fractions.

    Science.gov (United States)

    Ogunbusola, Moriyike Esther; Fagbemi, Tayo Nathaniel; Osundahunsi, Oluwatooyin Faramade

    2010-12-01

    Defatted seed flours of Lagenaria siceraria (calabash and bottle gourd) were fractionated into their major protein fractions. The amino acid composition of seed flours and their protein fractions were determined and the protein quality was evaluated. Glutamic acid (139-168 mg/g protein) was the most abundant amino acid followed by aspartic acid (89.0-116 mg/g protein) in both the seed flours and their protein fractions. The total essential amino acid ranged from 45.8 to 51.5%. The predicted protein efficiency ratio and the predicted biological value ranged from 2.4 to 2.9 and 8.7 to 44.0, respectively. Lysine and sulphur amino acids were mostly concentrated in the globulin fractions. The first and second limiting amino acids in seed flours and protein fractions were methionine and valine or threonine. The seed flours contained adequate essential amino acids required by growing school children and adults. The seed has potential as protein supplement in cereal based complementary diets or in the replacement of animal proteins in conventional foods.

  20. The sarcoplasmic calcium pump - a most efficient ion translocating system.

    Science.gov (United States)

    Hasselbach, W

    1977-04-21

    In contrast to the sodium-potassium transporting plasma membranes, the sarcoplasmic membranes (SR) are highly specialized structures into which only two major intrinsic proteins, a calcium transporting protein and a calcium binding protein are embedded. The calcium transporting protein is a highly asymmetric molecule. It binds two calcium ions with a very high affinity at its external, and two calcium ions with low affinity at the internal section of the molecule. ATP is bound with high afffinity to an external binding site, inducing a conformational change. When the vesicular membranes are exposed to solutions containing Ca++, Mg++ and ATP, ATP is hydrolyzed and simultaneously calcium ions are translocated from the external medium into the vesicular space. When calcium ions are translocated in the opposite direction, ATP is synthesized. The calcium-ATP ratio for ATP cleavage as well as for ATP synthesis is 2. Thus, the SR membranes can transform reversibly chemical into osmotical energy. Inward and outward movements of calcium ions are relatively slow processes connected with the appearance and disappearance of different phosphorylated intermediates. One phosphorylated intermediate is formed by phosphoryltransfer from ATP when calcium ions are present in the medium. In contrast, when calcium ions are absent from the external medium, two different intermediates can be formed by the incorporation of inorganic phosphate. Only when calcium ions present in the internal space of the vesicles are released, the incorporation of inorganic phosphate gives rise to an intermediate who phosphoryl group can be transferred to ADP.

  1. Minor sarcoplasmic reticulum membrane components that modulate excitation–contraction coupling in striated muscles

    Science.gov (United States)

    Treves, Susan; Vukcevic, Mirko; Maj, Marcin; Thurnheer, Raphael; Mosca, Barbara; Zorzato, Francesco

    2009-01-01

    In striated muscle, activation of contraction is initiated by membrane depolarisation caused by an action potential, which triggers the release of Ca2+ stored in the sarcoplasmic reticulum by a process called excitation–contraction coupling. Excitation–contraction coupling occurs via a highly sophisticated supramolecular signalling complex at the junction between the sarcoplasmic reticulum and the transverse tubules. It is generally accepted that the core components of the excitation–contraction coupling machinery are the dihydropyridine receptors, ryanodine receptors and calsequestrin, which serve as voltage sensor, Ca2+ release channel, and Ca2+ storage protein, respectively. Nevertheless, a number of additional proteins have been shown to be essential both for the structural formation of the machinery involved in excitation–contraction coupling and for its fine tuning. In this review we discuss the functional role of minor sarcoplasmic reticulum protein components. The definition of their roles in excitation–contraction coupling is important in order to understand how mutations in genes involved in Ca2+ signalling cause neuromuscular disorders. PMID:19403606

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

    Institute of Scientific and Technical Information of China (English)

    LiuXiaobing; LiWenxiong; 等

    1995-01-01

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

  3. The total protein content, protein fractions and proteases activities of drone prepupae of Apis mellifera due to varrosis.

    Science.gov (United States)

    Zółtowska, Krystyna; Lipiński, Zbigniew; Dmitryjuk, Małgorzata

    2005-01-01

    The proteins level and activities of acid and alkaline proteases in whole body extracts of drone prepupae of Apis mellifera naturally infested with Varroa destructor were studied. The infested and a non-infested group did not differ significantly in their total protein content. However, some differences in protein profiles were found. A lack of three protein fractions of moderate and lower molecular weight in infested prepupae was noted. Moreover, some differences in the quantity of protein in most of the fractions were observed. The activity of acid proteases from infested prepupae was lower (p drone had higher activity of alkaline proteases than non-infested but this difference was not statisticaly significant.

  4. Utilization of supercritical carbon dioxide to produce milk protein fractions

    Science.gov (United States)

    The nutritional, functional and bioactive properties of the individual whey proteins are appreciated by health-conscious consumers, yet few methods have been developed to produce these proteins to satisfy demand. The methods that are available are relatively new technologies that have not been prove...

  5. 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

  6. 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

    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.......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......H to 4.5 and the obtained supernatant was further fractionated by using ultrafiltration membranes with molecular weight cut offs of 50, 10 and 1kDa. The obtained >50kDa, 50–10kDa, 10–1kDa fractions and pH precipitated fraction were studied for their functional properties and antioxidant activity...

  7. Hydrolysis of Whey Protein Isolate with Bacillus licheniformis Protease: Aggregating Capacities of Peptide Fractions

    NARCIS (Netherlands)

    Creusot, N.P.; Gruppen, H.

    2008-01-01

    In a previous study, peptides aggregating at pH 7.0 derived from a whey protein hydrolysate made with Bacillus licheniformis protease were fractionated and identified. The objective of the present work was to investigate the solubility of the fractionated aggregating pepticles, as a function of conc

  8. 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.

  9. 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.

  10. 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.

  11. A fast method for the determination of fractional contributions to solvation in proteins

    Science.gov (United States)

    Talavera, David; Morreale, Antonio; Meyer, Tim; Hospital, Adam; Ferrer-Costa, Carles; Gelpi, Josep Lluis; de la Cruz, Xavier; Soliva, Robert; Luque, F. Javier; Orozco, Modesto

    2006-01-01

    A fast method for the calculation of residue contributions to protein solvation is presented. The approach uses the exposed polar and apolar surface of protein residues and has been parametrized from the fractional contributions to solvation determined from linear response theory coupled to molecular dynamics simulations. Application of the method to a large subset of proteins taken from the Protein Data Bank allowed us to compute the expected fractional solvation of residues. This information is used to discuss when a residue or a group of residues presents an uncommon solvation profile. PMID:17001031

  12. Listeria monocytogenes protein fraction induces dendritic cells maturation and T helper 1 immune responses.

    Directory of Open Access Journals (Sweden)

    Azad Saei

    2014-02-01

    Full Text Available Fully mature dendritic cells (DCs play pivotal role in inducing immune responses and converting naïve T lymphocytes into functional Th1 cells. We aimed to evaluate Listeria Monocytogenes-derived protein fractions to induce DC maturation and stimulating T helper (Th1 immune responses.In the present study, we fractionated Listeria Monocytogenes-derived proteins by adding of ammonium sulfate in a stepwise manner. DCs were also generated from C57BL/6 mice bone marrow precursor cells. Then, the effects of protein fractions on bone marrow derived DC (BMDC maturation were evaluated. In addition, we assessed the capacity of activated DCs to induce cytokine production and proliferation of lymphocytes.Listeria-derived protein fractions induced fully mature DCs expressing high costimulatory molecules such as CD80, CD86 and CD40. DCs that were activated by selected F3 fraction had low capacity to uptake exogenous antigens while secreted high levels of Interleukine (IL-12. Moreover, lymphocytes cultured with activated BMDCs produced high amounts of IFN-γ and showed higher proliferation than control. Listeria derived protein fractions differently influenced DC maturation.In conclusion, Listeria protein activated-BMDCs can be used as a cell based vaccine to induce anti-tumor immune responses.

  13. Composition and Molecular Weight Distribution of Carob Germ Proteins Fractions

    Science.gov (United States)

    Biochemical properties of carob germ proteins were analyzed using a combination of selective extraction, reversed-phase high performance liquid chromatography (RP-HPLC), size exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALS) and electrophoretic analysis. Using a mo...

  14. Arginine depletion by arginine deiminase does not affect whole protein metabolism or muscle fractional protein synthesis rate in mice.

    Science.gov (United States)

    Marini, Juan C; Didelija, Inka Cajo

    2015-01-01

    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 depletion can potentially exacerbate the progressive loss of body weight, and especially lean body mass, in cancer patients we determined the effect of arginine depletion by pegylated arginine deiminase (ADI-PEG 20) on whole body protein synthesis and fractional protein synthesis rate in multiple tissues of mice. ADI-PEG 20 successfully depleted circulating arginine (arginine, whole body protein synthesis and breakdown were maintained in the ADI-PEG 20 treated mice. The fractional protein synthesis rate of muscle was also not affected by arginine depletion. Most tissues (liver, kidney, spleen, heart, lungs, stomach, small and large intestine, pancreas) were able to maintain their fractional protein synthesis rate; however, the fractional protein synthesis rate of brain, thymus and testicles was reduced due to the ADI-PEG 20 treatment. Furthermore, these results were confirmed by the incorporation of ureido [14C]citrulline, which indicate the local conversion into arginine, into protein. In conclusion, the intracellular recycling pathway of citrulline is able to provide enough arginine to maintain protein synthesis rate and prevent the loss of lean body mass and body weight.

  15. Tissue subcellular fractionation and protein extraction for use in mass-spectrometry-based proteomics.

    Science.gov (United States)

    Cox, Brian; Emili, Andrew

    2006-01-01

    We have shown that sample fractionation is an effective method for increasing the detection coverage of the proteome of complex samples, such as organs, by mass-spectrometric techniques. Further fractionating a sample based on subcellular compartments can generate molecular information on the state of a tissue and the distribution of its protein components. Although many methods exist for fractionating proteins, the method described here can capture the majority of subcellular fractions simultaneously at reasonable purity. The scalability of this method makes it amenable to small samples, such as embryonic tissues, in addition to larger tissues. The protocol described is for the general fractionation and extraction of proteins from organs or tissues for subsequent analysis by mass spectrometry. It uses differential centrifugation in density gradients to isolate nuclear, cytosolic, mitochondrial and mixed microsomal (Golgi, endoplasmic reticulum, other vesicles and plasma membrane) fractions. Once the fractions are isolated, they are extracted for protein and the samples can then be frozen for processing and analysis at a later date. The procedure can typically be completed in 5 h.

  16. 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

  17. Amino acid composition, molecular weight distribution and gel electrophoresis of walnut (Juglans regia L.) proteins and protein fractionations.

    Science.gov (United States)

    Mao, Xiaoying; Hua, Yufei; Chen, Guogang

    2014-01-01

    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.

  18. 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.

  19. pH fractionation and identification of proteins: comparing column chromatofocusing versus liquid isoelectric focusing techniques.

    Science.gov (United States)

    Gunther, Nereus W; Paul, Moushumi; Nuñez, Alberto; Liu, Yanhong

    2012-06-01

    In proteomic investigations, a number of different separation techniques can be applied to fractionate whole cell proteomes into more manageable fractions for subsequent analysis. In this work, utilizing HPLC and mass spectrometry for protein identification, two different fractionation methods were compared and contrasted to determine the potential of each method for the simple and reproducible fractionation of a bacterial proteome. Column-based chromatofocusing and liquid-based isoelectric focusing both utilized pH gradients to produce similar results in terms of the numbers of proteins successfully identified (402 and 378 proteins) and the consistency of proteins identified from one experiment to the next (<10% change). However, there was limited overlap in the protein sets with <50% of the proteins identified as common between the sets of proteins identified by the different systems. In addition to the numbers of proteins identified and consistency of those identified, the reduced monetary costs of experimentation and increased assay flexibility produced by using isoelectric focusing was considered in order to adopt a system best suited for comparative proteomic projects. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Chromatofocusing fractionation and two-dimensional difference gel electrophoresis for low abundance serum proteins.

    Science.gov (United States)

    Qin, Shuzhen; Ferdinand, Angeline S; Richie, Jerome P; O'Leary, Michael P; Mok, Samuel C; Liu, Brian C-S

    2005-08-01

    The technical challenge to analysis of the serum proteome is that the serum proteins are present at unequal concentrations. A few are so dominant, such as serum albumin and immunoglobulins, that they mask detection of other proteins. Because of these high abundance proteins, current technologies, while theoretically capable of analyzing protein amounts spanning four orders of magnitude, are only able to analyze proteins ranging over two orders of magnitude and cannot analyze the lower abundance proteins that may be the next biomarkers and drug targets. To facilitate the identification of low abundance proteins, we fractionated serum samples from patients with prostate cancer and patients with benign prostate hyperplasia using anion displacement liquid chromatofocusing chromatography, which separates proteins by a pH gradient and a positively charged column. Differential expression of proteins from fractions was then determined and identified by IEF gels and 2-D DIGE. Results demonstrate improved resolution of proteins within the chosen pH gradient when compared to the unfractionated samples. Several proteins that were differentially expressed in serum from patients with prostate cancer were identified in the fractionated serum. Three of these proteins, squamous cell carcinoma antigen 1 (SCCA1), calgranulin B, and haptoglobin-related protein, are present in the serum at levels below the classical protein level of mg/mL. SCCA1 is normally expressed in serum at ng/mL levels, and calgranulin B is an intracellular protein. Our results demonstrate that the use of anion displacement liquid chromatofocusing chromatography may reduce the complexity of the serum proteome by separating proteins into distinct pH ranges, and facilitate the identification of low abundance proteins.

  1. Inhibition of platelet (/sup 3/H)- imipramine binding by human plasma protein fractions

    Energy Technology Data Exchange (ETDEWEB)

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

    1988-01-01

    Inhibition of high-affinity (/sup 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/sub 1/ acid glycoprotein, high density and low density lipoprotein, IgG and ..cap alpha../sub 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 (/sup 3/H)-imipramine binding site.

  2. An Economical High-Throughput Protocol for Multidimensional Fractionation of Proteins

    Directory of Open Access Journals (Sweden)

    David John Tooth

    2012-01-01

    Full Text Available A sequential protocol of multidimensional fractionation was optimised to enable the comparative profiling of fractions of proteomes from cultured human cells. Differential detergent fractionation was employed as a first step to obtain fractions enriched for cytosolic, membrane/organelle, nuclear, and cytoskeletal proteins. Following buffer exchange using gel-permeation chromatography, cytosolic proteins were further fractionated by 2-dimensional chromatography employing anion-exchange followed by reversed-phase steps. Chromatographic fractions were shown to be readily compatible with 1- and 2-dimensional gel electrophoresis or with direct analysis by mass spectrometry using linear-MALDI-TOF-MS. Precision of extraction was confirmed by reproducible SDS-PAGE profiles, MALDI-TOF-MS spectra, and quantitation of trypsinolytic peptides using LC-MS/MS (MRM analyses. Solid phases were immobilised in disposable cartridges and mobile-phase flow was achieved using a combination of centrifugation and vacuum pumping. These approaches yielded parallel sample handling which was limited only by the capacities of the employed devices and which enabled both high-throughput and experimentally precise procedures, as demonstrated by the processing of experimental replicates. Protocols were employed at 10 mg scale of extracted cell protein, but these approaches would be directly applicable to both smaller and larger quantities merely by adjusting the employed solid- and mobile-phase volumes. Additional potential applications of the fractionation protocol are briefly described.

  3. 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.

  4. Safety of protein hydrolysates, fractions thereof and bioactive peptides in human nutrition.

    Science.gov (United States)

    Schaafsma, G

    2009-10-01

    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 and evaluated. A new concept for the safety assessment of protein hydrolysates and fractions thereof is developed. Benchmarks for the evaluation are safety of total protein intake, safety of free amino acid intake, documented history of safe use, outcome of questionnaires in efficacy studies and safety studies. Similar to the intake of intact proteins with a history of safe use, the intake of hydrolysates made from them, does not raise concern about safety, provided the applied proteolytic enzymes are food grade and thus of suitable quality. The safety of hydrolysates and of fractions thereof, including the so-called bioactive peptides, should always be assessed by the company before market introduction (company safety assessment). Only when a novel protein source is used or a novel production process is applied, which results in significant changes in nutritional value, metabolic effect or increased level of undesirable substances, that products might fall under novel food regulations. This means that company safety assessment should be reviewed and approved by external independent experts (external safety evaluation) and the novel protein hydrolysate (fraction) is authorized by competent authorities before market introduction. It is argued that good judgement on the safety of hydrolysates and the fractions thereof can be obtained by comparing the anticipated intake of amino acids by these products with those levels to be reasonably expected to be ingested under normal conditions of consumption of a balanced and varied diet. The paper shows a decision tree that can be used for safety assessment.

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

    Directory of Open Access Journals (Sweden)

    Changyuan Wang

    2014-01-01

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

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

    Science.gov (United States)

    Dash, Priyanka; Ghosh, Goutam

    2017-04-10

    In the present study, a modified Osborne fractionation method was followed to isolate albumin (Calb), globulin (Cglo), prolamin (Cpro) and glutelin (Cglu) 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, Cpro was found to be most effective against A. baumannii followed by Calb and Cglo. The results showed that growth of inhibition of these protein fractions differ significantly from each other (p ≤ 0.05). In view of antioxidant potential, Cglo exhibited strongest antioxidant capacity while Cglu showed weakest antioxidant potential.

  7. Comparison of the kinetics of calcium transport in vesicular dispersions and oriented multilayers of isolated sarcoplasmic reticulum membranes.

    OpenAIRE

    Pierce, D H; Scarpa, A.; Trentham, D R; Topp, M. R.; Blasie, J K

    1983-01-01

    Knowledge of the functional properties of the protein in oriented multilayers, in addition to vesicular dispersions, of membranes such as the isolated sarcoplasmic reticulum (SR), extends the variety of techniques that can be effectively used in studies of the membrane protein's structure or structural changes associated with its function. One technique requiring the use of oriented multilayers to provide more direct time-averaged and time-resolved structural investigations of the SR membrane...

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

    Directory of Open Access Journals (Sweden)

    Andrew McAloon

    2011-12-01

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

  9. High resolution preparation of monocyte-derived macrophages (MDM protein fractions for clinical proteomics

    Directory of Open Access Journals (Sweden)

    Olivieri Oliviero

    2009-02-01

    Full Text Available Abstract Background Macrophages are involved in a number of key physiological processes and complex responses such as inflammatory, immunological, infectious diseases and iron homeostasis. These cells are specialised for iron storage and recycling from senescent erythrocytes so they play a central role in the fine tuning of iron balancing and distribution. The comprehension of the many physiological responses of macrophages implies the study of the related molecular events. To this regard, proteomic analysis, is one of the most powerful tools for the elucidation of the molecular mechanisms, in terms of changes in protein expression levels. Results Our aim was to optimize a protocol for protein fractionation and high resolution mapping using human macrophages for clinical studies. We exploited a fractionation protocol based on the neutral detergent Triton X-114. The 2D maps of the fractions obtained showed high resolution and a good level of purity. Western immunoblotting and mass spectrometry (MS/MS analysis indicated no fraction cross contamination. On 2D-PAGE mini gels (7 × 8 cm we could count more than five hundred protein spots, substantially increasing the resolution and the number of detectable proteins for the macrophage proteome. The fractions were also evaluated, with preliminary experiments, using Surface Enhanced Laser Desorption Ionization Time of Flight Mass Spectrometry (SELDI-TOF-MS. Conclusion This relatively simple method allows deep investigation into macrophages proteomics producing discrete and accurate protein fractions, especially membrane-associated and integral proteins. The adapted protocol seems highly suitable for further studies of clinical proteomics, especially for the elucidation of the molecular mechanisms controlling iron homeostasis in normal and disease conditions.

  10. In vitro antioxidant properties of chicken skin enzymatic protein hydrolysates and membrane fractions.

    Science.gov (United States)

    Onuh, John O; Girgih, Abraham T; Aluko, Rotimi E; Aliani, Michel

    2014-05-01

    Chicken thigh and breast skin proteins were hydrolysed using alcalase or a combination of pepsin and pancreatin (PP), each at concentrations of 1-4%. The chicken skin protein hydrolysates (CSPHs) were then fractionated by membrane ultrafiltration into different molecular weight peptides (antioxidant properties. Results showed that the CSPHs had a significantly (pskin hydrolysates had significantly higher DPPH scavenging activity than the chicken thigh skin hydrolysates. DPPH scavenging and metal ion chelation increased significantly (pantioxidant properties decreased as peptide size increased. We conclude that CSPHs and their peptide fractions may be used as ingredients in the formulation of functional foods and nutraceuticals for the control and management of oxidative stress-related diseases.

  11. Arginine depletion by arginine deiminase does not affect whole protein metabolism or muscle fractional protein synthesis rate in mice.

    Directory of Open Access Journals (Sweden)

    Juan C Marini

    Full Text Available 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 depletion can potentially exacerbate the progressive loss of body weight, and especially lean body mass, in cancer patients we determined the effect of arginine depletion by pegylated arginine deiminase (ADI-PEG 20 on whole body protein synthesis and fractional protein synthesis rate in multiple tissues of mice. ADI-PEG 20 successfully depleted circulating arginine (<1 μmol/L, and increased citrulline concentration more than tenfold. Body weight and body composition, however, were not affected by ADI-PEG 20. Despite the depletion of arginine, whole body protein synthesis and breakdown were maintained in the ADI-PEG 20 treated mice. The fractional protein synthesis rate of muscle was also not affected by arginine depletion. Most tissues (liver, kidney, spleen, heart, lungs, stomach, small and large intestine, pancreas were able to maintain their fractional protein synthesis rate; however, the fractional protein synthesis rate of brain, thymus and testicles was reduced due to the ADI-PEG 20 treatment. Furthermore, these results were confirmed by the incorporation of ureido [14C]citrulline, which indicate the local conversion into arginine, into protein. In conclusion, the intracellular recycling pathway of citrulline is able to provide enough arginine to maintain protein synthesis rate and prevent the loss of lean body mass and body weight.

  12. Enhanced biosynthetically directed fractional carbon-13 enrichment of proteins for backbone NMR assignments.

    Science.gov (United States)

    Wenrich, Broc R; Sonstrom, Reilly E; Gupta, Riju A; Rovnyak, David

    2015-11-01

    Routes to carbon-13 enrichment of bacterially expressed proteins include achieving uniform or positionally selective (e.g. ILV-Me, or (13)C', etc.) enrichment. We consider the potential for biosynthetically directed fractional enrichment (e.g. carbon-13 incorporation in the protein less than 100%) for performing routine n-(D)dimensional NMR spectroscopy of proteins. First, we demonstrate an approach to fractional isotope addition where the initial growth media containing natural abundance glucose is replenished at induction with a small amount (e.g. 10%(w/w)u-(13)C-glucose) of enriched nutrient. The approach considered here is to add 10% (e.g. 200mg for a 2g/L culture) u-(13)C-glucose at the induction time (OD600=0.8), resulting in a protein with enhanced (13)C incorporation that gives almost the same NMR signal levels as an exact 20% (13)C sample. Second, whereas fractional enrichment is used for obtaining stereospecific methyl assignments, we find that (13)C incorporation levels no greater than 20%(w/w) yield (13)C and (13)C-(13)C spin pair incorporation sufficient to conduct typical 3D-bioNMR backbone experiments on moderate instrumentation (600 MHz, RT probe). Typical 3D-bioNMR experiments of a fractionally enriched protein yield expected backbone connectivities, and did not show amino acid biases in this work, with one exception. When adding 10% u-(13)C glucose to expression media at induction, there is poor preservation of (13)Cα-(13)Cβ spin pairs in the amino acids ILV, leading to the absence of Cβ signals in HNCACB spectra for ILV, a potentially useful editing effect. Enhanced fractional carbon-13 enrichment provides lower-cost routes to high throughput protein NMR studies, and makes modern protein NMR more cost-accessible.

  13. Fractionation of sheep cheese whey by a scalable method to sequentially isolate bioactive proteins.

    Science.gov (United States)

    Pilbrow, Jodi; Bekhit, Alaa El-din A; Carne, Alan

    2016-07-15

    This study reports a procedure for the simultaneous purification of glyco(caseino)macropeptide, immunoglobulin, lactoperoxidase, lactoferrin, α-lactalbumin and β-lactoglobulin from sheep cheese sweet whey, an under-utilized by-product of cheese manufacture generated by an emerging sheep dairy industry in New Zealand. These proteins have recognized value in the nutrition, biomedical and health-promoting supplements industries. A sequential fractionation procedure using economical anion and cation exchange chromatography on HiTrap resins was evaluated. The whey protein fractionation is performed under mild conditions, requires only the adjustment of pH between ion exchange chromatography steps, does not require buffer exchange and uses minimal amounts of chemicals. The purity of the whey protein fractions generated were analyzed by reversed phase-high performance liquid chromatography and the identity of the proteins was confirmed by mass spectrometry. This scalable procedure demonstrates that several proteins of recognized value can be fractionated in reasonable yield and purity from sheep cheese whey in one streamlined process.

  14. Hydrolysis of whey protein isolate with Bacillus licheniformis protease: aggregating capacities of peptide fractions.

    Science.gov (United States)

    Creusot, Nathalie; Gruppen, Harry

    2008-11-12

    In a previous study, peptides aggregating at pH 7.0 derived from a whey protein hydrolysate made with Bacillus licheniformis protease were fractionated and identified. The objective of the present work was to investigate the solubility of the fractionated aggregating peptides, as a function of concentration, and their aggregating capacities toward added intact proteins. The amount of aggregated material and the composition of the aggregates obtained were measured by nitrogen concentration and size exclusion chromatography, respectively. The results showed that of the four fractions obtained from the aggregating peptides, two were insoluble, while the other two consisted of 1:1 mixture of low and high solubility peptides. Therefore, insoluble peptides coaggregated, assumedly via hydrophobic interactions, other relatively more soluble peptides. It was also shown that aggregating peptides could aggregate intact protein nonspecifically since the same peptides were involved in the aggregation of whey proteins, beta-casein, and bovine serum albumin. Both insoluble and partly insoluble peptides were required for the aggregation of intact protein. These results are of interest for the applications of protein hydrolysates, as mixtures of intact protein and peptides are often present in these applications.

  15. 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.

  16. 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.

    Science.gov (United States)

    Lima, Ana; Oliveira, Jennifer; Saúde, Filipe; Mota, Joana; Ferreira, Ricardo Boavida

    2017-02-27

    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.

  17. Fractionation of whey proteins with high-capacity superparamagnetic ion-exchangers

    DEFF Research Database (Denmark)

    Heebøll-Nielsen, Anders; Justesen, Sune; Thomas, Owen R. T.

    2004-01-01

    In this study we describe the design, preparation and testing of superparamagnetic anion-exchangers, and their use together with cation-exchangers in the fractionation of bovine whey proteins as a model study for high-gradient magnetic fishing. Adsorbents prepared by attachment of trimethyl amine...... to 337 mg g-1 with a dissociation constant of 0.042 µM. The latter anion-exchanger was selected for studies of whey protein fractionation. In these, crude bovine whey was treated with a superparamagnetic cation-exchanger to adsorb basic protein species, and the supernatant arising from this treatment......) was achieved with some simultaneous binding of immunoglobulins (Ig). The immunoglobulins were separated from the other two proteins by desorbing with a low concentration of NaCl (=0.4 M), whereas lactoferrin and lactoperoxidase were co-eluted in significantly purer form, e.g. lactoperoxidase was purified 28...

  18. Fractionation of whey proteins with high-capacity superparamagnetic ion-exchangers

    DEFF Research Database (Denmark)

    Heebøll-Nielsen, Anders; Justesen, S.F.L.; Thomas, Owen R. T.

    2004-01-01

    In this study we describe the design, preparation and testing of superparamagnetic anion-exchangers, and their use together with cation-exchangers in the fractionation of bovine whey proteins as a model study for high-gradient magnetic fishing. Adsorbents prepared by attachment of trimethyl amine...... to 337 mg g(-1) with a dissociation constant of 0.042 muM. The latter anion-exchanger was selected for studies of whey protein fractionation. In these, crude bovine whey was treated with a superparamagnetic cation-exchanger to adsorb basic protein species, and the supernatant arising from this treatment......) was achieved with some simultaneous binding of immunoglobulins (1g). The immunoglobulins were separated from the other two proteins by desorbing with a low concentration of NaCl (less than or equal to0.4 M), whereas lactoferrin and lactoperoxidase were co-eluted in significantly purer form, e...

  19. 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

  20. Denervation alters protein-lipid interactions in membrane fractions from electrocytes of Electrophorus electricus (L.).

    Science.gov (United States)

    Barriviera, M L; Louro, S R; Wajnberg, E; Hasson-Voloch, A

    2001-06-15

    Protein-lipid interactions are studied in normal and denervated electrocytes from Electrophorus electricus (L.). Structural modifications of the lipid micro-environment encircling integral membrane proteins in membrane fractions presenting Na(+),K(+)-ATPase activity are investigated using ESR spectroscopy of stearic acid spin labeled at the 14th carbon (14-SASL). The microsomal fraction derived from the innervated electric organ exhibits, on a discontinuous sucrose gradient, a bimodal distribution of the Na(+),K(+)-ATPase activity, bands a and b. Band b is almost absent in microsomes from the denervated organ, and band a', with the same density as band a has lower Na(+),K(+)-ATPase activity. Band a' presents a larger ratio of protein-interacting lipids than band a. Analysis of the lipid stoichiometry at the protein interface indicates that denervation causes at least a twofold average decrease on protein oligomerization. Physical inactivity and denervation have similar effects on protein-lipid interactions. Denervation also influences the selectivity of proteins for fatty acids. Experiments in decreasing pH conditions performed to verify the influence of stearic acid negative charge on protein interaction revealed that denervation produces loss of charge selectivity. The observed modifications on molecular interactions induced by denervation may have importance to explain modulation of enzyme activity.

  1. THE DISTRIBUTION OF ELECTROPHORETIC FRACTIONS OF PROTEIN ISOLATES FROM SUNFLOWER MEAL

    Directory of Open Access Journals (Sweden)

    Voronova N. S.

    2014-12-01

    Full Text Available The food status of Russians is characterized by deficiency of protein. Perspective sources of food protein are the secondary resources of the oil and fat industry received when processing seeds of sunflower, including sunflower meal. Unfortunately, the features of technological process at the oilextracting press exclude a possibility of receiving food protein-containing products from them without the additional processing increasing biological value and improving technical characteristics of proteins. On the basis of the above information, the researches of a protein complex of sunflower cake, development of ways of regulation of its functional and technological properties and increase of biological value is up-to-date. The article presents the analysis of the influence of enzymatic modification on the distribution of electrophoretic fractions of the modified protein isolates

  2. Nitrogen 15 abundance in protein fractions of beans fertilized with (15NH42SO4

    Directory of Open Access Journals (Sweden)

    Chaud Saula Goulart

    2002-01-01

    Full Text Available Studies evaluating the protein nutritive value of beans labelled with 15N, ussing 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 Piratã 1 with 1.394 atoms%15N, 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 15N, 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 L¹ 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.

  3. 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.

  4. Hydrophobic Fractionation Enhances Novel Protein Detection by Mass Spectrometry in Triple Negative Breast Cancer

    Science.gov (United States)

    Lu, Ming; Whitelegge, Julian P.; Whelan, Stephen A.; He, Jianbo; Saxton, Romaine E.; Faull, Kym F.; Chang, Helena R.

    2010-01-01

    It is widely believed that discovery of specific, sensitive and reliable tumor biomarkers can improve the treatment of cancer. The goal of this study was to develop a novel fractionation protocol targeting hydrophobic proteins as possible cancer cell membrane biomarkers. Hydrophobic proteins of breast cancer tissues and cell lines were enriched by polymeric reverse phase columns. The retained proteins were eluted and digested for peptide identification by nano-liquid chromatography with tandem mass spectrometry using a hybrid linear ion-trap Orbitrap. Hundreds of proteins were identified from each of these three specimens: tumors, normal breast tissue, and breast cancer cell lines. Many of the identified proteins defined key cellular functions. Protein profiles of cancer and normal tissues from the same patient were systematically examined and compared. Stem cell markers were overexpressed in triple negative breast cancer (TNBC) compared with non-TNBC samples. Because breast cancer stem cells are known to be resistant to radiation and chemotherapy, and can be the source of metastasis frequently seen in patients with TNBC, our study may provide evidence of molecules promoting the aggressiveness of TNBC. The initial results obtained using a combination of hydrophobic fractionation and nano-LC mass spectrometry analysis of these proteins appear promising in the discovery of potential cancer biomarkers. When sufficiently refined, this approach may prove useful for early detection and better treatment of breast cancer. PMID:20596302

  5. Conformational changes in the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum detected using phosphorescence polarization.

    Science.gov (United States)

    Restall, C J; Coke, M; Murray, E K; Chapman, D

    1985-02-28

    The technique of time-averaged phosphorescence has been used to study the interaction of calcium ions and ATP with the (Ca2+ + Mg2+)-ATPase in sarcoplasmic reticulum vesicles. The presence of excess calcium ions was found to cause a 20% decrease in the phosphorescence emission anisotropy. This is interpreted as being due to a conformational change in the protein and is supported by data from time-resolved phosphorescence measurements which also show a lowering of the anisotropy. This change in the decay of the emission anisotropy is associated with only minor changes in the rotational relaxation time of the protein and is again suggestive of a conformational change in the protein. In some cases ATP was also observed to lower the time-averaged phosphorescence anisotropy possibly via an interaction with the low-affinity regulatory site of the protein.

  6. Vitamins C and E attenuate apoptosis, beta-adrenergic receptor desensitization, and sarcoplasmic reticular Ca2+ ATPase downregulation after myocardial infarction.

    Science.gov (United States)

    Qin, Fuzhong; Yan, Chen; Patel, Ravish; Liu, Weimin; Dong, Erdan

    2006-05-15

    Oxidative stress plays an important role in mediating ventricular remodeling and dysfunction in heart failure (HF), but its mechanism of action has not been fully elucidated. In this study we determined whether a combination of antioxidant vitamins reduced myocyte apoptosis, beta-adrenergic receptor desensitization, and sarcoplasmic reticular (SR) Ca2+ ATPase downregulation in HF after myocardial infarction (MI) and whether these effects were associated with amelioration of left ventricular (LV) remodeling and dysfunction. Vitamins (vitamin C 300 mg and vitamin E 300 mg) were administered to rabbits 1 week after MI or sham operation for 11 weeks. The results showed that MI rabbits exhibited cardiac dilation and LV dysfunction measured by fractional shortening and the maximal rate of pressure rise (dP/dt), an index of contractility. These changes were associated with elevation of oxidative stress, decreases of mitochondrial Bcl-2 and cytochrome c proteins, increases of cytosolic Bax and cytochrome c proteins, caspase 9 and caspase 3 activities and myocyte apoptosis, and downregulation of beta-adrenergic receptor sensitivity and SR Ca2+ ATPase. Combined treatment with vitamins C and E diminished oxidative stress, increased mitochondrial Bcl-2 protein, decreased cytosolic Bax, prevented cytochrome c release from mitochondria to cytosol, reduced caspase 9 and caspase 3 activities and myocyte apoptosis, blocked beta-adrenergic receptor desensitization and SR Ca2+ ATPase downregulation, and attenuated LV dilation and dysfunction in HF after MI. The results suggest that antioxidant therapy may be beneficial in HF.

  7. Polymerization Degrees, Molecular Weights and Protein-Binding Affinities of Condensed Tannin Fractions from a Leucaena leucocephala Hybrid

    Directory of Open Access Journals (Sweden)

    Mookiah Saminathan

    2014-06-01

    Full Text Available Condensed tannins (CTs form insoluble complexes with proteins and are able to protect them from degradation, which could lead to rumen bypass proteins. Depending on their degrees of polymerization (DP and molecular weights, CT fractions vary in their capability to bind proteins. In this study, purified condensed tannins (CTs from a Leucaena leucocephala hybrid were fractionated into five different molecular weight fractions. The structures of the CT fractions were investigated using 13C-NMR. The DP of the CT fractions were determined using a modified vanillin assay and their molecular weights were determined using Q-TOF LC-MS. The protein-binding affinities of the respective CT fractions were determined using a protein precipitation assay. The DP of the five CT fractions (fractions F1–F5 measured by the vanillin assay in acetic acid ranged from 4.86 to 1.56. The 13C-NMR results showed that the CT fractions possessed monomer unit structural heterogeneity. The number-average molecular weights (Mn of the different fractions were 1265.8, 1028.6, 652.2, 562.2, and 469.6 for fractions F1, F2, F3, F4, and F5, respectively. The b values representing the CT quantities needed to bind half of the maximum precipitable bovine serum albumin increased with decreasing molecular weight—from fraction F1 to fraction F5 with values of 0.216, 0.295, 0.359, 0.425, and 0.460, respectively. This indicated that higher molecular weight fractions of CTs from L. leucocephala have higher protein-binding affinities than those with lower molecular weights.

  8. Polymerization degrees, molecular weights and protein-binding affinities of condensed tannin fractions from a Leucaena leucocephala hybrid.

    Science.gov (United States)

    Saminathan, Mookiah; Tan, Hui Yin; Sieo, Chin Chin; Abdullah, Norhani; Wong, Clemente Michael Vui Ling; Abdulmalek, Emilia; Ho, Yin Wan

    2014-06-12

    Condensed tannins (CTs) form insoluble complexes with proteins and are able to protect them from degradation, which could lead to rumen bypass proteins. Depending on their degrees of polymerization (DP) and molecular weights, CT fractions vary in their capability to bind proteins. In this study, purified condensed tannins (CTs) from a Leucaena leucocephala hybrid were fractionated into five different molecular weight fractions. The structures of the CT fractions were investigated using 13C-NMR. The DP of the CT fractions were determined using a modified vanillin assay and their molecular weights were determined using Q-TOF LC-MS. The protein-binding affinities of the respective CT fractions were determined using a protein precipitation assay. The DP of the five CT fractions (fractions F1-F5) measured by the vanillin assay in acetic acid ranged from 4.86 to 1.56. The 13C-NMR results showed that the CT fractions possessed monomer unit structural heterogeneity. The number-average molecular weights (Mn) of the different fractions were 1265.8, 1028.6, 652.2, 562.2, and 469.6 for fractions F1, F2, F3, F4, and F5, respectively. The b values representing the CT quantities needed to bind half of the maximum precipitable bovine serum albumin increased with decreasing molecular weight--from fraction F1 to fraction F5 with values of 0.216, 0.295, 0.359, 0.425, and 0.460, respectively. This indicated that higher molecular weight fractions of CTs from L. leucocephala have higher protein-binding affinities than those with lower molecular weights.

  9. Differences of protein fractions among fresh, frozen and powdered donkey milk.

    Science.gov (United States)

    Polidori, Paolo; Vincenzetti, Silvia

    2010-01-01

    Recently donkey milk has been the focus of several studies because of its special nutritional properties and composition, which is very close to human milk. When a mother cannot breastfeed, or chooses not to breastfeed, the use of a milk substitute must provide the best option to meet the nutritional and health needs of the infant. Donkey milk has been widely used in the past to replace human milk, because chemical composition and protein content are close to that of human milk, and also because the allergenicity of donkey milk is low. The recent studies of the paediatric scientists have demonstrated that infant formulae, which are based on dairy cows milk, are less adapted than donkey milk. In fact, donkey's milk digestibility is higher than cows' milk and similar to human milk, because of the high whey proteins content and the few casein content. Since donkey milk supply is related to its seasonal availability during the year, in this study were evaluated the effects of a specific technological treatment (spray-dryer) and a particular storage temperature (-20 degrees C) on the protein fractions of donkey milk. The results obtained in fresh, frozen and powdered donkey milk showed different values in total proteins, caseins, whey proteins and lysozyme content. The article presents some promising patents on protein fractions among fresh, frozen and powdered donkey milk.

  10. 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.

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

    Science.gov (United States)

    Balciunaite, Gabriele; Juodsnukyte, Jovita; Savickas, Arunas; Ragazinskiene, Ona; Siatkute, Luka; Zvirblyte, Gitana; Mistiniene, Edita; Savickiene, Nijole

    2015-12-01

    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.

  12. 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...

  13. 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 (P<0.05) improved in dispersions of hydrolysate fraction solutions (10 g protein L(-1)). Total iron 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.

  14. Biochemical evaluation of protein fractions from physic nut (Jatropha curcas L.

    Directory of Open Access Journals (Sweden)

    Chel-Guerrero, L.

    2012-09-01

    Full Text Available J. curcas seed proteins were fractioned according to the Osborne method and some biochemical properties were determined for these fractions. Glutelins (378 g kg–1 protein and globulins (201 g kg–1 protein were the main components. Albumins and prolamins were the minor components. Protein digestibility was highest in glutelins and globulins with values of 81 and 80% respectively. Electrophoresis analysis showed that globulins and glutelins exhibited similar polypeptide profiles. Electrophoresis patterns suggested that there could be a structural relationship among 2S, 7S and 11S storage proteins from plant sources. According to the FAO⁄WHO reference, the protein fractions had acceptable levels of most of the essential amino acids, but its globulins and glutelins were low in lysine and tryptophan.Las proteínas de semillas de J. curcas L. se fraccionaron empleando el método de Osborne y posteriormente se determinaron algunas de sus propiedades bioquímicas y nutricionales. Las fracciones mayoritarias resultaron ser glutelinas (378 g kg–1 de proteína y globulinas (201 g kg–1 de proteína mientras que las albúminas y prolaminas fueron las fracciones minoritarias. La digestibilidad de la proteína resultó ser más alta en las glutelinas y globulinas, con valores de 81 y 80% respectivamente. El análisis por electroforesis mostró que las globulinas y glutelinas presentaron perfiles similares, los resultados sugieren que podría existir una relación con proteínas de almacenamiento 2S, 7S y 11S de origen vegetal. Asimismo, de acuerdo con la FAO/WHO, las fracciones proteínicas tuvieron niveles aceptables para la mayoría de los aminoácidos esenciales, sin embargo, las globulinas y glutelinas fueron deficientes en Lys y Trp.

  15. 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.

  16. 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.

  17. 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.

  18. Fractionation, partial characterization and bioactivity of water-soluble polysaccharides and polysaccharide-protein complexes from Pleurotus geesteranus.

    Science.gov (United States)

    Zhang, Mei; Zhu, Lin; Cui, Steve W; Wang, Qi; Zhou, Ting; Shen, Hengsheng

    2011-01-01

    Fractionation and purification of mushroom polysaccharides is a critical process for mushroom clinical application. After a hot-water treatment, the crude Pleurotus geesteranus (PG) was further fractionated into four fractions (PG-1, -2, -3, -4) using gradient precipitation with water and ammonia sulphate. By controlling the initial polymer concentration and ratio of solvents, this process produced PG fractions with high chemical uniformity and narrow Mw distribution without free proteins. Structurally, PG-1 and PG-2 are pure homopolysaccharide mainly composed of glucose; and PG-3 and PG-4 are heteropolysaccharide-protein complexes. PG-2, a high M(w) fraction mainly composed of glucose presented significant cytotoxicity at the concentration of 200 and 100 μg/ml to human breast cancer cells. Here, we report a new mushroom polysaccharides extraction and fractionation method, with which we produced four fractions of PG with PG-2 appearing effective anti-tumour activity.

  19. Exercise & NSAID: Effect on muscle protein synthesis in knee osteoarthritis patients?

    DEFF Research Database (Denmark)

    Petersen, S.G.; Miller, Ben F; Hansen, M

    2011-01-01

    a flooding dose of 13C/12C-proline.RESULTS:Circulating levels of prostaglandin F2α were lower in the NSAID group compared with the placebo group (P ...:In elderly patients with knee OA, an acute bout of moderate exercise significantly increases FSR of muscle myofibrillar and sarcoplasmic protein, but not tendon collagen, 24 h after exercise. NSAID administration in patients with knee OA reduced the level of circulating prostaglandin F2α but did not diminish...... the contralateral leg remained rested. Twenty-four hours after exercise, we determined circulating concentrations of inflammatory parameters and measured FSR of myofibrillar and sarcoplasmic protein fractions of vastus lateralis muscle and patellar tendon collagen protein by the direct incorporation method using...

  20. 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

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

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    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

  2. Coupling a detergent lysis/cleanup methodology with intact protein fractionation for enhanced proteome characterization.

    Science.gov (United States)

    Sharma, Ritin; Dill, Brian D; Chourey, Karuna; Shah, Manesh; VerBerkmoes, Nathan C; Hettich, Robert L

    2012-12-07

    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 detergent cleanup methods (trichloroacetic acid (TCA) precipitation, chloroform/methanol/water (CMW) extraction, a commercial detergent removal spin column method (DRS) and filter-aided sample preparation (FASP)) to provide efficiency benchmarks with respect to protein, peptide, and spectral identifications in each case. Our results show that for protein-limited samples, FASP outperforms the other three cleanup methods, while at high protein amounts, all the methods are comparable. This information was used to investigate and contrast molecular weight-based fractionated with unfractionated lysates from three increasingly complex samples ( Escherichia coli K-12, a five microbial isolate mixture, and a natural microbial community groundwater sample), all of which were prepared with an SDS-FASP approach. The additional fractionation step enhanced the number of protein identifications by 8% to 25% over the unfractionated approach across the three samples.

  3. Biochemical and morphological characterization of light and heavy sarcoplasmic reticulum vesicles. Volume I

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, Kevin Peter [Univ. of Rochester, NY (United States)

    1978-01-01

    Light (30 to 32.5% sucrose) and heavy (38.5 to 42% sucrose) sarcoplasmic reticulum vesicles (LSR, HSR) were isolated from rabbit leg muscle. They were then diluted and washed with sucrose or KCl and referred to as sucrose or KCl washed vesicles. Thin-section electron microscopy of LSR vesicles reveals empty vesicles of various sizes and shapes where as the HSR vesicles appear as rounded vesicles of uniform size filled with electron dense material. The LSR consists of predominantly Ca2+ + Mg2+ ATPase (80 to 90%), a small amount of the high affinity Ca binding protein (5%), and a 5000 dalton proteolipid. The sucrose HSR vesicles contain the Ca2+ + Mg2+ ATPase (50%), Calsequestrin (25%), high affinity Ca binding protein (5%), one extrinsic 34,000 dalton protein (3%), one intrinsic 30,000 dalton protein (3%), a 9000 dalton proteolipid, and a 5000 dalton proteolipid. The sucrose--washed HSR vesicles contain greater than three times the calcium content of the sucrose washed LSR vesicles where as the KCl--washed vesicles contain less than 15 nmoles Ca2+ mg of protein each. The light and heavy sarcoplasmic reticulum vesicles were both able to accumulate calcium in the presence of ATP. Exchange of methanesulfonate for chloride resulted in the release of calcium from both the light and heavy SR vesicles. Sucrose causes a slight inhibition of chloride--induced calcium release from the heavy SR vesicles but it greatly reduces the release of calcium from the light SR vesicles. Sodium dantrolene (20 uM) has no effect on the release of calcium from the light SR vesicles but it inhibits the release of calcium from the heavy SR vesicles. The results indicate that the chloride--induced release of calcium may be acting by two mechanisms, osmotic swelling and depolarization.

  4. H+ countertransport and electrogenicity of the sarcoplasmic reticulum Ca2+ pump in reconstituted proteoliposomes.

    Science.gov (United States)

    Yu, X; Carroll, S; Rigaud, J L; Inesi, G

    1993-04-01

    The Ca2+ transport adenosine triphosphatase of sarcoplasmic reticulum was reconstituted in unilamellar liposomes prepared by reverse-phase evaporation. The size of the resulting proteoliposomes was similar to that of native sarcoplasmic reticulum vesicles, but their protein content was much lower, with a protein/lipid ratio (wt/wt) of 1:40-160, as compared with 1:1 in the native membrane. The proteoliposomes sustained adenosine triphosphate-dependent Ca2+ uptake at rates proportional to the protein content (1-2 mumol Ca2+/mg protein/min), reaching asymptotic levels corresponding to a lumenal calcium concentration of 10-20 mM. The low permeability of the proteoliposomes permitted direct demonstration of Ca2+/H+ countertransport and electrogenicity by parallel measurements in the same experimental system. Countertransport of one H+ per one Ca2+ was demonstrated, and inhibition of the Ca2+ pump by lumenal alkalinization was relieved by the H+ ionophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone. Consistent with the countertransport stoichiometry, net positive charge displacement was produced by Ca2+ transport, as revealed by a rapid oxonol VI absorption rise. The initial rise and the following steady-state level of oxonol absorption were highest when SO4(2-) was the prevalent anion and lowest in the presence of the lipophilic anion SCN-. The influence of anions was attributed to potential driven counterion compensation. The absorption rise was rapidly collapsed by addition of valinomycin in the presence of K+. Experimentation with Ca2+ and H+ ionophores was consistent with a primary role of Ca2+ and H+ in net charge displacement. The estimated value of the steady-state electrical potential observed under optimal conditions was approximately 50 mV and was accounted for by the estimated charge transfer associated with Ca2+ and H+ countertransport under the same conditions.

  5. ALTERATIONS IN TOTAL PROTEIN CONCENTRATION, SERUM PROTEIN FRACTIONS AND ALBUMIN/GLOBULIN RATIO IN HEALTHY RABBITS

    Directory of Open Access Journals (Sweden)

    Nuzhat Sultana

    2013-08-01

    Full Text Available This study assessed the effect of oral administration of Aloe vera and was to evaluate total serum protein, albumin and globulin concentrations as well as albumin / globulin (A / G ratio. Twenty rabbits weighing 1000 – 1800 g were divided into 2 groups. Each group consisted of ten animals. One served as control and other group served as experimental group. Results show that animals after 07, 15 and 30 days dosing of Aloe vera showed highly significant decrease in total protein and globulin and highly significant decrease in Albumin after 15 and 30 days of dosing of Aloe vera in comparison to control animals group. It is concluded that the long-term use of Aloe vera may cause hypoglobinemia and hypoalbuminemia at 30 days of dosing and it could be due to the liver diseases, evidence of hepatotoxicity induced Aloe vera also reported in previous studies.

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

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    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.

  7. Selecting protein N-terminal peptides by combined fractional diagonal chromatography.

    Science.gov (United States)

    Staes, An; Impens, Francis; Van Damme, Petra; Ruttens, Bart; Goethals, Marc; Demol, Hans; Timmerman, Evy; Vandekerckhove, Joël; Gevaert, Kris

    2011-07-14

    In recent years, procedures for selecting the N-terminal peptides of proteins with analysis by mass spectrometry have been established to characterize protease-mediated cleavage and protein α-N-acetylation on a proteomic level. As a pioneering technology, N-terminal combined fractional diagonal chromatography (COFRADIC) has been used in numerous studies in which these protein modifications were investigated. Derivatization of primary amines--which can include stable isotope labeling--occurs before trypsin digestion so that cleavage occurs after arginine residues. Strong cation exchange (SCX) chromatography results in the removal of most of the internal peptides. Diagonal, reversed-phase peptide chromatography, in which the two runs are separated by reaction with 2,4,6-trinitrobenzenesulfonic acid, results in the removal of the C-terminal peptides and remaining internal peptides and the fractionation of the sample. We describe here the fully matured N-terminal COFRADIC protocol as it is currently routinely used, including the most substantial improvements (including treatment with glutamine cyclotransferase and pyroglutamyl aminopeptidase to remove pyroglutamate before SCX, and a sample pooling scheme to reduce the overall number of liquid chromatography-tandem mass spectrometry analyses) that were made since its original publication. Completion of the N-terminal COFRADIC procedure takes ~5 d.

  8. Discontinuity of sarcoplasmic reticulum in the mid-sarcomere region in flight muscle of dragonflies.

    Science.gov (United States)

    de Eguileor, M; Valvassori, R; Lanzavecchia, G

    1980-01-01

    The sarcoplasmic reticulum organization of dragonfly flight muscles is analyzed, with particular reference to the doubling existing at H-band level. This doubling could be explained as a consequence of a regular discontinuity in the sarcoplasmic reticulum covering myofibrils. In each sarcomere, two sleeves of the sarcoplasmic reticulum seem to overlap forming a telescopic system which can slide outside each other during the lengthening and shortening movements of the fiber.

  9. 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

  10. 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.

  11. Chemical characteristics and fractionation of proteins from Moringa oleifera Lam. leaves.

    Science.gov (United States)

    Teixeira, Estelamar Maria Borges; Carvalho, Maria Regina Barbieri; Neves, Valdir Augusto; Silva, Maraíza Apareci; Arantes-Pereira, Lucas

    2014-03-15

    Moringa oleifera Lam. is a leguminous plant, originally from Asia, which is cultivated in Brazil because of its low production cost. Although some people have used this plant as food, there is little information about its chemical and nutritional characteristics. The objective of this study was to characterise the leaves of M. oleifera in terms of their chemical composition, protein fractions obtained by solubility in different systems and also to assess their nutritional quality and presence of bioactive substances. The whole leaf flour contained 28.7% crude protein, 7.1% fat, 10.9% ashes, 44.4% carbohydrate and 3.0mg 100g(-1) calcium and 103.1mg 100g(-1) iron. The protein profile revealed levels of 3.1% albumin, 0.3% globulins, 2.2% prolamin, 3.5% glutelin and 70.1% insoluble proteins. The hydrolysis of the protein from leaf flour employing sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (ME) resulted in 39.5% and 29.5%, respectively. The total protein showed low in vitro digestibility (31.8%). The antinutritional substances tested were tannins (20.7 mg g(-1)), trypsin inhibitor (1.45TIU mg g(-1)), nitrate (17 mg g(-1)) and oxalic acid (10.5 mg g(-1)), besides the absence of cyanogenic compounds. β-Carotene and lutein stood out as major carotenoids, with concentrations of 161.0 and 47.0 μg g(-1) leaf, respectively. Although M. oleifera leaves contain considerable amount of crude protein, this is mostly insoluble and has low in vitro digestibility, even after heat treatment and chemical attack. In vivo studies are needed to better assess the use of this leaf as a protein source in human feed.

  12. Inositol (1,4,5)-trisphosphate activates a calcium channel in isolated sarcoplasmic reticulum membranes.

    Science.gov (United States)

    Suárez-Isla, B. A.; Irribarra, V.; Oberhauser, A.; Larralde, L.; Bull, R.; Hidalgo, C.; Jaimovich, E.

    1988-01-01

    Sarcoplasmic reticulum membrane vesicles isolated from frog skeletal muscle display high conductance calcium channels when fused into phospholipid bilayers. The channels are selective for calcium and barium over Tris. The fractional open time was voltage-independent (-40 to +25 mV), but was steeply dependent on the free cis [Ca2+] (P0 = 0.02 at 10 microM cis Ca2+ and 0.77 at 150 microM Ca2+; estimated Hill coefficient: 1.6). Addition of ATP (1 mM; cis) further increased P0 from 0.77 to 0.94. Calcium activation was reversed by addition of EGTA to the cis compartment. Magnesium (2 mM) increased the frequency of rapid closures and 8 mM magnesium decreased the current amplitude from 3.4 to 1.2 pA at 0 mV, suggesting a reversible fast blockade. Addition of increasing concentrations of inositol (1, 4, 5)-triphosphate (cis), increased P0 from 0.10 +/- 0.01 (mean +/- SEM) in the control to 0.85 +/- 0.02 at 50 microM in an approximately sigmoidal fashion, with an apparent half-maximal activation at 15 microM inositol (1, 4, 5)-trisphosphate in the presence of 40 microM cis Ca2+. Lower concentrations of this agonist were required to produce a significant increase in P0 when 10 microM or less cis Ca2+ were used. The channel was blocked by the addition to the cis compartment of either 0.5 mM lanthanum, 0.5 microM ruthenium red, or 200 nM ryanodine, all known inhibitors of Ca2+ release from sarcoplasmic reticulum vesicles. These results demonstrate the presence of calcium channels in the sarcoplasmic reticulum from frog skeletal muscle with a pharmacological profile consistent with a role in excitation contraction coupling and with the hypothesis that inositol ( 1,4,5)-trisphosphate is a physiological agonist in this process. PMID:2852037

  13. Binding of aflatoxin M1 to different protein fractions in ovine and caprine milk.

    Science.gov (United States)

    Barbiroli, A; Bonomi, F; Benedetti, S; Mannino, S; Monti, L; Cattaneo, T; Iametti, S

    2007-02-01

    The affinity of aflatoxin M1 toward the main milk protein fractions in ewe and goat milk was investigated by using an ELISA. This study took into account the possible effects of common dairy processes such as ultrafiltration, acidic or rennet curding, and production of ricotta from acidic or rennet whey. Treatments that allowed the separation of casein from whey proteins under conditions that do not alter the physical or chemical status of the proteins (such as ultracentrifugation) were used as a reference. None of the treatments used in typical dairy processes caused significant release of the toxin, in spite of the relevant changes they induced in the interactions among proteins. Only the combined heat and acidic treatment used for production of ricotta cheese altered the structure of whey proteins to the point where they lost their ability to bind the toxin. This study also showed that, regardless of the physical state of the sample, a commercial electronic nose device, in combination with appropriate statistical tools, was able to discriminate among different levels of sample contamination.

  14. 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.

  15. Effect of source and sex on blood protein fractions of West African Dwarf Goats (WADG

    Directory of Open Access Journals (Sweden)

    J. C. Okonkwo,

    2011-03-01

    Full Text Available Source and sex effects on the total blood protein and its various fractions were studied using juvenile West African Dwarf goats derived from Southern Nigeria. The goats were sourced from three distinct towns in the humid tropics namely, South-East (Umuahia, South-South (Ugheli and South-West (Akure at the rate of 6 males and 18 females per location. The mean values of the total blood plasma protein and its fractions obtained for the WADGs from different zones are 10.01±0.07 g/100ml, 10.07±0.08 g/100ml and 10.16±0.35 g/100ml (total plasma protein; 9.62±0.10 g/100ml, 9.68±0.08 g/100ml and 9.68±0.09 g/100ml (total serum protein, 0.38±0.03 g/100ml, 0.39±0.01 g/100ml, and 0.38±0.04 g/100ml (plasma fibrinogen, 5.62±0.23 g/100ml, 5.78±0.24 g/100ml and 5.45±0.26 g/100ml (serum albumin, 4.00±0.19 g/100ml, 3.89±0.29 g/100ml, and 4.12±0.25 g/100ml (serum globulin, and 1.41±0.27, 1.49±0.15 and 1.34±0.12 (albumin/globulin ratio for the goats from South-East (Umuahia, South-South (Ugheli and South-West (Akure respectively. The studies also indicate that albumin accounts for 53-58% of the total serum protein; globulin accounts for 42-47% serum protein, and the plasma fibrinogen 3.6-4% of the total plasma protein. sex and source interaction had no significant (P>0.05 effects on serum proteins; plasma fibrinogen is sex dependent, and the source of goat affects the proportions of the serum albumin, globulin, and albumin/globulin ratio characteristics of the experimental goats.

  16. Alterations in the protein pattern of subcellular fractions isolated from Paramecium cells suppressed in phagocytosis.

    Science.gov (United States)

    Surmacz, L; Wiejak, J; Wyroba, E

    2001-01-01

    SDS-PAGE and quantitative densitometric analysis revealed alterations in the protein pattern of subcellular fractions (100,000 x g) isolated from Paramecium aurelia (299s axenic) cells suppressed in phagocytosis as compared with the control. Two different agents were used to block phagocytosis: the beta-adrenergic antagonist-1-propranolol (200 microM) and inhibitor of calmodulin-dependent processes--trifluoperazine (20 microM). More than 40 polypeptides were identified in the cytosolic (soluble) fractions S1 and S2. A considerable decrease in band intensity was found for three polypeptides: by 60% for 87 kDa band, 52% for 75 kDa and 37% for 42 kDa in comparison to the control, when S2 fractions from propranolol-treated cells of equal load were quantified. TFP treatment evoked only a small decrease in the intensity of the same bands: 9%, 10% and 6%, respectively. The 42 kDa band was identified by Western blot analysis and chemiluminiscent detection to be actin. This result suggests that actin may be a primary target of pharmacological agents used in this study to inhibit Paramecium phagocytic activity.

  17. Separation of parasite antigens by molecular exclusion, anion exchange, and chromatofocusing utilizing FPLC protein fractionation systems.

    Science.gov (United States)

    Zimmerman, G L; Clark, C R

    1986-03-01

    Excretory-secretory products (ESP) were harvested from balanced salt solutions in which adult Fasciola hepatica had been incubated for 4-6 h at 37 degrees C. The ESP was fractionated by standard low pressure molecular exclusion chromatography and FPLC (fast protein liquid chromatography) using the principles of molecular exclusion, anion exchange, and chromatofocusing. The dot-enzyme-linked immunosorbent assay (Dot-ELISA) was used to demonstrate the immunoreactivity of eluted fractions. Compared to Sephacryl S-200, separation by Superose-6 (FPLC) was faster and resolved more peaks (four with Sephacryl S-200 and nine with Superose-6). Peaks from Sephacryl S-200 were resolved by the first anion exchange (Mono Q) separation into seven peaks; when these peaks were subjected to a second anion exchange, 15 peaks were resolved. Thirty-eight peaks were resolved by chromatofocusing (Mono P) in the pH range 7-4. Immunoreactive fractions from narrow-range (single pH unit) chromatofocusing were identified by the Dot-ELISA. The FPLC system proved to be a means of rapid and high resolution separation of F. hepatica antigens.

  18. The Key Factors Affecting Tuber Development of Potato in vitro and the Relation with Protein Fractions

    Institute of Scientific and Technical Information of China (English)

    WANG Da-yong; LIAN Yong; ZHU De-wei

    2002-01-01

    According to previous analysis, some properties bounding up with tuber yield were investigated.The results showed that tuber average weight, plastid Mg2+ -ATPase activity, plastid Ca2+ -ATPase activity,mitochondria Mg2+-ATPase activity, total soluble protein content, tuber average diameter, and Q-enzyme activity were important factors determining the tuber yield. The linear regression equation was:Y = 0.5211 +0.0595X(1) + 0.8389X(2) + 0.0882X(3) - 0. 0073X(4) + 0. 1449X(5) + 0. 3510X(6) + 0. 0031X(7) -0.00003X(8) + 0.3412X(9)+ 0.0127X(10) + 0.2904X(11) + 0.0570X(12) + 0.0159X(13) + 0.3585X(14)+ 0.0134X(15) -0.1012X(16). At the same time, the relation between several important properties and soluble protein fractions were analyzed.

  19. Solution-blown nanofiber mats from fish sarcoplasmic protein

    DEFF Research Database (Denmark)

    Sett, S.; Boutrup Stephansen, Karen; Yarin, A.L.

    2016-01-01

    that the production rate of solution-blowing was increased 30-fold in relation to electrospinning. Overall, this study reveals FSP as an interesting biopolymeric alternative to synthetic polymers, and the introduction of FSP to nylon 6 provides a composite with controlled properties....

  20. Probing and quantifying DNA-protein interactions with asymmetrical flow field-flow fractionation.

    Science.gov (United States)

    Ashby, Jonathan; Schachermeyer, Samantha; Duan, Yaokai; Jimenez, Luis A; Zhong, Wenwan

    2014-09-05

    Tools capable of measuring binding affinities as well as amenable to downstream sequencing analysis are needed for study of DNA-protein interaction, particularly in discovery of new DNA sequences with affinity to diverse targets. Asymmetrical flow field-flow fractionation (AF4) is an open-channel separation technique that eliminates interference from column packing to the non-covalently bound complex and could potentially be applied for study of macromolecular interaction. The recovery and elution behaviors of the poly(dA)n strand and aptamers in AF4 were investigated. Good recovery of ssDNAs was achieved by judicious selection of the channel membrane with consideration of the membrane pore diameter and the radius of gyration (Rg) of the ssDNA, which was obtained with the aid of a Molecular Dynamics tool. The Rg values were also used to assess the folding situation of aptamers based on their migration times in AF4. The interactions between two ssDNA aptamers and their respective protein components were investigated. Using AF4, near-baseline resolution between the free and protein-bound aptamer fractions could be obtained. With this information, dissociation constants of ∼16nM and ∼57nM were obtained for an IgE aptamer and a streptavidin aptamer, respectively. In addition, free and protein-bound IgE aptamer was extracted from the AF4 eluate and amplified, illustrating the potential of AF4 in screening ssDNAs with high affinity to targets. Our results demonstrate that AF4 is an effective tool holding several advantages over the existing techniques and should be useful for study of diverse macromolecular interaction systems. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Multidimensional LC–MS/MS analysis of liver proteins in rat, mouse and human microsomal and S9 fractions

    Directory of Open Access Journals (Sweden)

    Makan Golizeh

    2015-03-01

    Full Text Available Liver plays a key role in metabolism and detoxification, therefore analysis of its proteome is relevant for toxicology and drug discovery studies. To optimize for high proteome coverage, protein and peptide-level ion exchange fractionation were assessed using rat liver microsomes and S9 fractions. 2D-(SCX-RP-LC–MS/MS analysis with peptide fractionation was subsequently employed for rat, mouse and human samples, yielding between 1400 and 1939 identified proteins, 58% of which were shared between species, and with relatively high sequence coverage. This rich dataset is specifically interesting for the toxicology community, and could serve as an excellent source for targeted assay development.

  2. 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.

  3. Conventional and alternative principles for stabilization of protein and polyphenol fractions in beer

    Directory of Open Access Journals (Sweden)

    Marković Romeo S.

    2003-01-01

    Full Text Available Beer haze is primarily formed through complexation of protein and polyphenolic beer ingredients. The problem of reducing susceptibility of beer haze formation can be done either by lowering protein and/or polyphenol levels, or by minimizing the molecular size of protein/polyphenols. In experimental part of this work the shelf life of unstabilized beer is being compared with beer stabilized with various standard products, such as PVPP and silica gel. Furthermore, the trials have been made to prove the functionality of a new product consisting of carrageenan and cross-linked PVPP. The method used to determine shelf life was haze forcing test (0/60°C. Extract, alcohol, bitterness, foam, haze, color and pH were also monitored. The test results showed expectedly that combined treatment of beer ensures the highest level of product stability. Through selective stripping of polyphenols and protein fractions it is possible to improve shelf life of beer to a significant extent.

  4. Characterization of protein fractions and antioxidant activity of Chia seeds (Salvia hispanica L.

    Directory of Open Access Journals (Sweden)

    Kvetoslava Kačmárová

    2016-01-01

    Full Text Available Chia seed (Salvia hispanica L. is an annual herbaceous plant categorized under Lamiaceae family. Chia seeds were investigated as a source of proteins and natural antioxidants. It is a potential alternative source of high quality protein, fats, carbohydrates, high dietary fibre, vitamins and mineral elements. The objective of this study was to evaluate chia seed from protein content and antioxidant acivity and highlight the quality of this pseudocereal. A crude protein, moisture content, content of protein fractions, total antioxidant capacity (TAC and superoxide dismutase activity of chia seeds and food products containing chia seeds were determined. The protein content of chia seeds ranged from 2.9% to 4.6% dry matter from that albumins and globulins ranged from 54.6% to 62.8%. Chia is poor in a prolamines (<15%. Various chia seeds showed differences in their SOD activity and exhibited the high antiradical activity against 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS. The highest antioxidant capacity was found in sample chia seeds from Bolivia (1.46 mM TEAC.g-1 in the dry matter and the lowest values of antioxidant activity was estimated in sample chia seeds from Argentina (1.05 mM TEAC.g-1 in the dry matter. The highest SOD activity was determined in sample chia from Argentina (2191.8 U.g-1 in the dry matter. The lowest SOD activity was found in sample chia-bio from Argentina (754.0 U.g-1 in the dry matter.. It makes them potentially suitable for use in the gluten-free diet of coeliac people and it can be used as a potential ingredient in health food because of its high antioxidant activity.

  5. Evaluation of protein fractionation and ruminal and intestinal digestibility of corn milling co-products.

    Science.gov (United States)

    Kelzer, J M; Kononoff, P J; Tedeschi, L O; Jenkins, T C; Karges, K; Gibson, M L

    2010-06-01

    Novel corn milling co-products developed from technological advancements in ethanol production vary widely in chemical composition and nutrient availability. The objectives of this study were to characterize feed protein fractions and evaluate differences in rumen-undegradable protein (RUP) and its digestible fraction (dRUP), amino acid concentration, and in vitro gas production of 7 corn milling co-products. The crude protein (CP; % of dry matter) of co-products was 12.7 for germ, 26.9 for dried distillers grains plus solubles that had no heat exposure before fermentation (DDGS1), 45.4 for high-protein dried distillers grains (HPDDG), 12.7 for bran, 30.2 for wet distillers grains plus solubles (WDGS), 23.1 for wet corn gluten feed (WCGF), and 26.0 for dried distillers grains plus solubles that had heat exposure before fermentation (DDGS2). Two ruminally and duodenally fistulated Holstein steers weighing 663+/-24 kg were used to determine RUP and dRUP with the in situ and mobile bag techniques. Samples of each feed were ruminally incubated for 16 h, and mobile bags were exposed to simulated abomasal digestion before insertion into the duodenum and subsequent collection in the feces. Protein fractions A, B(1), B(2), B(3), and C were characterized as follows (% CP): germ=30.0, 15.0, 38.1, 13.5, 3.4; DDGS1=17.0, 7.0, 67.0, 4.8, 4.2; HPDDG=7.4, 0.6, 82.4, 8.8, 0.8; bran=33.5, 4.0, 54.3, 6.0, 2.2; WDGS=18.6, 2.4, 53.1, 11.0, 14.9; WCGF=36.6, 15.9, 33.2, 10.1, 4.1; and DDGS2=17.9, 2.1, 41.1, 11.1, 27.9. The proportions of RUP and dRUP were different and are reported as follows (% CP): DDGS2=56.3, 91.9; HPDDG=55.2, 97.7; WDGS=44.7, 93.1; DDGS1=33.2, 92.1; bran=20.7, 65.8; germ=16.5, 66.8; and WCGF=11.5, 51.1. The concentrations of Lys and Met in the RUP were different and are listed as follows (% CP): germ=2.9, 2.0; DDGS1=1.9, 2.0; HPDDG=2.0, 3.2; bran=3.2, 1.5; WDGS=1.9, 2.3; WCGF=3.5, 1.6; and DDGS2=1.9, 2.4. In vitro gas production (mL/48h) was highest for germ (52

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

    NARCIS (Netherlands)

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

    2012-01-01

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

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

    NARCIS (Netherlands)

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

    2012-01-01

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

  8. Milk from different species: Relationship between protein fractions and inflammatory response in infants affected by generalized epilepsy.

    Science.gov (United States)

    Albenzio, M; Santillo, A; Ciliberti, M G; Figliola, L; Caroprese, M; Marino, R; Polito, A N

    2016-07-01

    The present study was undertaken to evaluate the effect of protein fractions from bovine, caprine, and ovine milk on production of cytokines and reactive oxygen species (ROS) and reactive nitrogen species (RNS) by cultured peripheral blood mononuclear cells (PMBC) from infants with generalized epilepsy. Bovine, caprine, and ovine bulk milks were pasteurized and analyzed for chemical composition. Then, PBMC were isolated from 10 patients with generalized epilepsy (5 males; mean age 33.6±5.4mo). Production of tumor necrosis factor-α (TNF-α), IL-10, IL-6, and IL-1β was studied in cultured PBMC (from infants with epilepsy and controls) stimulated by bovine, caprine, and ovine milk and casein and whey protein fractions, and levels of ROS and RNS were measured in the culture supernatant. The ability of PBMC to secrete cytokines in response to milk and protein fraction stimulation may predict the secretion of soluble factor TNF-α in the bloodstream of challenged patients. Bovine, caprine, and ovine bulk milks induced low-level production of IL-10 by cultured PBMC in at least 50% of cases; the same behavior was observed in both casein and whey protein fractions for all species studied. Bovine and ovine milk and their casein fractions induced production of lower levels of IL-1β in 80% of patients, whereas caprine milk and its casein fraction induced the highest levels in 80% of patients. The amount of IL-6 detected after stimulation of PBMC by milk and its fractions for all species was lower than that of other proinflammatory cytokines. In the bovine, total free radicals were higher in bulk milk and lower in the casein fraction, whereas the whey protein fraction showed an intermediate level; in caprine, ROS/RNS levels were not different among milk fractions, whereas ovine had higher levels for bulk milk and casein than the whey protein fraction. Lower levels of ROS/RNS detected in PBMC cultured with caprine milk fraction could be responsible for the lower levels of

  9. Preliminary evaluation of total protein concentration and electrophoretic protein fractions in fresh and frozen serum from wild Horned Vipers (Vipera ammodytes ammodytes).

    Science.gov (United States)

    Proverbio, Daniela; de Giorgi, Giada Bagnagatti; Della Pepa, Alessandra; Baggiani, Luciana; Spada, Eva; Perego, Roberta; Comazzi, Carlo; Belloli, Angelo

    2012-12-01

    Determination of the health status of reptiles is based on physical examination and evaluation of hematologic and biochemical values. Evaluation of serum total protein (TP) concentration and protein fractions plays an important role in health assessment; however, little is known about references value for these analytes in wild viperoid snakes. In addition, studies evaluating the stability of proteins in frozen viperoid serum are lacking. The aims of this study were to establish preliminary reference values for concentrations of TP and protein fractions in serum from wild vipers and to evaluate the stability of serum proteins in frozen serum samples from viperoid snakes. Blood samples were collected from wild Horned Vipers (Vipera ammodytes ammodytes). Using fresh serum, TP concentrations were determined using the biuret method and protein fractions were analyzed using agarose gel electrophoresis (AGE); albumin/globulin ratios were calculated. Analyses were also performed on serum frozen at -20°C for 70 days and then thawed. Pre- and post-storage results were compared using the Mann-Whitney U-test. Five adult wild Horned Vipers were sampled and comprised 4 males and 1 female. The female snake had higher TP concentrations than the male snakes. The electrophoretic patterns demonstrated 6 protein fractions that were similar for all 5 snakes. There were no significant changes in the concentrations of the 6 protein fractions post-storage; the percentage of the alpha-1 fraction was increased in frozen/thawed serum. Total protein concentrations in serum from Vipera ammodytes ammodytes were in agreement with published reference intervals for healthy reptiles and viperoid snakes. Serum protein fractions were easy to identify using AGE electrophoresis. © 2012 American Society for Veterinary Clinical Pathology.

  10. A neutrophil GTP-binding protein that regulates cell free NADPH oxidase activation is located in the cytosolic fraction.

    Science.gov (United States)

    Gabig, T G; Eklund, E A; Potter, G B; Dykes, J R

    1990-08-01

    The dormant O2(-)-generating oxidase in plasma membranes from unstimulated neutrophils becomes activated in the presence of arachidonate and a multicomponent cytosolic fraction. This process is stimulated by nonhydrolyzable GTP analogues and may involve a pertussis toxin insensitive GTP-binding protein. Our studies were designed to characterize the putative GTP-binding protein, localizing it to either membrane or cytosolic fraction in this system. Exposure of the isolated membrane fraction to guanosine-5'-(3-O-thio)triphosphate (GTP gamma S), with or without arachidonate, had no effect on subsequent NADPH oxidase activation by the cytosolic fraction. Preexposure of the cytosolic fraction to GTP gamma S alone did not enhance activation of the membrane oxidase. However, preexposure of the cytosol to GTP gamma S then arachidonate caused a four-fold enhancement of its ability to activate the membrane oxidase. This enhancement was evident after removal of unbound GTP gamma S and arachidonate, and was not augmented by additional GTP gamma S during membrane activation. A reconstitution assay was developed for cytosolic component(s) responsible for the GTP gamma S effect. Cytosol preincubated with GTP gamma 35S then arachidonate was fractionated by anion exchange chromatography. A single peak of protein-bound GTP gamma 35S was recovered that had reconstitutive activity. Cytosol preincubated with GTP gamma 35S alone was similarly fractionated and the same peak of protein-bound GTP gamma 35S was observed. However, this peak had no reconstitutive activity. We conclude that the GTP-binding protein regulating this cellfree system is located in the cytosolic fraction. The GTP gamma S-liganded form of this protein may be activated or stabilized by arachidonate.

  11. Proteomic analysis of processing by-products from canned and fresh tuna: identification of potentially functional food proteins.

    Science.gov (United States)

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

    2012-09-15

    Proteomic approaches have been used to identify the main proteins present in processing by-products generated by the canning tuna-industry, as well as in by-products derived from filleting of skeletal red muscle of fresh tuna. Following fractionation by using an ammonium sulphate precipitation method, three proteins (tropomyosin, haemoglobin and the stress-shock protein ubiquitin) were identified in the highly heterogeneous and heat-treated material discarded by the canning-industry. Additionally, this fractionation method was successful to obtain tropomyosin of high purity from the heterogeneous starting material. By-products from skeletal red muscle of fresh tuna were efficiently fractionated to sarcoplasmic and myofibrillar fractions, prior to the identification based mainly on the combined searching of the peptide mass fingerprint (MALDI-TOF) and peptide fragment fingerprinting (MALDI LIFT-TOF/TOF) spectra of fifteen bands separated by 1D SDS-PAGE. Thus, the sarcoplasmic fraction contained myoglobin and several enzymes that are essential for efficient energy production, whereas the myofibrillar fraction had important contractile proteins, such as actin, tropomyosin, myosin or an isoform of the enzyme creatine kinase. Application of proteomic technologies has revealed new knowledge on the composition of important by-products from tuna species, enabling a better evaluation of their potential applications. Copyright © 2012 Elsevier Ltd. All rights reserved.

  12. Protein from the fraction remaining after RNA extraction is useful for proteomics but care must be exercised in its application.

    Science.gov (United States)

    Yamaguchi, Hiromi; Hasegawa, Kiyoshi; Esumi, Mariko

    2013-08-01

    Simultaneous isolation of mRNA and proteins from a single small biopsy specimen can be useful for integrated omics studies. Here, we have improved the method for extracting protein from the fraction remaining after RNA isolation by TRIzol reagent, for application in protein and proteome analyses. Protein yield was reduced by half, but the patterns developed on 2D gels were equivalent to conventional urea extractions. Thus, although quantitative profiles of individual proteins were different from conventionally-isolated samples, overall profiles were similar. Therefore, this particular protein source is useful for proteomics but care must be exercised in its application.

  13. Mass-spectrometric identification of binding proteins of Mr 25,000 protein, a part of vitellogenin B1, detected in particulate fraction of Xenopus laevis oocytes.

    Science.gov (United States)

    Sugimoto, Isamu; Li, Zhijun; Yoshitome, Satoshi; Ito, Susumu; Hashimoto, Eikichi

    2004-10-01

    A phosphorylated protein with molecular mass of 25,000 (pp25) is a component of Xenopus laevis vitellogenin B1. Our previous report showed the existence of several binding proteins of pp25 in the particulate fraction of Xenopus oocytes. In an attempt to elucidate the function of pp25, two of these binding proteins were purified, analyzed by mass-spectrometry, and identified as ribosomal proteins S13 and S14. Other binding proteins in the particulate fraction mostly corresponded to those derived from purified 40S and 60S ribosomal subunits, as shown by the overlay assay method. However, pp25 did not show any effect on protein synthesis in the rabbit reticulocyte lysate system. A model in which pp25 connects a type of serpin (serine protease inhibitor), the only pp25-binding protein detected in the cytoplasm, to the endoplasmic reticulum through two serine clusters is proposed to explain a possible function of this protein.

  14. A multi-channel gel electrophoresis and continuous fraction collection apparatus for high throughput protein separation and characterization

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Megan; Nordmeyer, Robert A.; Cornell, Earl; Dong, Ming; Biggin, Mark D.; Jin, Jian

    2009-10-02

    To facilitate a direct interface between protein separation by PAGE and protein identification by mass spectrometry, we developed a multichannel system that continuously collects fractions as protein bands migrate off the bottom of gel electrophoresis columns. The device was constructed using several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A Counter Free-Flow elution technique then allows continuous and simultaneous fraction collection from multiple channels at low cost. We demonstrate that rapid, high-resolution separation of a complex protein mixture can be achieved on this system using SDS-PAGE. In a 2.5 h electrophoresis run, for example, each sample was separated and eluted into 48-96 fractions over a mass range of 10-150 kDa; sample recovery rates were 50percent or higher; each channel was loaded with up to 0.3 mg of protein in 0.4 mL; and a purified band was eluted in two to three fractions (200 L/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 g per channel and reduced resolution.

  15. A multichannel gel electrophoresis and continuous fraction collection apparatus for high-throughput protein separation and characterization.

    Science.gov (United States)

    Choi, Megan; Nordmeyer, Robert A; Cornell, Earl; Dong, Ming; Biggin, Mark D; Jin, Jian

    2010-01-01

    To facilitate a direct interface between protein separation by PAGE and protein identification by mass spectrometry, we developed a multichannel system that continuously collects fractions as protein bands migrate off the bottom of gel electrophoresis columns. The device was constructed using several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A "Counter Free-Flow" elution technique then allows continuous and simultaneous fraction collection from multiple channels at low cost. We demonstrate that rapid, high-resolution separation of a complex protein mixture can be achieved on this system using SDS-PAGE. In a 2.5 h electrophoresis run, for example, each sample was separated and eluted into 48-96 fractions over a mass range of approximately 10-150 kDa; sample recovery rates were 50% or higher; each channel was loaded with up to 0.3 mg of protein in 0.4 mL; and a purified band was eluted in two to three fractions (200 microL/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 microg per channel and reduced resolution.

  16. 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.

  17. 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 ...

  18. CaMKII inhibition targeted to the sarcoplasmic reticulum inhibits frequency dependent acceleration of relaxation and Ca2+ current facilitation

    OpenAIRE

    Picht, Eckard; DeSantiago, Jaime; Huke, Sabine; Kaetzel, Marcia A.; Dedman, John R.; Bers, Donald M.

    2006-01-01

    Cardiac Ca2+/calmodulin-dependent protein kinase II (CaMKII) in heart has been implicated in Ca2+ current (ICa) facilitation, enhanced sarcoplasmic reticulum (SR) Ca2+ release and frequency dependent acceleration of relaxation (FDAR) via enhanced SR Ca2+ uptake. However, questions remain about how CaMKII may work in these three processes. Here we tested the role of CaM-KII in these processes using transgenic mice (SR-AIP) that express four concatenated repeats of the CaMKII inhibitory peptide...

  19. PROTEIN FRACTIONATION AND UTILIZATION OF SOYBEAN AND REDBEAN AS AFFECTED BY DIFFERENT DRYING TEMPERATURE

    Directory of Open Access Journals (Sweden)

    Anuraga Jayanegara

    2017-02-01

    Full Text Available The objective of this study was to investigate the influence of different drying temperature on chemical composition, in vitro rumen fermentation and digestibility of soybean and redbean. Soybean and redbean were dried in an oven set at four different drying temperatures, i.e. 50, 60, 70 and 80 oC for 24 h in three replicates. Dried samples were then milled and used further for chemical composition determination (proximate analysis, Van Soest analysis and protein fraction and in vitro rumen fermentation assay. Parameters measured in the in vitro assay were gas production, digestibility, pH, ammonia and volatile fatty acids (VFA. Data obtained were analyzed by using analysis of variance and a posthoc test namely Duncan’s multiple range test. Results revealed that neutral detergent insoluble crude protein (NDICP content increased at higher drying temperature (70 or 80 oC for both soybean and redbean (P<0.05 but at different magnitude. As with NDICP, higher temperature led to a higher acid detergent insoluble crude protein (ADICP both in soybean and redbean (P<0.05. Higher temperature decreased gas production rate (GPR of both beans (P<0.05. Drying of soybean at 70 or 80 oC decreased crude protein digestibility (CPD of soybean than dried at 50 or 60 oC (P<0.05. Higher drying temperature resulted in a lower NH3 concentration (P<0.05. It can be concluded that drying temperature at 50 or 60 oC is safe to maintain nutritional quality of soybean and redbean.

  20. 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...

  1. Improving basic and membrane protein MS detection of the culture filtrate proteins from Mycobacterium tuberculosis H37Rv by biomimetic affinity pre-fractionation.

    Science.gov (United States)

    Ma, Guorong; Zhou, Fuqiang; Gao, Lina; Sun, Zhanqiang; Deng, Li; Zhang, Shulin; Li, Rongxiu

    2017-04-09

    The culture filtrate proteins (CFPs) from Mycobacterium tuberculosis (MTB) have been shown to induce protective immune responses in human and animal models, making them a promising source of candidate targets for tuberculosis drugs, vaccines and diagnostics. The constituents of the MTB CFP proteome are complex and vary with growth conditions. To effectively profile CFPs, gel-based pre-fractionation is usually performed before MS analysis. In this study, we describe a novel pre-fractionation approach by which the proteome is divided into seven partially overlapping fractions by biomimetic affinity chromatography (BiAC) using a six-column cascade. The LC-MS/MS analysis of individual fractions identified a total of 541 CFPs, including 61 first-time identifications. Notably, ∼1/3 (20/61) of these novel CFPs are membrane proteins, among which 9 proteins have 2-14 transmembrane domains (TMD). In addition, ∼1/4 (14/61) of the CFPs are basic proteins with pI values greater than 9.0. Our data demonstrate that BiAC pre-fractionation markedly improves protein detection by LC-MS/MS, and the coverage of basic and hydrophobic proteins in particular is remarkably increased. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  2. 大鼠 H9c2心肌细胞缺氧/复氧损伤模型肌浆网钙调控相关蛋白表达及当归补血汤的干预作用%Danggui Buxue Decoction on expression of sarcoplasmic reticulum calcium regulatory protein in H9c2 myocardial cell model with hypoxia/reoxygenation damage

    Institute of Scientific and Technical Information of China (English)

    周春刚; 汤加; 李卿; 徐辰; 张志斌

    2016-01-01

    Objective To establish the model of hypoxia / reoxygenation damage in H9c2 rat myocardial cell and observe the expression of myocardial sarcoplasmic reticulum calcium regulatory protein and the effect of Danggui Bux-ue Decoction on calcium overload in H9c2 cells with hypoxia / reoxygenation injury. Methods JC-1 staining was used to detect the mitochondrial membrane potential by flow cytometry,Fluo-3 AM calcium fluorescence probe for the detection of intracellular calcium concentration,The expression level of sarcoplasmic reticulum Ca transport ATP en-zyme(SERCA2a),L type calcium channel(CAV1. 3),ryanodine receptor(RyR1,RyR2),phospholamban(PLB), calsequestrin(CASQ)mRNA were detected by RT-PCR. Results Compared with the normal control group,the early apoptosis rate and intracellular calcium concentration in model group increased significantly(P 0. 05),compared with the model group. Con-clusion There are obvious calcium overload and sarcoplasmic reticulum calcium regulating proteins adjustment disor-der in the model of H9c2 rat myocardial cell with hypoxia / reoxygenation damage,Danggui Buxue Decoction can in-crease the expression of the sarcoplasmic reticulumcalcium regulating protein SERCA2a and CASQ mRNA after reoxy-genation,alleviate calcium overload,and significantly reduce the early apoptosis rate of reoxygenation damage cells.%目的:建立缺氧/复氧大鼠 H9c2心肌细胞损伤模型,观察心肌细胞肌浆网钙调控相关蛋白表达及当归补血汤对 H9c2细胞缺氧/复氧损伤钙超载的影响。方法 JC-1染色流式细胞仪检测细胞线粒体膜电位, Fluo-3 AM 钙离子荧光探针检测细胞内钙离子浓度,RT-PCR 荧光相对定量检测大鼠心肌肌浆网 Ca 转运 ATP酶(SERCA2a)、L 型钙通道(CAV1.3)、兰尼碱受体(RyR1,RyR2)、受磷蛋白(PLB)、肌集钙蛋白( CASQ)的mRNA 表达水平。结果与正常对照组比较,模型组细胞早期凋亡显著增加(P <0.05),细胞内

  3. Reverse-phase HPLC separation of hemp seed (Cannabis sativa L.) protein hydrolysate produced peptide fractions with enhanced antioxidant capacity.

    Science.gov (United States)

    Girgih, Abraham T; Udenigwe, Chibuike C; Aluko, Rotimi E

    2013-03-01

    Hemp seed protein hydrolysate (HPH) was produced through simulated gastrointestinal tract (GIT) digestion of hemp seed protein isolate followed by partial purification and separation into eight peptide fractions by reverse-phase (RP)-HPLC. The peptide fractions exhibited higher oxygen radical absorbance capacity as well as scavenging of 2,2-diphenyl-1-picrylhydrazyl, superoxide and hydroxyl radicals when compared to HPH. Radical scavenging activities of the fractionated peptides increased as content of hydrophobic amino acids or elution time was increased, with the exception of hydroxyl radical scavenging that showed decreased trend. Glutathione (GSH), HPH and the RP-HPLC peptide fractions possessed low ferric ion reducing ability but all had strong (>60 %) metal chelating activities. Inhibition of linoleic acid oxidation by some of the HPH peptide fractions was higher at 1 mg/ml when compared to that observed at 0.1 mg/ml peptide concentration. Peptide separation resulted in higher concentration of some hydrophobic amino acids (especially proline, leucine and isoleucine) in the fractions (mainly F5 and F8) when compared to HPH. The elution time-dependent increased concentrations of the hydrophobic amino acids coupled with decreased levels of positively charged amino acids may have been responsible for the significantly higher (p < 0.05) antioxidant properties observed for some of the peptide fractions when compared to the unfractionated HPH. In conclusion, the antioxidant activity of HPH after simulated GIT digestion is mainly influenced by the amino acid composition of some of its peptides.

  4. Occurrence of major whey proteins in the pH 4.6 insoluble protein fraction from UHT-treated milk.

    Science.gov (United States)

    Pizzano, Rosa; Manzo, Carla; Nicolai, Maria Adalgisa; Addeo, Francesco

    2012-08-15

    A clear picture of the protein rearrangement in milk following UHT-treatment was drawn by a comparative analysis of the pH 4.6 soluble protein fraction (SPF) and the pH 4.6 insoluble protein fraction (IPF) recovered from raw and UHT-treated milk samples. The two protein fractions were analyzed by mono- or bidimensional gel electrophoresis under reducing and nonreducing conditions, and protein bands were identified by specific immunostaining. Results showed that bovine serum albumin, β-lactoglobulin, and, to a lesser extent, α-lactalbumin coprecipitated with caseins in UHT-treated milk samples at pH 4.6. These proteins were almost exclusively involved in high molecular weight aggregates held together by disulfide bonds. Partition of α-lactalbumin and bovine serum albumin in the protein fractions obtained upon acidification of milk at pH 4.6 was evaluated by competitive immunoassays. The ELISA-based results suggested the possibility of using pH 4.6 insoluble α-lactalbumin and bovine serum albumin, in addition to pH 4.6 insoluble β-lactoglobulin, as indicators of the intensity of the heat treatment applied to milk.

  5. Gluten Protein Composition in Several Fractions Obtained by Shear Induced Separation of Wheat Flour

    NARCIS (Netherlands)

    Zalm, van der E.E.J.; Grabowska, K.J.; Strubel, M.; Goot, van der A.J.; Hamer, R.J.; Boom, R.M.

    2010-01-01

    Recently, it was found that applying curvilinear shear flow in a cone-cone shearing device to wheat flour dough induces separation, resulting in a gluten-enriched fraction in the apex of the cone and gluten-depleted fraction at the outer part. This article describes whether fractionation of the vari

  6. Identification of canine platelet proteins separated by differential detergent fractionation for nonelectrophoretic proteomics analyzed by Gene Ontology and pathways analysis

    Directory of Open Access Journals (Sweden)

    Trichler SA

    2014-01-01

    Full Text Available Shauna A Trichler,1,* Sandra C Bulla,1,* Nandita Mahajan,1 Kari V Lunsford,2 Ken Pendarvis,3 Bindu Nanduri,4,5 Fiona M McCarthy,3 Camilo Bulla1 1Department of Pathobiology and Population Medicine, 2Department of Clinical Sciences and Animal Health Center, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS, 3Department of Veterinary Science and Microbiology, University of Arizona, Tucson, AZ, 4Department of Biological Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS, 5Institute for Genomics, Biocomputing and Biotechnology, Starkville, MS, USA *These authors contributed equally to this work Abstract: During platelet development, proteins necessary for the many functional roles of the platelet are stored within cytoplasmic granules. Platelets have also been shown to take up and store many plasma proteins into granules. This makes the platelet a potential novel source of biomarkers for many disease states. Approaches to sample preparation for proteomic studies for biomarkers search vary. Compared with traditional two-dimensional polyacrylamide gel electrophoresis systems, nonelectrophoretic proteomics methods that employ offline protein fractionation methods such as the differential detergent fractionation method have clear advantages. Here we report a proteomic survey of the canine platelet proteome using differential detergent fractionation coupled with mass spectrometry and functional modeling of the canine platelet proteins identified. A total of 5,974 unique proteins were identified from platelets, of which only 298 (5% had previous experimental evidence of in vivo expression. The use of offline prefractionation of canine proteins by differential detergent fractionation resulted in greater proteome coverage as compared with previous reports. This initial study contributes to a broader understanding of canine platelet biology and aids functional research

  7. 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

  8. Interactions of vanadate oligomers with sarcoplasmic reticulum Ca(2+)-ATPase.

    Science.gov (United States)

    Aureliano, M; Mdeira, V M

    1994-04-28

    Upon addition of sarcoplasmic reticulum (SR), the line width of tetrameric vanadate signal of 51V-NMR spectra narrowed in the presence of ATP and Ca2+, whereas monomeric vanadate line widths were broadened. Thus, ATP decreases the affinity of the enzyme for tetravanadate whereas it induces the interaction with monomeric vanadate. In the presence of Ca2+ it was observed that tetrameric and decameric vanadate bind to SR ATPase whereas monomeric vanadate only binds to SR when ATP is present. However, decameric vanadate clearly differs from vanadate oligomers present in monovanadate solutions in preventing the accumulation of Ca2+ by sarcoplasmic reticulum (SR) vesicles coupled to ATP hydrolysis. Mg2+ increased the inhibitory effect promoted by decavanadate whereas a slight enhancement of Ca2+ uptake was observed in the presence of monovanadate. For 5 mM Mg2+, a nominal 2 mM vanadium 'decavanadate' solution containing about 190 to 200 microM decameric and less than 100 microM monomeric species depressed the rate of Ca2+ uptake by 50% whereas a nominal 2 mM monovanadate solution containing about 662 microM monomeric, 143 microM dimeric and 252 microM tetrameric species had no effect on the rate of Ca2+ accumulation. However, 2 mM 'decavanadate' inhibits by 75% the SR Ca(2+)-ATPase activity whereas the presence of 2 mM 'monovanadate' produces an inhibitory effect below 50%. Therefore, the Ca:ATP stoichiometry of Ca2+ transport is enhanced by monovanadate. In the presence of oxalate, inhibition of SR Ca(2+)-ATPase activity by these solutions is enhanced to 97% and 86% whereas in the presence of the ionophore lasalocid, the inhibitory values were 87% and 19% for 2 mM decavanadate and 2 mM monovanadate solutions, respectively. Apparently, the increase of vesicular Ca2+ concentration counteracts monovanadate inhibition of SR Ca(2+)-ATPase activity but it does not significantly affect decavanadate inhibition.

  9. 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, J; Hartvigsen, M L

    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...... in the incremental area under the curve over the 480-min period.Conclusions:A supplement of four different whey protein fractions to a fat-rich meal had similar effects on postprandial triglyceride responses in type 2 diabetic subjects. Whey isolate and whey hydrolysate caused a higher insulin response...... on postprandial lipid and hormone responses added to a high-fat meal in type 2 diabetic subjects.Subjects/Methods:A total of 12 type 2 diabetic subjects ingested four isocaloric test meals in randomized order. The test meals contained 100¿g of butter and 45¿g of carbohydrate in combination with 45¿g of whey...

  10. Complexation of bovine beta-lactoglobulin with 11S protein fractions of soybean (Glycine max) and sesame (Sesamum indicum).

    Science.gov (United States)

    Anuradha, S N; Prakash, V

    2009-01-01

    Beta-lactoglobulin (beta-Lg) comprises 50% of the whey component of bovine milk. Protein-protein interactions between bovine beta-Lg and 11S protein fractions of soybean and sesame were investigated by turbidity, solubility behaviour and by evaluation of functional properties in the mixed systems. In this work, the aggregation behaviour of soybean and the whey protein (beta-Lg) showed the formation of soluble complexes. Turbidity and solubility studies showed that the proteins interacted at temperatures between 60 and 90+/-5 degrees C. Heating a mixture of beta-Lg and 11S proteins of soybean at higher temperatures formed soluble complexes with beta-Lg. It also reduced the self aggregation behaviour, especially that of 11S protein fraction of soybean. This reduced the precipitation of soybean proteins at higher temperature. The complex formed was resolved by gel filtration using high-performance liquid chromatography. Upon heating beta-Lg at neutral pH, native dimer starts to dissociate into monomers leading to the exposure of previously buried hydrophobic amino acids and the free thiol group. The soluble complex is formed by the exposed thiol groups. But interaction of beta-Lg with sesame 11S protein fractions did not form any soluble complexes. The mechanism of interaction indicates that hydrophobic interactions were preferred over disulfide linkages at the high salt concentrations of the buffer used. During thermal treatment the molecules are unfolded, leading to an exposure of the hydrophobic groups that further enhance the protein-protein interactions that are entropically driven hydrophobic interactions.

  11. 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.

  12. Identification of the major proteins of an immune modulating fraction from adult Fasciola hepatica released by Nonidet P40.

    Science.gov (United States)

    Morphew, Russell M; Hamilton, Clare M; Wright, Hazel A; Dowling, David J; O'Neill, Sandra M; Brophy, Peter M

    2013-01-31

    Fasciola hepatica NP-40 released protein extract (FhNPE) exhibits potent Th1 immunosuppressive properties in vitro and in vivo. However, the protein composition of this active fraction, responsible for Th1 immune modulatory activity, has yet to be resolved. Therefore, FhNPE, a Nonidet P-40 extract, was subjected to a proteomic analysis in order to identify individual protein components. This was performed using an in house F. hepatica EST database following 2D electrophoresis combined with de novo sequencing based mass spectrometry. The identified proteins, a mixture of excretory/secretory and membrane-associated proteins, are associated with stress response and chaperoning, energy metabolism and cytoskeletal components. The immune modulatory properties of these identified protein(s) are discussed and HSP70 from F. hepatica is highlighted as a potential host immune modulator for future study.

  13. Prediction of laboratory and in situ protein fractions in legume and grass silages using near-infrared reflectance spectroscopy.

    Science.gov (United States)

    Hoffman, P C; Brehm, N M; Bauman, L M; Peters, J B; Undersander, D J

    1999-04-01

    Legume and grass silage samples (n = 121) were collected from commercial forage testing laboratories (trial 1). Samples were dried at 55 degrees C for 48 h, ground, scanned on a near-infrared reflectance spectrophotometer, and analyzed for crude protein (CP), soluble CP, acid detergent fiber (ADF) CP, and neutral detergent fiber (NDF) CP by wet chemistry methods. Sixty samples were selected for calibration development, and the remaining samples were used for equation validation. Near-infrared reflectance spectroscopy accurately predicted the CP content of the silages (R2 = 0.96), but prediction of soluble CP, ADF CP, and NDF CP was markedly less accurate. The coefficients of determination and standard errors of calibration for CP, ADF CP, NDF CP (percentage of DM), and soluble CP (percentage of CP) were as follows (0.96 and 0.80, 0.77 and 0.24, 0.72 and 0.71, and 0.82 and 4.40). In a second study, legume and grass silage samples (n = 32) were dried at 55 degrees C and ground (2 mm). Duplicate dacron bags containing 5 g of silage were incubated in the ventral rumen of three ruminally cannulated cows for 0, 3, 6, 12, 24, 48, and 72 h. In situ protein fractions, including rapidly degraded protein, slowly degraded protein, undegradable protein, degradation rate, and rumen-undegradable protein, were determined. Original samples were reground (1 mm) and scanned. Previously defined near-infrared spectroscopy calibration procedures were conducted. Coefficients of determination for in situ CP fractions were R2 > 0.92 with the exception of degradation rate (R2 = 0.87). Data suggest that in situ protein fractions are better predicted by near-infrared reflectance spectroscopy than by laboratory protein fractions.

  14. Identification of the major proteins of an immune modulating fraction from adult Fasciola hepatica released by Nonidet P40

    OpenAIRE

    Morphew, R.M.; Hamilton, C M; Wright, H. A.; Dowling, D. J.; O’Neill, S. M.; Brophy, P.M.

    2012-01-01

    Fasciola hepatica NP-40 released antigens (FhTeg) exhibit potent Th1 immunosuppressive properties in vitro and in vivo. However, the protein composition of this active fraction, responsible for Th1 immune modulatory activity, has yet to be resolved. Therefore, FhTeg, a Nonidet P-40 extract, was subjected to a proteomic analysis in order to identify individual protein components. This was performed using an in house F. hepatica EST database following 2D electrophoresis combined with de novo se...

  15. 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...

  16. Oily fraction of Semecarpus anacardium Linn nuts involves protein kinase C activation for its pro-inflammatory response.

    Science.gov (United States)

    Tripathi, Yamini B; Pandey, Nidhi; Tripathi, Deepshikha; Tripathi, Pratibha

    2010-12-01

    The oily fraction (non polar fraction-NPF) of S. anacardium (SA) significantly increased the expression of protein kinase C-delta (PKC-delta) in macrophages in concentration dependent manner, which was similar to phorbol myristate acetate (PMA) response. Further, H-7 (1-(5-isoquinolinesulphonyl)-2-methylpiperazine), an inhibitor of PKC significantly inhibited this NPF mediated response in a concentration dependent manner. In the post treatment kinetics, H-7 showed this inhibition only up to 6 min post NPF/PMA addition, but in similar condition, quercetin, a flavone with reported antioxidant property, showed this inhibition only up to 2 min. The results clearly suggest that oily fraction of SA nuts enhances the expression of PKC protein, which may be responsible for its reported pro-inflammatory property.

  17. Emulsifying and Foaming Properties of Different Protein Fractions Obtained from a Novel Lupin Variety AluProt-CGNA(®) (Lupinus luteus).

    Science.gov (United States)

    Burgos-Díaz, César; Piornos, José A; Wandersleben, Traudy; Ogura, Takahiro; Hernández, Xaviera; Rubilar, Mónica

    2016-07-01

    The use of vegetable proteins as food ingredient is becoming increasingly important due to their high versatility and environmental acceptability. This work describes a chemical characterization and techno-functional properties (emulsifying and foaming properties) of 3 protein fractions obtained from a protein-rich novel lupin variety, AluProt-CGNA(®) . This nongenetically modified variety have a great protein content in dehulled seeds (60.6 g protein/100 g, dry matter), which is higher than soybean and other lupin varieties. A simple procedure was utilized to obtain 3 different fractions by using alkali solubilization and isoelectric precipitation. Fractions 1 and 3 were mainly composed of protein and polysaccharides (NNE), whereas fraction 2 was mainly composed by protein (97%, w/w). Fraction 3 presented interesting and potential foaming properties in comparison to the other fractions evaluated in the study. Besides, its solubility, foaming and emulsifying capacity were practically not affected by pH variations. The 3 fractions also presented good emulsion stability, reaching values above a 95%. SDS-PAGE showed that fractions 1 and 2 contained mainly conglutin α, β, and δ, but in different ratios, whereas fraction 3 contained mainly conglutin γ and albumins. The results of this work will provide better understanding for the utilization of each protein fractions as potential ingredients in food industry.

  18. Protein viscosity, mineral fraction and staggered architecture cooperatively enable the fastest stress wave decay in load-bearing biological materials.

    Science.gov (United States)

    Qwamizadeh, Mahan; Zhang, Zuoqi; Zhou, Kun; Zhang, Yong Wei

    2016-07-01

    One of the key functions of load-bearing biological materials, such as bone, dentin and sea shell, is to protect their inside fragile organs by effectively damping dynamic impact. How those materials achieve this remarkable function remains largely unknown. Using systematic finite element analyses, we study the stress wave propagation and attenuation in cortical bone at the nanoscale as a model material to examine the effects of protein viscosity, mineral fraction and staggered architecture on the elastic wave decay. It is found that the staggered arrangement, protein viscosity and mineral fraction work cooperatively to effectively attenuate the stress wave. For a typical mineral volume fraction and protein viscosity, an optimal staggered nanostructure with specific feature sizes and layouts is able to give rise to the fastest stress wave decay, and the optimal aspect ratio and thickness of mineral platelets are in excellent agreement with experimental measurements. In contrary, as the mineral volume fraction or the protein viscosity goes much higher, the structural arrangement is seen having trivial effect on the stress wave decay, suggesting that the damping properties of the composites go into the structure-insensitive regime from the structure-sensitive regime. These findings not only significantly add to our understanding of the structure-function relationship of load-bearing biological materials, and but also provide useful guidelines for the design of bio-inspired materials with superior resistance to impact loading.

  19. Thyroid hormones differentially affect sarcoplasmic reticulum function in rat atria and ventricles.

    Science.gov (United States)

    Kaasik, A; Minajeva, A; Paju, K; Eimre, M; Seppet, E K

    1997-11-01

    The present study was undertaken to compare the effects of hypothyroidism and hyperthyroidism on sarcoplasmic reticulum (SR) Ca(2+)-pump activity, together with assessment of the functional role of SR in providing activator Ca2+ under these altered thyroid states. In response to a shift from hypothyroid to hyperthyroid state, a 10 fold and 2 fold increase in SR Ca(2+)-pump activity in atria and ventricles, respectively, were observed. This was associated with the 8-9 fold increases in atrial contractility (+dT/dt) and relaxation (-dT/dt), but only with a 3-4 fold increase in their ventricular counterparts. Also, the recirculation fraction of activator Ca2+ (RFA) increased to a far greater extent in atria (4 fold) than in papillary muscles, and the relative increment in inhibition of developed tension by ryanodine became 3 times larger in atria than in papillary muscles. A positive force-frequency relationship (FFR) was observed in hypothyroid atria, whereas the hyperthyroid atria, hypothyroid and hyperthyroid papillary muscles showed a negative FFR. These results suggest the greater role of transsarcolemmal (SL) Ca2+ and smaller role of SR Ca2+ in activating contraction in hypothyroid atria compared to other preparations. Thyroid hormones decrease the contribution of SL and increase that of SR in providing activator Ca2+ to the greater extent in atria than in ventricles. This effect of thyroid hormones is based on larger stimulation of SR Ca(2+)-pump in atria compared to ventricles.

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

    Science.gov (United States)

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

    1994-01-01

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

  1. The reaction of N-(1-pyrene)maleimide with sarcoplasmic reticulum.

    Science.gov (United States)

    Papp, S; Kracke, G; Joshi, N; Martonosi, A

    1986-01-01

    The excimer fluorescence of the adduct of N-(1-pyrene)maleimide (PMI) with the Ca2+-ATPase was proposed as a probe of ATPase-ATPase interactions in sarcoplasmic reticulum (Lüdi and Hasselbach, Eur. J. Biochem., 1983, 130:5-8). We tested this proposition by analyzing the spectral properties and stoichiometry of the adducts of pyrenemaleimide with sarcoplasmic reticulum and with dithiothreitol and by comparing the effects of various detergents on the excimer fluorescence of the two adducts, with their influence on the sedimentation characteristics, ATPase activity, and light scattering of the pyrenemaleimide-labeled sarcoplasmic reticulum. These studies indicate that pyrenemaleimide reacts nearly randomly with several SH groups on the Ca2+-ATPase, and suggest that the observed excimer fluorescence of pyrenemaleimide-labeled sarcoplasmic reticulum may reflect intramolecular phenomena rather than ATPase-ATPase interactions. Further work is required to establish the relative contribution of intra- and intermolecular mechanisms to the excimer fluorescence. PMID:2937461

  2. Multiparametric investigation of competitive and noncompetitive sorption characteristics of SMP fractions (carbohydrate and protein) on activated carbon.

    Science.gov (United States)

    Dizge, Nadir; Tansel, Berrin

    2011-01-30

    Sorption characteristics of soluble microbial products (SMPs) as carbohydrate and protein on activated carbon were investigated. Batch experiments were conducted to evaluate the sorption kinetics and the equilibrium conditions. The parameters studied included initial SMP concentration (50-200mg/L), activated carbon dosage (0.25-50 g/L), contact time (0.02-4h), particle size of activated carbon used (5-75 μm, 75-850 μm, and 850-1000 μm), and presence of one or both SMP fractions. The equilibrium sorption of carbohydrate and protein were significantly affected by the presence of the second SMP fraction in the solutions. Adsorption isotherms were expressed by the Langmuir and Freundlich models. The adsorption rates under noncompetitive and competitive conditions were analyzed with kinetics-based Lagergren pseudo-first order and pseudo-second order models; and diffusion-based external diffusion and Weber-Morris intraparticle models. Both SMP fractions were removed effectively, however, sorption of protein was significantly better than that of carbohydrate in all cases. The relatively significant effect of particle size on sorption of protein indicates that protein is most likely adsorbed as a single layer on the carbon surface. For the carbohydrate, the increase in particle size did not decrease the sorption significantly indicating that carbohydrate may be adsorbed in multiple layers or may diffuse into the porous matrix more effectively.

  3. Inhibition of protein glycation by procyanidin-B2 enriched fraction of cinnamon: delay of diabetic cataract in rats.

    Science.gov (United States)

    Muthenna, Puppala; Raghu, Ganugula; Akileshwari, Chandrasekhar; Sinha, Sukesh Narayana; Suryanarayana, Palla; Reddy, Geereddy Bhanuprakash

    2013-11-01

    Accumulation of advanced glycation endproducts (AGE) from nonenzymatic glycation of proteins has been implicated in several diabetic complications including diabetic cataract. Previously, we have reported that extracts of dietary agents such as cinnamon have the potential to inhibit AGE formation. In this study, we have shown procyanidin-B2 as the active component of cinnamon that is involved in AGE inhibition using bioassay-guided fractionation of eye lens proteins under in vitro conditions. The data indicate that procyanidin-B2 enriched fraction scavenges dicarbonyls. Further, procyanidin-B2 fraction of cinnamon inhibited the formation of glycosylated hemoglobin in human blood under ex vivo conditions. We have also demonstrated the physiological significance of procyanidin-B2 fraction in terms of delay of diabetic cataract through inhibition of AGE in diabetic rats. These findings establish the antiglycating potential of procyanidin-B2 fraction of cinnamon which suggests a scope for controlling AGE-mediated diabetic complications by food sources that are rich in proanthocyanidins like procyanidin-B2.

  4. Effects of prior exercise and a low-carbohydrate diet on muscle sarcoplasmic reticulum function during cycling in women.

    Science.gov (United States)

    Duhamel, T A; Green, H J; Perco, J G; Ouyang, J

    2006-09-01

    The effects of exercise and diet on sarcoplasmic reticulum Ca(2+)-cycling properties in female vastus lateralis muscle were investigated in two groups of women following four different conditions. The conditions were 4 days of a low-carbohydrate (Lo CHO) and glycogen-depleting exercise plus a Lo CHO diet (Ex + Lo CHO) (experiment 2) and 4 days of normal CHO (Norm CHO) and glycogen-depleting exercise plus Norm CHO (Ex + Norm CHO) (experiment 1). Peak aerobic power (Vo2peak)) was 38.1 +/- 1.4 (SE); n = 9 and 35.6 +/- 1.4 ml.kg(-1).min(-1); n = 9, respectively. Sarcoplasmic reticulum properties measured in vitro in homogenates (micromol.g protein(-1).min(-1)) indicated exercise-induced reductions (P 30, 60 min > fatigue), Ca(2+) uptake (0 > 30 > 60 min, fatigue), and Ca(2+) release, both phase 1 (0, 30 > 60 min, fatigue) and phase 2 (0 > 30, 60 min, fatigue; 30 min > fatigue) in Norm CHO. Exercise was without effect in altering the Hill slope (n(H)), defined as the slope of relationship between Ca(2+)-ATPase activity and Ca(2+) concentration. No differences were observed between Norm CHO and Ex+Norm CHO. Compared with Norm CHO, Lo CHO resulted in a lower (P cycling and that, with the exception of Ca(2+) release, a glycogen-depleting session of exercise before Lo CHO can reverse the effects.

  5. Cylindrical Spirals in Skeletal Muscles Originate From the Longitudinal Sarcoplasmic Reticulum.

    Science.gov (United States)

    Xu, Jing-Wen; Liu, Fu-Chen; Li, Wei; Zhao, Yu-Ying; Zhao, Dan-Dan; Luo, Yue-Bei; Lu, Jian-Qiang; Yan, Chuan-Zhu

    2016-02-01

    Cylindrical spirals (CSs) are rare but distinct subsarcolemmal accumulations in skeletal muscle fibers. To date, CSs have been reported in only 16 patients with a variety of neuromuscular conditions. The origin and composition of CSs are unknown, although there are some morphologic similarities between CSs and tubular aggregates (TAs). To clarify the nature of CSs, we characterized the sarcoplasmic reticulum (SR) and other intracellular membrane system proteins in CSs of muscle biopsies from 2 adult Chinese siblings. Immunohistochemical studies revealed subsarcolemmal immunoreactivity for sarco/endoplasmic reticulum Ca2þ-ATPase 1 (SERCA 1) in the longitudinal SR, but no immunoreactivity for calsequestrin in the terminal cisternae or type 1 ryanodine receptor (RYR1) in the junctional SR. Muscles biopsied from 2 patients with TAs showed immunoreactivity not only for SERCA1 but also for other SR proteins, including calsequestrin and RYR1. CSs exhibited no immunoreactivity for the Golgi apparatus marker GM130, the nuclear membrane emerin, desmin, the autophagosome marker LC3, the lysosomal membrane marker LAMP2, dystrophin, or myosin. Our results suggest CSs may originate only from the longitudinal SR, whereas TAs are composed of both the junctional and longitudinal SR. Immunochemical staining with antibodies against calsequestrin and RYR1 help to distinguish these 2 pathological alterations.

  6. Re-fraction: a machine learning approach for deterministic identification of protein homologues and splice variants in large-scale MS-based proteomics.

    Science.gov (United States)

    Yang, Pengyi; Humphrey, Sean J; Fazakerley, Daniel J; Prior, Matthew J; Yang, Guang; James, David E; Yang, Jean Yee-Hwa

    2012-05-04

    A key step in the analysis of mass spectrometry (MS)-based proteomics data is the inference of proteins from identified peptide sequences. Here we describe Re-Fraction, a novel machine learning algorithm that enhances deterministic protein identification. Re-Fraction utilizes several protein physical properties to assign proteins to expected protein fractions that comprise large-scale MS-based proteomics data. This information is then used to appropriately assign peptides to specific proteins. This approach is sensitive, highly specific, and computationally efficient. We provide algorithms and source code for the current version of Re-Fraction, which accepts output tables from the MaxQuant environment. Nevertheless, the principles behind Re-Fraction can be applied to other protein identification pipelines where data are generated from samples fractionated at the protein level. We demonstrate the utility of this approach through reanalysis of data from a previously published study and generate lists of proteins deterministically identified by Re-Fraction that were previously only identified as members of a protein group. We find that this approach is particularly useful in resolving protein groups composed of splice variants and homologues, which are frequently expressed in a cell- or tissue-specific manner and may have important biological consequences.

  7. Protein and hordein fraction content in barley seeds as affected by sowing date and their relations to malting quality

    Institute of Scientific and Technical Information of China (English)

    QI Jun-cong; CHEN Jin-xin; WANG Jun-mei; WU Fei-bo; CAO Lian-pu; ZHANG Guo-ping

    2005-01-01

    The effect of sowing date on grain protein, hordein fraction content and malting quality of two-rowed spring barley was investigated by using ten commercial cultivars with different grain protein content and the relationships among these traits were examined. The results showed that grain protein content and B hordein content increased as the sowing date postponed and were significantly affected by sowing date, while C and D hordein contents were less influenced by sowing date. There were significant differences in grain protein and hordein fraction content among the ten cultivars. The coefficient of variation of D hordein content was much larger than that of B and C hordein contents, suggesting its greater variation caused by different sowing dates. Beta-amylase activity and diastatic power were also significantly affected by sowing date, with malt extract being less affected. Significant differences in measured malt quality were found among the ten cultivars. Grain protein was significantly correlated with B hordein and malt extract positively and negatively, respectively. There was no significant correlation between beta-amylase activity or diastatic power and grain protein content. B hordein was negatively and significantly correlated with malt extract, but no significant correlations between C hordein, D hordein and malting quality traits.

  8. The calcium uptake of the rat heart sarcoplasmic reticulum is altered by dietary lipid.

    Science.gov (United States)

    Taffet, G E; Pham, T T; Bick, D L; Entman, M L; Pownall, H J; Bick, R J

    1993-01-01

    Small amounts of dietary n-3 fatty acids can have dramatic physiological effects, including the reduction of plasma triglycerides and an elevation of cellular eicosapentanoic (EPA) and docosahexanoic acids (DHA) at the expense of arachidonic acid (AA). We investigated the effects of alterations in the fatty acid compositions of cardiac sarcoplasmic reticulum (CSR) produced by dietary manipulation on the calcium pump protein that is required for energy dependent calcium transport. CSR was isolated from rats fed menhaden oil, which is rich in n-3 fatty acids, and from control animals that were given corn oil. Relative to control membranes, those isolated from rats fed menhaden oil, had a lower content of saturated phospholipids, an increased DHA/AA ratio, and an increased ratio of n-3 to n-6 fatty acids. These changes were associated with a 30% decrease in oxalate-facilitated, ATP-dependent calcium uptake and concomitant decreased Ca-ATPase activity in the membranes from the animals fed menhaden oil. In contrast, there was no alteration in active pump sites as measured by phosphoenzyme formation. Thus, the CSR Ca-ATPase function can be altered by dietary interventions that change the composition, and possibly structure, of the phospholipid membranes thereby affecting enzyme turnover.

  9. Alteration of Sarcoplasmic Reticulum Ca2+ Release in Skeletal Muscle from Calpain 3-Deficient Mice

    Directory of Open Access Journals (Sweden)

    Govindan Dayanithi

    2009-01-01

    Full Text Available Mutations of Ca2+-activated proteases (calpains cause muscular dystrophies. Nevertheless, the specific role of calpains in Ca2+ signalling during the onset of dystrophies remains unclear. We investigated Ca2+ handling in skeletal cells from calpain 3-deficient mice. [Ca2+]i responses to caffeine, a ryanodine receptor (RyR agonist, were decreased in −/− myotubes and absent in −/− myoblasts. The −/− myotubes displayed smaller amplitudes of the Ca2+ transients induced by cyclopiazonic acid in comparison to wild type cells. Inhibition of L-type Ca2+ channels (LCC suppressed the caffeine-induced [Ca2+]i responses in −/− myotubes. Hence, the absence of calpain 3 modifies the sarcoplasmic reticulum (SR Ca2+ release, by a decrease of the SR content, an impairment of RyR signalling, and an increase of LCC activity. We propose that calpain 3-dependent proteolysis plays a role in activating support proteins of intracellular Ca2+ signalling at a stage of cellular differentiation which is crucial for skeletal muscle regeneration.

  10. High-Throughput Liquid-Liquid Fractionation of Multiple Protein Post-Translational Modifications*

    Science.gov (United States)

    DeFord, James H.; Nuss, Jonathan E.; Amaning, James; English, Robert D.; Tjernlund, Don; Papaconstantinou, John

    2009-01-01

    Post-translational protein modifications have contributed significantly to the identification of macromolecular biomarkers of biological processes. We have modified a 2-dimensional HPLC system (Beckman Coulter PF2D ProteomeLab) to create proteome maps of post-translational protein modifications. This system resolves complex protein mixtures by anion exchange chromatofocusing in the first dimension and hydrophobicity (reverse phase chromatography) in the second dimension. The simultaneous identification of multiple protein modifications, accomplished by incorporating a photo diode array (PDA) detector into the PF2D system, facilitates the simultaneous production of three dimensional proteome maps and visualization of both unmodified and post-translationally modified (PTM) proteins at their signature wavelengths within the proteome. We describe procedures for the simultaneous resolution of proteome maps, the identification of proteins modified by nitration, carbonylation, and phosphorylation, and proteins with unique spectra such as the heme containing proteins. PMID:19099502

  11. Characterization of protein fractions and antioxidant activity of Chia seeds (Salvia hispanica L.)

    OpenAIRE

    Kvetoslava Kačmárová; Blažena Lavová; Peter Socha; Dana Urminská

    2016-01-01

    Chia seed (Salvia hispanica L.) is an annual herbaceous plant categorized under Lamiaceae family. Chia seeds were investigated as a source of proteins and natural antioxidants. It is a potential alternative source of high quality protein, fats, carbohydrates, high dietary fibre, vitamins and mineral elements. The objective of this study was to evaluate chia seed from protein content and antioxidant acivity and highlight the quality of this pseudocereal. A crude protein, moisture...

  12. Analysis of the electromobility of different protein fractions of Bovicola bovis females (Mallophaga: Insecta).

    Science.gov (United States)

    Muñoz Parra, S; Soler Cruz, M D; Benítez Rodríguez, R

    1994-09-01

    The authors characterize in females of Bovicola bovis the total proteins of this species by means of SDS-page determining the R(mb), Rx, Rf and mW of each protein in particular and studying finally the number of protein bands in function with their mW.

  13. 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

  14. 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.

  15. Automated 2D-HPLC method for characterization of protein aggregation with in-line fraction collection device.

    Science.gov (United States)

    Williams, Abasha; Read, Erik K; Agarabi, Cyrus D; Lute, Scott; Brorson, Kurt A

    2017-03-01

    Monoclonal antibodies are mainly produced by mammalian cell culture, which due to its complexity, results in a wide range of product variants/isoforms. With the growing implementation of Quality by Design (QbD) and Process Analytical Technology (PAT) in drug manufacturing, monitoring and controlling quality attributes within a predefined range during manufacturing may provide added consistency to product quality. To implement these concepts, more robust analytical tools could reduce the time needed for monitoring quality attributes during upstream processing. The formation of protein aggregates is one such quality attribute that can lead to safety and efficacy issues in the final drug product. Described in this study is a fully automated two-dimensional high performance liquid chromatography (2D-HPLC) method for characterizing protein aggregation of crude in-process bioreactor samples. It combines protein A purification and separation by size exclusion into a single analytical module that has the potential to be employed at-line within a bioprocessing system. This method utilizes a novel in-line fraction collection device allowing for the collection of up to twelve fractions from a single sample or peak which facilitates the subsequent linked analysis of multiple protein peaks of interest in one chromatography module. Published by Elsevier B.V.

  16. Functional properties of protein from frozen mantle and fin of jumbo squid Dosidicus gigas in function of pH and ionic strength.

    Science.gov (United States)

    Rocha-Estrada, J G; Córdova-Murueta, J H; García-Carreño, F L

    2010-10-01

    Functional properties of protein from mantle and fin of the jumbo squid Dosidicus gigas were explained based on microscopic muscle fiber and protein fractions profiles as observed in SDS-PAGE. Fin has higher content of connective tissue and complex fiber arrangement, and we observed higher hardness of fin gels as expected. Myosin heavy chain (MHC) was found in sarcoplasmic, myofibril and soluble-in-alkali fractions of mantle and only in sarcoplasmic and soluble-in-alkali fractions of fin. An additive effect of salt concentration and pH affected the solubility and foaming properties. Fin and mantle proteins yielded similar results in solubility tests, but significant differences occurred for specific pH and concentrations of salt. Foaming capacity was proportional to solubility; foam stability was also affected by pH and salt concentration. Hardness and fracture strength of fin gels were significantly higher than mantle gels; gels from proteins of both tissues reached the highest level in the folding test. Structural and molecular properties, such as MHC and paramyosin solubility, arrangement of muscle fibers and the content of connective tissue were useful to explain the differences observed in these protein properties. High-strength gels can be formed from squid mantle or fin muscle. Fin displayed similar or better properties than mantle in all tests.

  17. Proteomic characterization of specific minor proteins in the human milk casein fraction.

    Science.gov (United States)

    Liao, Yalin; Alvarado, Rudy; Phinney, Brett; Lönnerdal, Bo

    2011-12-02

    Human milk contains many bioactive proteins that are likely to support the early development of the newborn. The aim of this study was to identify whether there are specific minor proteins associated with the human milk casein micelle prepared by the acid precipitation method. Protein identification was performed by liquid chromatography tandem mass spectrometry analysis. Eighty-two proteins were identified in the casein micelle, 18 of which are not present in their whey compartment. Thirty-two of these proteins specifically associated with the casein micelle have not previously been identified in human milk or colostrum. Proteins involved in immune function comprised the major part (28%) of total proteins, and another significant part is involved in metabolism/energy production (22%). Most of the proteins were of extracellular or cytoplasmic origin (accounting for 50 and 29%, respectively). This study indicates that various soluble proteins should be considered as part of the casein compartment, prepared by the acid precipitation method. The data provide new insight not only into the proteomic profile of the human milk casein micelle and its physiological significance, but also into the proper proportion of casein and casein-associated proteins to use in infant formula.

  18. Thermal and physicochemical properties and nutritional value of the protein fraction of Mexican chia seed (Salvia hispanica L.).

    Science.gov (United States)

    Olivos-Lugo, B L; Valdivia-López, M Á; Tecante, A

    2010-02-01

    Thermal, functional and nutritional properties of the main protein fractions and a protein isolate of chia seed from the state of Jalisco, Mexico, were studied by differential scanning calorimetry, gelling, foaming, water-holding capacity (WHC) and oil-holding capacity, amino acid profile, chemical score and in vitro digestibility tests. The protein isolate showed good WHC (4.06 g/g) and excellent oil-retention capacities (4.04 g/g), making it attractive as an additive in bakery products and food emulsions. It also contained high amounts of glutamic acid (123 g/kg raw protein), arginine (80.6 g/kg raw protein) and aspartic acid (61.3 g/kg raw protein). However, its essential amino acid profile showed deficiencies with respect to the 1985 standard of the FAO/WHO/UNU for pre-school children. Therefore, its use as a sole protein source is not recommended; supplementation with a lysine-rich source would be necessary, as this was the limiting amino acid.

  19. Isolation of rat cardiac sarcoplasmic reticulum with improved Ca2+ uptake and ryanodine binding.

    Science.gov (United States)

    Feher, J J; Davis, M D

    1991-03-01

    The instability of the oxalate-supported Ca2+ uptake activity of rat cardiac sarcoplasmic reticulum (CSR) in ventricular homogenates most likely accounts for the low specific activity of the rate of oxalate-supported Ca2+ uptake in previously reported fractions of isolated rat CSR. We have found that CSR vesicles with improved Ca2+ transport capabilities can be isolated if 1 M KCl is used to stabilize the CSR activity and to allow the extraction of the CSR from the cellular debris. The average rate of Ca2+ uptake by the isolated rat CSR in the presence of 10 mM oxalate at 37 degrees C was 0.45 mumols/min-mg in the absence of CSR Ca2+ channel blockers and 0.87 mumols/min-mg in the presence of 10 microM ruthenium red. The Ca(2+)-dependent ATPase activity under the conditions of oxlate-supported uptake was 1.25 mumols/min-mg and 0.84 mumols/min-mg in the absence and presence of 10 microM ruthenium red, respectively. The rat CSR vesicles bound 3H-ryanodine with a Kd of 1.45 nM and a Bmax of 3.7 pmol mg. The level of phosphorylated intermediate was 0.30 nmol/mg. The values Bmax, EP and Ca(2+)-ATPase activity are from one-third to one-half of those previously reported for isolated canine CSR vesicles. These results suggest that the isolated rat CSR may be quite similar to dog CSR.

  20. Conventional and alternative principles for stabilization of protein and polyphenol fractions in beer

    OpenAIRE

    Marković Romeo S.; Grujić Olgica S.; Pejin Jelena D.

    2003-01-01

    Beer haze is primarily formed through complexation of protein and polyphenolic beer ingredients. The problem of reducing susceptibility of beer haze formation can be done either by lowering protein and/or polyphenol levels, or by minimizing the molecular size of protein/polyphenols. In experimental part of this work the shelf life of unstabilized beer is being compared with beer stabilized with various standard products, such as PVPP and silica gel. Furthermore, the trials have been made to p...

  1. Potential effect of striatin (DLBS0333), a bioactive protein fraction isolated from Channa striata for wound treatment

    Institute of Scientific and Technical Information of China (English)

    Puji Rahayu; Faustine Marcelline; Erna Sulistyaningrum; Maggy Thenawidjaja Suhartono; Raymond Rubianto Tjandrawinata

    2016-01-01

    Objective: To characterize proteins and other nutrients in striatin (DLBS0333), a bioactive protein fraction isolated from snakehead fish (Channa striata) and to investigate its wound healing activity. Methods: Proteins and other constituents in striatin were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two dimension electrophoresis, immunoblotting assay, spectroscopy and high performance liquid chromatography. The wound healing activity of striatin was studied in vitro using 3T3 fibroblast cells and in vivo using wound-induced animal model. Various parameters related to wound healing process were evaluated. Results: Striatin contained four major bioactive proteins with approximate molecular weight of 8.3, 10.9, 15.4 and 16.7 kDa. In addition to proteins, striatin also contained amino acids (10 essential and 7 non essential amino acids), fatty acids (palmitic acid, oleic acid, stearic acid, linoleic acid, arachidonic acid), vitamins (vitamin A, vitamin B6) and other nutrients (carbohydrate, dietary fiber, biotin, choline, inositol, L-carnitine, selenium) which are potential for wound healing and increasing serum albumin level. Treatment with striatin both in vitro and in vivo indicated that striatin enhanced cell proliferation. Wound-induced animal model treated with striatin showed significantly faster wound healing process, as confirmed by wound size and faster serum albumin level recovery. Although striatin does not contain albumin, this bioactive protein fraction may lead to enhanced albumin synthesis in the liver thereby maintaining the blood albumin level. Thus, consuming striatin is a better option to improve albumin levels in diseased condition and the condition of being injured. Conclusions: Striatin (DLBS0333) is a potential natural compound for accelerating wound healing in conditions such as post surgery and post partum, and increasing albumin level.

  2. 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 separ

  3. 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

  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. Effect of the Ethyl Acetate Fraction of Eugenia uniflora on Proteins Global Expression during Morphogenesis in Candida albicans

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    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

  6. Improved recovery of proteome-informative, protein N-terminal peptides by combined fractional diagonal chromatography (COFRADIC).

    Science.gov (United States)

    Staes, An; Van Damme, Petra; Helsens, Kenny; Demol, Hans; Vandekerckhove, Joël; Gevaert, Kris

    2008-04-01

    We previously described a proteome-wide, peptide-centric procedure for sorting protein N-terminal peptides and used these peptides as readouts for protease degradome and xenoproteome studies. This procedure is part of a repertoire of gel-free techniques known as COmbined FRActional DIagonal Chromatography (COFRADIC) and highly enriches for alpha-amino-blocked peptides, including alpha-amino-acetylated protein N-terminal peptides. Here, we introduce two additional steps that significantly increase the fraction of such proteome-informative, N-terminal peptides: strong cation exchange (SCX) segregation of alpha-amino-blocked and alpha-amino-free peptides and an enzymatic step liberating pyroglutamyl peptides for 2,4,6-trinitrobenzenesulphonic acid (TNBS) modification and thus COFRADIC sorting. The SCX step reduces the complexity of the analyte mixture by enriching N-terminal peptides and depleting alpha-amino-free internal peptides as well as proline-starting peptides prior to COFRADIC. The action of pyroglutamyl aminopeptidases prior to the first COFRADIC peptide separation results in greatly diminishing numbers of contaminating pyroglutamyl peptides in peptide maps. We further show that now close to 95% of all COFRADIC-sorted peptides are alpha-amino-acetylated and, using the same amount of starting material, our novel procedure leads to an increased number of protein identifications.

  7. 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.

  8. Differential phosphoprotein mapping in cancer cells using protein microarrays produced from 2-D liquid fractionation.

    Science.gov (United States)

    Pal, Manoj; Moffa, Allison; Sreekumar, Arun; Ethier, Stephen P; Barder, Timothy J; Chinnaiyan, Arul; Lubman, David M

    2006-02-01

    A combination of protein microarrays and two-dimensional liquid-phase separation of proteins has been used for global profiling of the phosphoproteome in human breast cancer cells. This method has been applied to study changes in phosphorylation profile resulting from treatment of the cancer cells with PD173074, a known receptor tyrosine kinase inhibitor. The proteins separated by 2-D liquid-phase separation were arrayed on epoxy-coated glass slides and first screened for phosphorylation using fluorescent Pro-Q Diamond stain. The candidate proteins were then identified using MALDI/ESI MS/MS analysis. Further, validation was achieved by immunoblot analysis using anti-phosphotyrosine antibodies. A dynamic range of approximately 100 was achieved on the microarray when beta-casein was used as a standard protein for obtaining quantitative data. Importantly, the power of this method lies in its ability to identify a large group of proteins in a single experiment that are coregulated in their posttranslational modifications, upon treatment with the inhibitor. Since proteins are known to form interacting circuits that eventually lead to various signaling events, detection of such global phosphorylation profiles might enable delineation of functional pathways that play an important role during cancer initiation and progression.

  9. SynProt: A Comprehensive Database for Proteins of the Detergent-Resistant Synaptic Junctions Fraction

    Directory of Open Access Journals (Sweden)

    Rainer ePielot

    2012-06-01

    Full Text Available Chemical synapses are highly specialized cell-cell contacts for communication between neurons in the CNS characterized by complex and dynamic protein networks at both synaptic membranes. The cytomatrix at the active zone (CAZ organizes the apparatus for the regulated release of transmitters from the presynapse. At the postsynaptic side, the postsynaptic density constitutes the machinery for detection, integration and transduction of the transmitter signal. Both pre- and postsynaptic protein networks represent the molecular substrates for synaptic plasticity. Their function can be altered both by regulating their composition and by post-translational modification of their components. For a comprehensive understanding of synaptic networks the entire ensemble of synaptic proteins has to be considered. To support this, we established a comprehensive database for synaptic junction proteins (SynProt database primarily based on proteomics data obtained from biochemical preparations of detergent-resistant synaptic junctions. The database currently contains 2,788 non-redundant entries of rat, mouse and some human proteins, which mainly have been manually extracted from twelve proteomic studies and annotated for synaptic subcellular localization. Each dataset is completed with manually added information including protein classifiers as well as automatically retrieved and updated information from public databases (UniProt and PubMed. We intend that the database will be used to support modeling of synaptic protein networks and rational experimental design.

  10. Protein Molecular Structures and Protein Fraction Profiles of New Co-Products of BioEthanol Production: A Novel Approach

    Energy Technology Data Exchange (ETDEWEB)

    Yu, P.; Niu, Z; Damiran, D

    2010-01-01

    The objectives of this study were to determine the protein molecular structures of the new coproducts from bioethanol production, quantify protein structure amide I to II and {alpha}-helix to {beta}-sheet spectral peak intensity ratio, and illustrate multivariate molecular spectral analyses as a novel research tool for rapid characterization of protein molecular structures in bioethonal bioproducts. The study demonstrated that the grains had a significantly higher ratio of {alpha}-helix to {beta}-sheet in the protein structure than their coproducts produced from bioethanol processing (1.38 vs 1.03, P < 0.05). There were significant differences between wheat and corn (1.47 vs 1.29, P < 0.05) but no difference between wheat dried distiller grains with solubles (DDGS) and corn DDGS (1.04 vs 1.03, P > 0.05). The grains had a significantly higher ratio of protein amide I to II in the protein structure than their coproducts produced from bioethanol processing (4.58 vs 2.84, P < 0.05). There were no significant differences between wheat and corn (4.61 vs 4.56, P > 0.05), but there were significant differences between wheat DDGS and corn DDGS (3.08 vs 2.21, P < 0.05). This preliminary study indicated that bioethanol processing changes protein molecular structures, compared with original grains. Further study is needed with a large set of the new bioethanol coproducts to quantify protein molecular structures ({alpha}-helix to {beta}-sheet ratio; amide I to II ratio) of the bioethanol coproducts in relation to nutrient supply and availability in animals.

  11. Study of the Changes in Protein Fractions and Amino Acids of an ...

    African Journals Online (AJOL)

    We report results in this respect as observed for South African dried sausage. Results ... Keywords: Protein, unfermented sausage, South Africa, proteolytic enzyme, processing conditions. J Food Tech in Africa (2002) 7, 101-. Article Metrics.

  12. Protein pre-fractionation with a mixed-bed ion exchange column in 3D LC-MS/MS proteome analysis.

    Science.gov (United States)

    Zhang, Luofu; Yao, Ling; Zhang, Yan; Xue, Ting; Dai, Guangchen; Chen, Keying; Hu, Xiaofang; Xu, Lisa X

    2012-09-15

    The fractionation of complex samples at the protein level prior to shotgun proteomics analysis is an efficient means to more comprehensive analysis of samples. A mixed-bed ion-exchange (IEX) column, packed with both weak anion exchange (WAX) and weak cation exchange (WCX) materials, was used for the first dimensional separation of complex samples at the protein level using volatile solvents. The peptides from digestion of each fraction were then identified by 2D SCX-RP-LC-MS/MS. We applied this 3D strategy to mouse mammary tumor 4T1 cell lysate and identified a total of 3084 proteins in a typical experiment. The moderate separation performance of the mixed-bed IEX column facilitated the in-depth identification of the proteins in the complex sample. There were some acceptable inter-fraction overlaps. Nearly half (45.8%) of the proteins were only identified in single fractions, while 82.3% were identified in no more than 3 fractions. The identified proteins covered a broad range of pI, size and grand average hydrophobicity (GRAVY) values. Detailed analysis of proteins identified in each fraction elucidated the separation characteristics of mixed-bed IEX. Retention on mixed-bed IEX was associated, but not restricted to the extreme pI values (pI10) and to the percentage of charged residues of both signs. In conclusion, we have exploited the mixed-bed IEX column to establish an efficient and comprehensive identification method for complex samples.

  13. Inhibition of haemoglobin-mediated lipid oxidation in washed cod muscle and cod protein isolates by Fucus vesiculosus extract and fraction

    DEFF Research Database (Denmark)

    Wang, Tao; Jonsdottir, Rosa; Kristinsson, Hordur

    2010-01-01

    The effects of Fucus vesiculosus extract and fractions towards haemoglobin- (Hb-) catalysed lipid oxidation in washed cod muscle system and cod protein isolates during ice storage were examined. The extract and fractions were characterised in terms of total phlorotannin content (TPC), 2,2-diphenyl...

  14. Inhibition of haemoglobin-mediated lipid oxidation in washed cod muscle and cod protein isolates by Fucus vesiculosus extract and fraction

    DEFF Research Database (Denmark)

    Wang, Tao; Jonsdottir, Rosa; Kristinsson, Hordur

    2010-01-01

    The effects of Fucus vesiculosus extract and fractions towards haemoglobin- (Hb-) catalysed lipid oxidation in washed cod muscle system and cod protein isolates during ice storage were examined. The extract and fractions were characterised in terms of total phlorotannin content (TPC), 2,2-diphenyl...

  15. 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.

  16. Plasma protein fractions in healthy blood donors quantitated by an automated multicapillary electrophoresis system.

    Science.gov (United States)

    Larsson, Anders; Hansson, Lars-Olof

    2006-09-01

    During the last decade, capillary electrophoresis (CE) has emerged as an important alternative to traditional analysis of serum and plasma proteins by agarose or celluloseacetate electrophoresis. CE analysis of plasma proteins can now be fully automated and also includes bar-code identification of samples, preseparation steps, and direct post-separation quantitation of individual peaks, which permits short assay times and high throughput. For laboratory work, it is important to have reference values from healthy individuals. Therefore, plasma samples from 156 healthy blood donors (79 females and 77 males) have been analyzed with the Capillarys instrument and the new high resolution buffer, which yields higher resolution than the beta1-beta2+ buffer. Albumin concentrations in samples are measured using nephelometry in order to assign protein concentrations to each peak. The 2.5 and 97.5 percentiles for both the percentages of different peaks and the protein concentrations in the peaks are calculated according to the recommendations of the International Federation of Clinical Chemistry on the statistical treatment of reference values. The Capillarys instrument is a reliable system for plasma protein analysis, combining advantages of full automation with high analytical performances and throughput.

  17. 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.

  18. 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...

  19. Monitoring the fractionation of a whey protein isolate during dead-end membrane filtration using fluorescence and chemometric methods.

    Science.gov (United States)

    Elshereef, Rand; Budman, Hector; Moresoli, Christine; Legge, Raymond L

    2010-01-01

    During membrane-based separation of proteins, changes in protein concentration of the permeate and retentate streams occurs over time. The current work proposes a new approach for monitoring the changes in concentrations of proteins in both permeate and retentate by making use of data collected using fluorescence spectroscopy and intrinsic protein fluorescence analyzed by multivariate statistical techniques. Whey protein isolate consists mainly of alpha-lactalbumin (alpha-LA), beta-lactoglobulin (beta-LG), and small proportion of bovine serum albumin (BSA) and was used as a model system in this study. A fiber optic probe (FOP) was used to acquire multiwavelength fluorescence spectra for permeate and retentate streams at different times during UF-based separation of the components from a multicomponent solution. Multivariate regression models were developed for predicting the concentrations of alpha-LA, beta-LG, and BSA by establishing a calibration model between data acquired using the FOP and the corresponding protein concentration levels measured by size-exclusion chromatography. The model was validated using FOP data that were not previously used for calibration of the regression models. This comparison showed that concentrations of alpha-LA, beta-LG, and BSA could be predicted directly from FOP data within reasonable accuracy by making use of multivariate calibration tools. This approach has several attractive features including that it is nondestructive, fast, and relatively simple to perform. This technique has potential practical applications as it could offer the opportunity for in situ monitoring of membrane filtration processes by tracking individual protein transmission and selectivity of fractionation.

  20. Isolation of mRNAs encoding peroxisomal proteins from yeast using a combined cell fractionation and affinity purification procedure.

    Science.gov (United States)

    Zipor, Gadi; Brocard, Cecile; Gerst, Jeffrey E

    2011-01-01

    Targeted mRNA localization to distinct subcellular sites occurs throughout the eukaryotes and presumably allows for the localized translation of proteins near their site of function. Specific mRNAs have been localized in cells using a variety of reliable methods, such as fluorescence in situ hybridization with labeled RNA probes, mRNA tagging using RNA aptamers and fluorescent proteins that recognize these aptamers, and quenched fluorescent RNA probes that become activated upon binding to mRNAs. However, fluorescence-based RNA localization studies can be strengthened when coupled with cell fractionation and membrane isolation techniques in order to identify mRNAs associated with specific organelles or other subcellular structures. Here we describe a novel method to isolate mRNAs associated with peroxisomes in the yeast, Saccharomyces cerevisiae. This method employs a combination of density gradient centrifugation and affinity purification to yield a highly enriched peroxisome fraction suitable for RNA isolation and reverse transcription-polymerase chain reaction detection of mRNAs bound to peroxisome membranes. The method is presented for the analysis of peroxisome-associated mRNAs; however it is applicable to studies on other subcellular compartments.

  1. 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 sta

  2. 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...

  3. Molecular spectroscopic investigation on fractionation-induced changes on biomacromolecule of co-products from bioethanol processing to explore protein metabolism in ruminants

    Science.gov (United States)

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

    2014-03-01

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

  4. 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.

  5. Reduced sarcoplasmic reticulum content of releasable Ca2+ in rat soleus muscle fibres after eccentric contractions

    DEFF Research Database (Denmark)

    Nielsen, J S; Sahlin, K; Ørtenblad, N

    2007-01-01

    AIM: The purpose was to evaluate the effects of fatiguing eccentric contractions (EC) on calcium (Ca2+) handling properties in mammalian type I muscles. We hypothesized that EC reduces both endogenous sarcoplasmic reticulum (SR) content of releasable Ca2+ (eSRCa2+) and myofibrillar Ca2+ sensitivity...

  6. Sub-sarcolemmal swelling of sarcoplasmic reticulum after isometric contractions in rat semimembranosus lateralis muscle

    NARCIS (Netherlands)

    Willems, M.E.T.; Huijing, P.A.J.B.M.; Friden, J.

    1999-01-01

    The decline in isometric force, swelling of sarcoplasmic reticulum and loss of desmin was measured in semimembranosus lateralis muscle of male Wistar rats immediately after a short series of brief (500 ms) maximal isometric contractions. For the active muscle, the series ended below (protocol A) and

  7. Effects of boldine on mouse diaphragm and sarcoplasmic reticulum vesicles isolated from skeletal muscle.

    Science.gov (United States)

    Kang, J J; Cheng, Y W

    1998-02-01

    The effects of boldine [(S)-2,9-dihydroxy-1,10-dimethoxyaporphine], a major alkaloid in the leaves and bark of boldo (Peumus boldus Mol.), on skeletal muscle were studied using mouse diaphragm and isolated sarcoplasmic reticulum membrane vesicles. Boldine, at 10-200 microM, has little effect on the muscle-evoked twitches; however, the ryanodine-induced contracture was potentiated dose-dependently. At higher concentrations of 300 microM, boldine by itself induced muscle contracture of two phases, which were caused by the influx of extracellular Ca2+ and induction of Ca2+ release from the internal Ca2+ storage site, the sarcoplasmic reticulum, respectively. When tested with isolated sarcoplasmic reticulum membrane vesicles, boldine dose-dependently induced Ca2+ release from actively loaded sarcoplasmic reticulum vesicles isolated from skeletal muscle of rabbit or rat which was inhibited by ruthenium red, suggesting that the release was through the Ca2+ release channel, also known as the ryanodine receptor. Boldine also dose-dependently increased apparent [3H]-ryanodine binding with the EC50 value of 50 microM. In conclusion, we have shown that boldine could sensitize the ryanodine receptor and induce Ca2+ release from the internal Ca2+ storage site of skeletal muscle.

  8. Study of the protein-bound fraction of calcium, iron, magnesium and zinc in bovine milk

    Science.gov (United States)

    Silva, Fernando V.; Lopes, Gisele S.; Nóbrega, Joaquim A.; Souza, Gilberto B.; Nogueira, Ana Rita A.

    2001-10-01

    Two approaches were used to study the interaction of Ca, Fe, Mg and Zn with bovine milk proteins by inductively coupled plasma optical emission spectrometry (ICPOES). Selective separations in bovine milk samples were accomplished employing an acid protein precipitation using 100 g l -1 trichloroacetic acid (TCA), and an enzymatic protein hydrolysis using 50 g l -1 pepsin (PEP) solution, respectively. The results were compared with total mineral contents determined after microwave-assisted acid digestion. The results obtained by enzymatic and acid precipitation evidenced the different interaction forms of Ca, Fe, Mg and Zn in the system formed by milk components. Iron was not solubilized by the TCA treatment, but was recovered completely after the enzymatic treatment. Quantitative recoveries of Ca, Mg and Zn were obtained using both approaches, showing that these analytes were bound to milk compounds affected by either treatment. Calcium, Mg and Zn are mainly associated with colloidal calcium phosphate and Fe is bound to the backbone of the casein polypeptide chain, cleaved by pepsin enzyme. The proposed approaches could be used to assess the complexity of these chemical interactions.

  9. Isotopic fractionation in proteins as a measure of hydrogen bond length

    CERN Document Server

    McKenzie, Ross H; Ramesh, Sai

    2015-01-01

    If a deuterated molecule containing strong intramolecular hydrogen bonds is placed in a hydrogenated solvent it may preferentially exchange deuterium for hydrogen. This preference is due to the difference between the vibrational zero-point energy for hydrogen and deuterium. It is found that the associated fractionation factor $\\Phi$ is correlated with the strength of the intramolecular hydrogen bonds. This correlation has been used to determine the length of the H-bonds (donor-acceptor separation) in a diverse range of enzymes and has been argued to support the existence of short low-barrier H-bonds. Starting with a potential energy surface based on a simple diabatic state model for H-bonds we calculate $\\Phi$ as a function of the proton donor-acceptor distance $R$. For numerical results, we use a parameterization of the model for symmetric O-H.... O bonds. We consider the relative contributions of the O-H stretch vibration, O-H bend vibrations (both in plane and out of plane), tunnelling splitting effects at...

  10. Isotopic fractionation in proteins as a measure of hydrogen bond length

    Energy Technology Data Exchange (ETDEWEB)

    McKenzie, Ross H., E-mail: r.mckenzie@uq.edu.au [School of Mathematics and Physics, University of Queensland, Brisbane 4072 (Australia); Athokpam, Bijyalaxmi; Ramesh, Sai G. [Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore 560 012 (India)

    2015-07-28

    If a deuterated molecule containing strong intramolecular hydrogen bonds is placed in a hydrogenated solvent, it may preferentially exchange deuterium for hydrogen. This preference is due to the difference between the vibrational zero-point energy for hydrogen and deuterium. It is found that the associated fractionation factor Φ is correlated with the strength of the intramolecular hydrogen bonds. This correlation has been used to determine the length of the H-bonds (donor-acceptor separation) in a diverse range of enzymes and has been argued to support the existence of short low-barrier H-bonds. Starting with a potential energy surface based on a simple diabatic state model for H-bonds, we calculate Φ as a function of the proton donor-acceptor distance R. For numerical results, we use a parameterization of the model for symmetric O–H⋯O bonds [R. H. McKenzie, Chem. Phys. Lett. 535, 196 (2012)]. We consider the relative contributions of the O–H stretch vibration, O–H bend vibrations (both in plane and out of plane), tunneling splitting effects at finite temperature, and the secondary geometric isotope effect. We compare our total Φ as a function of R with NMR experimental results for enzymes, and in particular with an earlier model parametrization Φ(R), used previously to determine bond lengths.

  11. Angiotensin I-Converting Enzyme (ACE Inhibitory Activity and ACE Inhibitory Peptides of Salmon (Salmo salar Protein Hydrolysates Obtained by Human and Porcine Gastrointestinal Enzymes

    Directory of Open Access Journals (Sweden)

    Małgorzata Darewicz

    2014-08-01

    Full Text Available The objectives of the present study were two-fold: first, to detect whether salmon protein fractions possess angiotensin I-converting enzyme (ACE inhibitory properties and whether salmon proteins can release ACE inhibitory peptides during a sequential in vitro hydrolysis (with commercial porcine enzymes and ex vivo digestion (with human gastrointestinal enzymes. Secondly, to evaluate the ACE inhibitory activity of generated hydrolysates. A two-step ex vivo and in vitro model digestion was performed to simulate the human digestion process. Salmon proteins were degraded more efficiently by porcine enzymes than by human gastrointestinal juices and sarcoplasmic proteins were digested/hydrolyzed more easily than myofibrillar proteins. The ex vivo digested myofibrillar and sarcoplasmic duodenal samples showed IC50 values (concentration required to decrease the ACE activity by 50% of 1.06 and 2.16 mg/mL, respectively. The in vitro hydrolyzed myofibrillar and sarcoplasmic samples showed IC50 values of 0.91 and 1.04 mg/mL, respectively. Based on the results of in silico studies, it was possible to identify 9 peptides of the ex vivo hydrolysates and 7 peptides of the in vitro hydrolysates of salmon proteins of 11 selected peptides. In both types of salmon hydrolysates, ACE-inhibitory peptides IW, IY, TVY and VW were identified. In the in vitro salmon protein hydrolysates an ACE-inhibitory peptides VPW and VY were also detected, while ACE-inhibitory peptides ALPHA, IVY and IWHHT were identified in the hydrolysates generated with ex vivo digestion. In our studies, we documented ACE inhibitory in vitro effects of salmon protein hydrolysates obtained by human and as well as porcine gastrointestinal enzymes.

  12. Functional properties of human embryonic stem cell-derived cardiomyocytes: intracellular Ca2+ handling and the role of sarcoplasmic reticulum in the contraction.

    Science.gov (United States)

    Dolnikov, Katya; Shilkrut, Mark; Zeevi-Levin, Naama; Gerecht-Nir, Sharon; Amit, Michal; Danon, Asaf; Itskovitz-Eldor, Joseph; Binah, Ofer

    2006-02-01

    Since cardiac transplantation is limited by the small availability of donor organs, regeneration of the diseased myocardium by cell transplantation is an attractive therapeutic modality. To determine the compatibility of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) (7 to 55 days old) with the myocardium, we investigated their functional properties regarding intracellular Ca2+ handling and the role of the sarcoplasmic reticulum in the contraction. The functional properties of hESC-CMs were investigated by recording simultaneously [Ca2+]i transients and contractions. Additionally, we performed Western blot analysis of the Ca2+-handling proteins SERCA2, calsequestrin, phospholamban, and Na+/Ca2+ exchanger (NCX). Our major findings are, first, that hESC-CMs displayed temporally related [Ca2+]i transients and contractions, negative force-frequency relations, and lack of post-rest potentiation. Second, ryanodine, thapsigargin, and caffeine did not affect the [Ca2+]i transient and contraction, indicating that at this developmental stage, contraction depends on transsarcolemmal Ca2+ influx rather than on sarcoplasmic reticulum Ca2+ release. Third, in agreement with the notion that a voltage-dependent Ca2+ current is present in hESC-CMs and contributes to the mechanical function, verapamil completely blocked contraction. Fourth, whereas hESC-CMs expressed SERCA2 and NCX at levels comparable to those of the adult porcine myocardium, calsequestrin and phospholamban were not expressed. Our study shows for the first time that functional properties related to intracellular Ca2+ handling of hESC-CMs differ markedly from the adult myocardium, probably due to immature sarcoplasmic reticulum capacity.

  13. Diagnosis of Fasciola infection by SDS–PAGE eluted excretory secretory (ES protein fractions using dot-ELISA

    Directory of Open Access Journals (Sweden)

    M.A. Sabry

    2014-12-01

    Full Text Available Fascioliasis is now recognized as an emerging zoonotic disease in Egypt. Diagnosis in suspected patients still needs some degree of accuracy. In the present study, three Fasciola gigantica execratory secretory (ES protein bands of molecular weight (MW ranging from 14 to 20 kDa, 25 to 32 kDa and 45 to 65 kDa were eluted after fractionation of the parasite antigen using SDS–PAGE. The extracted kDa protein bands were concentrated and evaluated in diagnosis of Fasciola infection. Moreover the level of their cross reaction with other parasitic infections in infected and suspected patients of known parasite eggs/gram stool was evaluated using the dot-ELISA technique. Protein bands in the range of 14–20 kDa and that of 25–32 kDa were markedly specific and sensitive in diagnosis of different levels of anti-Fasciola antibodies (Ab in sera of infected cases. These two groups of bands were able to exclude cross-reaction between anti-Fasciola Ab and other parasites recorded in stool of selected patients suffering from Schistosoma mansoni, Ascaris, and Giardia, either in single or mixed conditions with Fasciola eggs. While that of 45–65 kDa appeared less specific than the other previously mentioned bands. Protein bands in the range of 25–32 kDa appeared more sensitive than the other protein bands in detection of anti-Fasciola Ab at higher serum dilutions. The Dot-ELISA technique was proved to be more economic and easy in application. The dotted very small amount of antigens can be stored in a freezer and used at request in diagnosis of large numbers of samples.

  14. Insulin fails to enhance mTOR phosphorylation, mitochondrial protein synthesis, and ATP production in human skeletal muscle without amino acid replacement.

    Science.gov (United States)

    Barazzoni, Rocco; Short, Kevin R; Asmann, Yan; Coenen-Schimke, Jill M; Robinson, Matthew M; Nair, K Sreekumaran

    2012-11-01

    Systemic insulin administration causes hypoaminoacidemia by inhibiting protein degradation, which may in turn inhibit muscle protein synthesis (PS). Insulin enhances muscle mitochondrial PS and ATP production when hypoaminoacidemia is prevented by exogenous amino acid (AA) replacement. We determined whether insulin would stimulate mitochondrial PS and ATP production in the absence of AA replacement. Using l-[1,2-¹³C]leucine as a tracer, we measured the fractional synthetic rate of mitochondrial as well as sarcoplasmic and mixed muscle proteins in 18 participants during sustained (7-h) insulin or saline infusion (n = 9 each). We also measured muscle ATP production, mitochondrial enzyme activities, mRNA levels of mitochondrial genes, and phosphorylation of signaling proteins regulating protein synthesis. The concentration of circulating essential AA decreased during insulin infusion. Mitochondrial, sarcoplasmic, and mixed muscle PS rates were also lower during insulin (2-7 h) than during saline infusions despite increased mRNA levels of selected mitochondrial genes. Under these conditions, insulin did not alter mitochondrial enzyme activities and ATP production. These effects were associated with enhanced phosphorylation of Akt but not of protein synthesis activators mTOR, p70(S6K), and 4EBP1. In conclusion, sustained physiological hyperinsulinemia without AA replacement did not stimulate PS of mixed muscle or protein subfractions and did not alter muscle mitochondrial ATP production in healthy humans. These results support that insulin and AA act in conjunction to stimulate muscle mitochondrial function and mitochondrial protein synthesis.

  15. Effects of motor patterns on water-soluble and membrane proteins and cholinesterase activity in subcellular fractions of rat brain tissue

    Science.gov (United States)

    Pevzner, L. Z.; Venkov, L.; Cheresharov, L.

    1980-01-01

    Albino rats were kept for a year under conditions of daily motor load or constant hypokinesia. An increase in motor activity results in a rise in the acetylcholinesterase activity determined in the synaptosomal and purified mitochondrial fractions while hypokinesia induces a pronounced decrease in this enzyme activity. The butyrylcholinesterase activity somewhat decreases in the synaptosomal fraction after hypokinesia but does not change under the motor load pattern. Motor load causes an increase in the amount of synaptosomal water-soluble proteins possessing an intermediate electrophoretic mobility and seem to correspond to the brain-specific protein 14-3-2. In the synaptosomal fraction the amount of membrane proteins with a low electrophoretic mobility and with the cholinesterase activity rises. Hypokinesia, on the contrary, decreases the amount of these membrane proteins.

  16. [Protective effect of serum containing n-butanol fraction from Gynostemma pentaphyllum on NG108-15 cell apoptosis induced by Aβ25.35 protein].

    Science.gov (United States)

    Wu, Yan-chun; Zhong, Zhen-guo; Liu, Shu-ling; Wang, Chun-ling; Zhang, Wen-yan; Cui, Wen

    2014-06-01

    To investigate the inhibition effect of serum containing n-butanol fractions from Gynostemma pentaphyllum on NG108-15 cell apoptosis induced by Aβ25-35 protein in vitro. The apoptosis of NG108-15 cells induced by Aβ25-35 protein in vitro was evaluated by immunofluorescence and flow cytometry assay. The cellular Caspase-3 level during the apoptosis was determined by ELISA. The serum containing n-butanol fractions from Gynostemma pentaphyllum significantly inhibited the NG108-15 cells apoptosis induced by Aβ25-35 protein in vitro,and decreased the cellular Caspase-3 level. The inhibition effect of n-butanol fractions from Gynostemma pentaphyllum on NG108-15 cell apoptosis induced by Aβ25.35 protein is likely related to its potency on reducing of cellular Caspase-3 level.

  17. The changes of proteins fractions shares in milk and fermented milk drinks

    Directory of Open Access Journals (Sweden)

    Genowefa Bonczar

    2016-12-01

    Full Text Available Background. The aim of this research was to observe the changes which take place in the electrophoretic picture of milk proteins after pasteurisation and inoculation with different starter cultures (both traditional and probiotic. After incubation, the yoghurt, kefir, acidified milk, fermented Bifidobacterium bifidum drink and Lactobacillus acidophillus drink were chilled for 14 days to observe the changes which occurred. Materials and methods. The research materials were raw and pasteurised milk, as well as fermented milk- based drinks. The raw milk used for research came from Polish Holstein-Fresian black and white cows. The milk was sampled 3 times and divided into 5 parts, each of which was pasteurised at 95°C for 10 min and then cooled for inoculation: yoghurt to 45°C, kefir and acidified milk to 22°C and drinks with Bifidobacterium bifidum and Lactobacillus acidophillus to 38°C. Milk was inoculated with lyophilised, direct vat starter cul- tures, in an amount equal to 2% of the working starter. For the production of fermented drinks, the subsequent starters were applied: “YC-180” Christian Hansen for yoghurt, “D” Biolacta-Texel-Rhodia for kefir, CH-N--11 Christian Hansen for acidified milk, starter by Christian Hansen for the probiotic Bifidobacterium bifidum milk, starter by Biolacta-Texel-Rhodia for the probiotic Lactobacillus acidophillus milk. The analyses were conducted in raw, pasteurised and freshly fermented milk as well as in milk drinks stored for 14 days. The total solid content was estimated by the drying method; the fat content by the Gerber method; the lactose content by the Bertrand method; the protein content by the Kjeldahl method with Buchi apparatus; the density of milk was measured with lactodensimeter; acidity with a pH-meter; and potential acidity by Soxhlet-Henkl method (AOAC, 1990. The electrophoretic separation of proteins in raw and pasteurised milk, as well as in freshly produced milk drinks

  18. Evaluation of some residual bioactivities of microencapsulated Phaseolus lunatus protein fraction with carboxymethylated flamboyant (Delonix regia gum/sodium alginate

    Directory of Open Access Journals (Sweden)

    Mukthar Sandovai-Peraza

    2014-12-01

    Full Text Available Recent studies have shown the beneficial effect of peptides, an unexploited source could be Phaseolus lunatus being an important raw material for those functional products in order to improve their utilization. In addition to improve the beneficial effect of bioactive peptides the microencapsulation could be a way to protect the peptides against the environment to which they are exposed. P. lunatus protein fraction (<10 kDa of weight was encapsulated using a blend of carboxymethylated flamboyant gum (CFG and sodium alginate (SA at different concentrations of CaCl2 and hardening times. After in vitro digestion of microcapsules the residual activity, in the intestinal system, both inhibition of agiotensin-converting enzyme (I-ACE and antioxidant activity obtained were in a range of 0.019-0.136 mg/mL and 570.64-813.54 mM of TEAC respectively. The microencapsulation employed CFG/SA blends could be used controlled delivery of peptide fractions with potential use as a nutraceutical or therapeutic agents.

  19. Process steps for the preparation of purified fractions of alpha-lactalbumin and beta-lactoglobulin from whey protein concentrates.

    Science.gov (United States)

    Gésan-Guiziou, G; Daufin, G; Timmer, M; Allersma, D; van der Horst, C

    1999-05-01

    Fractions enriched with alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg) were produced by a process comprising the following successive steps: clarification-defatting of whey protein concentrate, precipitation of alpha-lactalbumin, separation of soluble beta-lactoglobulin, washing the precipitate, solubilization of the precipitate, concentration and purification of alpha-la. The present study evaluated the performance of the process, firstly on a laboratory scale with acid whey and then on a pilot scale with Gouda cheese whey. In both cases soluble beta-lg was separated from the precipitate using diafiltration or microfiltration and the purities of alpha-la and beta-lg were in the range 52-83 and 85-94% respectively. The purity of the beta-lg fraction was higher using acid whey, which does not contain caseinomacropeptide, than using sweet whey. With the pilot scale plant, the recoveries (6% for alpha-la; 51% for beta-lg) were disappointing, but ways of improving each step in the process are discussed.

  20. The effect of particle size of wheat bran fractions on bread quality - Evidence for fibre-protein interactions

    NARCIS (Netherlands)

    Noort, M.W.J.; Haaster, D. van; Hemery, Y.; Schols, H.A.; Hamer, R.J.

    2010-01-01

    The nature of the adverse effects of wheat bran fractions on bread-making quality was studied. Two fractions of bran, representing different tissue layers and having different compositions, were used. The particle size of the bran fractions was varied by various milling techniques. All fractions wer

  1. Protective and inhibitory effects of various types of amphipols on the Ca2+-ATPase from sarcoplasmic reticulum: a comparative study.

    Science.gov (United States)

    Picard, Martin; Dahmane, Tassadite; Garrigos, Manuel; Gauron, Carole; Giusti, Fabrice; le Maire, Marc; Popot, Jean-Luc; Champeil, Philippe

    2006-02-14

    Amphipols are amphipathic polymers designed to replace or supplement detergents in membrane protein solution studies. Previous work has suggested both advantages and disadvantages to the use of a polyacrylate-based amphipol, A8-35, for studying the sarcoplasmic reticulum Ca2+-ATPase (SERCA1a). We investigated this issue further using a set of four amphipols with different chemical structures. Previous size exclusion chromatography experiments had shown that A8-35 and SERCA1a/A8-35 complexes aggregate under certain conditions. We show here that aggregation can be prevented by omitting calcium from buffers or by using a sulfonated version of A8-35. A8-35 had previously been shown to protect Ca2+-ATPase from irreversible denaturation, while inhibiting its activity in a reversible manner. We show here that the other three amphipols tested also display these properties and that all four amphipols slow down backward calcium dissociation from the nonphosphorylated solubilized enzyme, a priori an unrelated step. As this calcium dissociation involves the opening up of the bundle of transmembrane ATPase segments, the slowing of this process may indicate that multipoint attachment of the polymers to the hydrophobic transmembrane surface damps protein dynamics ("Gulliver" effect). Damping might be the reason why amphipols also simultaneously protect membrane proteins against irreversible denaturation and may inhibit the activity of those of them that display large rearrangements of their transmembrane surface during their catalytic cycle.

  2. Short and long range functions of amino acids in the transmembrane region of the sarcoplasmic reticulum ATPase. A mutational study.

    Science.gov (United States)

    Chen, L; Sumbilla, C; Lewis, D; Zhong, L; Strock, C; Kirtley, M E; Inesi, G

    1996-05-01

    Mutational analysis of several amino acids in the transmembrane region of the sarcoplasmic reticulum ATPase was performed by expressing wild type ATPase and 32 site-directed mutants in COS-1 cells followed by functional characterization of the microsomal fraction. Four different phenotype characteristics were observed in the mutants: (a) functions similar to those sustained by the wild type ATPase; (b) Ca2+ transport inhibited to a greater extent than ATPase hydrolytic activity; (c) inhibition of transport and hydrolytic activity in the presence of high levels of phosphorylated enzyme intermediate; and (d) total inhibition of ATP utilization by the enzyme while retaining the ability to form phosphoenzyme by utilization of P(i). Analysis of experimental observations and molecular models revealed short and long range functions of several amino acids within the transmembrane region. Short range functions include: (a) direct involvement of five amino acids in Ca2+ binding within a channel formed by clustered transmembrane helices M4, M5, M6, and M8; (b) roles of several amino acids in structural stabilization of the helical cluster for optimal channel function; and (c) a specific role of Lys297 in sealing the distal end of the channel, suggesting that the M4 helix rotates to allow vectorial flux of Ca2+ upon enzyme phosphorylation. Long range functions are related to the influence of several transmembrane amino acids on phosphorylation reactions with ATP or P(i), transmitted to the extramembranous region of the ATPase in the presence or in the absence of Ca2+.

  3. Interactions of vanadium(V)-citrate complexes with the sarcoplasmic reticulum calcium pump.

    Science.gov (United States)

    Aureliano, Manuel; Tiago, Teresa; Gândara, Ricardo M C; Sousa, Andrea; Moderno, A; Kaliva, M; Salifoglou, A; Duarte, Rui O; Moura, José J G

    2005-12-01

    Among the biotargets interacting with vanadium is the calcium pump from the sarcoplasmic reticulum (SR). To this end, initial research efforts were launched with two vanadium(V)-citrate complexes, namely (NH(4))(6)[V(2)O(4)(C(6)H(4)O(7))(2)].6H(2)O and (NH(4))(6)[V(2)O(2)(O(2))(2)(C(6)H(4)O(7))(2)].4H(2)O, potentially capable of interacting with the SR calcium pump by combining kinetic studies with (51)V NMR spectroscopy. Upon dissolution in the reaction medium (concentration range: 4-0.5mM), both vanadium(V):citrate (VC) and peroxovanadium(V):citrate (PVC) complexes are partially converted into vanadate oligomers. A 1mM solution of the PVC complex, containing 184microM of the PVC complex, 94microM oxoperoxovanadium(V) (PV) species, 222microM monomeric (V1), 43microM dimeric (V2) and 53microM tetrameric (V4) species, inhibits Ca(2+) accumulation by 75 %, whereas a solution of the VC complex of the same vanadium concentration, containing 98microM of the VC complex, 263microM monomeric (V1), 64microM dimeric (V2) and 92microM tetrameric (V4) species inhibits the calcium pump activity by 33 %. In contrast, a 1 mM metavanadate solution, containing 460microM monomeric (V1), 90.2microM dimeric (V2) and 80microM tetrameric (V4) species, has no effect on Ca(2+) accumulation. The NMR signals from the VC complex (-548.0ppm), PVC complex (-551.5ppm) and PV (-611.1ppm) are broadened upon SR vesicle addition (2.5mg/ml total protein). The relative order for the half width line broadening of the NMR signals, which reflect the interaction with the protein, was found to be V4>PVC>VC>PV>V2=V1=1, with no effect observed for the V1 and V2 signals. Putting it all together the effects of two vanadium(V)-citrate complexes on the modulation of calcium accumulation and ATP hydrolysis by the SR calcium pump reflected the observed variable reactivity into the nature of key species forming upon dissolution of the title complexes in the reaction media.

  4. Carboxypeptidase I from triticale grains and the hydrolysis of salt-soluble fractions of storage proteins.

    Science.gov (United States)

    Drzymała, Adam; Prabucka, Beata; Bielawski, Wiesław

    2012-09-01

    Carboxypeptidase I was purified from triticale grains (×Triticosecale Wittm.) by a 5-step purification procedure including gel filtration, cation-exchange chromatography and affinity chromatography. The enzyme was purified 595.9 fold with a 1.58% recovery. Triticale carboxypeptidase I is a homodimer with a molecular weight of 124.2 kDa and a subunit weight of 55.2 kDa. Each subunit is composed of two polypeptide chains (33.4 and 21.3 kDa). Serine was found in the active site of triticale carboxypeptidase I; DFP (diisopropylflourophosphate) and other applied inhibitors of serine proteases inhibited the enzyme activity. Triticale carboxypeptidase I hydrolyzes N-CBZ-dipeptide (N-carbobenzoxy-dipeptide) substrates at low pH. N-CBZ-Phe-Ala, N-CBZ-Phe-Leu and N-CBZ-Ala-Met were hydrolyzed with the highest rates. The lowest K(m) value and the highest k(cat)/K(m) ratio were observed for hydrolysis of N-CBZ-Phe-Ala. Studies on the amino acid sequence revealed that the purified enzyme is homologous to carboxypeptidase I from barley. Analyses of conserved regions in the sequence of triticale carboxypeptidase I revealed the presence of Ser, Asp and His that compose the catalytic triad. Intact storage proteins were poor substrates for carboxypeptidases. Carboxypeptidase I together with carboxypeptidase III effectively degraded albumins proteolytically modified by endopeptidase EP8. Modified globulins were degraded at a slower rate, and all three carboxypeptidases were required for a significantly increased activity. Studies of the expression of the carboxypeptidase I gene revealed that the synthesis of the enzyme occurs mainly in the scutellum of the grain. The enzyme is also expressed in the aleurone layer of the grains, although its function in this tissue is unknown.

  5. Effect of membrane length, membrane resistance, and filtration conditions on the fractionation of milk proteins by microfiltration.

    Science.gov (United States)

    Piry, A; Heino, A; Kühnl, W; Grein, T; Ripperger, S; Kulozik, U

    2012-04-01

    We investigated the fractionation of casein micelles and the whey protein β-lactoglobulin (β-LG) of skim milk by crossflow microfiltration (0.1 μm) for the first time by a novel approach as a function of membrane length and membrane resistance. A special module was constructed with 4 sections and used to assess the effects of membrane length by measuring flux and β-LG permeation (or transmission) as a function of transmembrane pressure and membrane length. Depending on the position, the membranes were partly controlled by a deposit layer. A maximum for β-LG mass flow through the various membrane sections was found, depending on the position along the membrane. To study the effect of convective flow toward the membrane, membranes with 4 different intrinsic permeation resistances were assessed in terms of the permeation and fouling effects along the flow channel. From these findings, we derived a ratio between transmembrane pressure and membrane resistance, which was useful in reducing the effect of deposit formation and, thus, to optimize the protein permeation. In addition, the fouling effect was investigated in terms of reversible and irreversible fouling and, in addition, by differentiation between pressure-induced fouling and adsorption-induced (pressure-independent) fouling, again as a function of membrane length.

  6. Purif ied Protein Fraction of Garlic Extract Modulates Cellular Immune Response against Breast Transplanted Tumors in BALB/c Mice Model

    Directory of Open Access Journals (Sweden)

    Narges Zare Mehrjardi

    2013-01-01

    Full Text Available Objective: Garlic (Allium sativum has anti-inflammatory, anti-mutagenesis, and immunomodulatory properties that modulate anti-tumor immunity and inhibit tumor growth. In this study we have examined the effect of a protein fraction isolated from fresh garlic on anti-tumor response and intra-tumor lymphocyte infiltration.Materials and Methods: In this experimental study a protein fraction was purified from fresh garlic bulbs using ultra-filtration, followed by chromatofocusing, and SDS-PAGE analysis. Anti-tumor activity was assessed by intra-tumor injection of the protein fraction and garlic extract, itself, into groups of 5 mice each. The percentage of peripheral blood and intra-tumor CD4+ and CD8+ cells were assessed by flow cytometry. Unpaired student’s t test using the SPSS program was applied for all statistical analyses.Results: Garlic extract included different type of proteins with different molecular weight. One of protein’s fraction was immunomodeulator and was composed of three single polypeptides, with molecular masses of ~10-13 kDa and different isoelectric points (pI. These molecules augmented the delayed type hypersensitivity (DTH response compared to the control group. Intra-tumor injection of the fraction provoked a significant increase in the CD8+ subpopulation of T-lymphocytes, as well as a decrease in tumor size. The fraction increased peripheral blood CD8+ T-lymphocytes in treated animals.Conclusion: The data confirms that protein fractions purified from fresh garlic bulbs augment CD8+ T-cell infiltration into the tumor site, inhibiting tumor growth more efficiently than garlic extract. These fi ndings provide a basis for further investigations on the purified polypeptide as a useful candidate for immunomodulation and tumor treatment.

  7. Purif ied Protein Fraction of Garlic Extract Modulates Cellular Immune Response against Breast Transplanted Tumors in BALB/c Mice Model

    Science.gov (United States)

    Ebrahimi, Marzieh; Mohammad Hassan, Zuhair; Mostafaie, Ali; Zare Mehrjardi, Narges; Ghazanfari, Tooba

    2013-01-01

    Objective: Garlic (Allium sativum) has anti-inflammatory, anti-mutagenesis, and immunomodulatory properties that modulate anti-tumor immunity and inhibit tumor growth. In this study we have examined the effect of a protein fraction isolated from fresh garlic on anti-tumor response and intra-tumor lymphocyte infiltration. Materials and Methods: In this experimental study a protein fraction was purified from fresh garlic bulbs using ultra-filtration, followed by chromatofocusing, and SDS-PAGE analysis. Anti-tumor activity was assessed by intra-tumor injection of the protein fraction and garlic extract, itself, into groups of 5 mice each. The percentage of peripheral blood and intra-tumor CD4+ and CD8+ cells were assessed by flow cytometry. Unpaired student’s t test using the SPSS program was applied for all statistical analyses. Results: Garlic extract included different type of proteins with different molecular weight. One of protein’s fraction was immunomodeulator and was composed of three single polypeptides, with molecular masses of ~10-13 kDa and different isoelectric points (pI). These molecules augmented the delayed type hypersensitivity (DTH) response compared to the control group. Intra-tumor injection of the fraction provoked a significant increase in the CD8+ subpopulation of T-lymphocytes, as well as a decrease in tumor size. The fraction increased peripheral blood CD8+ T-lymphocytes in treated animals. Conclusion: The data confirms that protein fractions purified from fresh garlic bulbs augment CD8+ T-cell infiltration into the tumor site, inhibiting tumor growth more efficiently than garlic extract. These findings provide a basis for further investigations on the purified polypeptide as a useful candidate for immunomodulation and tumor treatment. PMID:23700562

  8. Optimization of iTRAQ labelling coupled to OFFGEL fractionation as a proteomic workflow to the analysis of microsomal proteins of Medicago truncatula roots

    Directory of Open Access Journals (Sweden)

    Abdallah Cosette

    2012-06-01

    Full Text Available Abstract Background Shotgun proteomics represents an attractive technical framework for the study of membrane proteins that are generally difficult to resolve using two-dimensional gel electrophoresis. The use of iTRAQ, a set of amine-specific isobaric tags, is currently the labelling method of choice allowing multiplexing of up to eight samples and the relative quantification of multiple peptides for each protein. Recently the hyphenation of different separation techniques with mass spectrometry was used in the analysis of iTRAQ labelled samples. OFFGEL electrophoresis has proved its effectiveness in isoelectric point-based peptide and protein separation in solution. Here we describe the first application of iTRAQ-OFFGEL-LC-MS/MS on microsomal proteins from plant material. The investigation of the iTRAQ labelling effect on peptide electrofocusing in OFFGEL fractionator was carried out on Medicago truncatula membrane protein digests. Results In-filter protein digestion, with easy recovery of a peptide fraction compatible with iTRAQ labelling, was successfully used in this study. The focusing quality in OFFGEL electrophoresis was maintained for iTRAQ labelled peptides with a higher than expected number of identified peptides in basic OFFGEL-fractions. We furthermore observed, by comparing the isoelectric point (pI fractionation of unlabelled versus labelled samples, a non-negligible pI shifts mainly to higher values. Conclusions The present work describes a feasible and novel protocol for in-solution protein digestion in which the filter unit permits protein retention and buffer removal. The data demonstrates an impact of iTRAQ labelling on peptide electrofocusing behaviour in OFFGEL fractionation compared to their native counterpart by the induction of a substantial, generally basic pI shift. Explanations for the occasionally observed acidic shifts are likewise presented.

  9. A method for protein extraction from different subcellular fractions of laticifer latex in Hevea brasiliensis compatible with 2-DE and MS

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    Guo Anping

    2010-06-01

    Full Text Available Abstract Background Proteomic analysis of laticifer latex in Hevea brasiliensis has been received more significant attentions. However, the sticky and viscous characteristic of rubber latex as cytoplasm of laticifer cells and the complication of laticifer latex membrane systems has made it challenge to isolate high-quality proteins for 2-DE and MS. Results Based on the reported Borax/PVPP/Phenol (BPP protocol, we developed an efficient method for protein preparation from different latex subcellular fractions and constructed high-resolution reference 2-DE maps. The obtained proteins from both total latex and C-serum fraction with this protocol generate more than one thousand protein spots and several hundreds of protein spots from rubber particles as well as lutoid fraction and its membranes on the CBB stained 2-DE gels. The identification of 13 representative proteins on 2-DE gels by MALDI TOF/TOF MS/MS suggested that this method is compatible with MS. Conclusion The proteins extracted by this method are compatible with 2-DE and MS. This protein preparation protocol is expected to be used in future comparative proteomic analysis for natural rubber latex.

  10. Luteolin improves cardiac dysfunction in heart failure rats by regulating sarcoplasmic reticulum Ca2+-ATPase 2a

    Science.gov (United States)

    Hu, Wenjing; Xu, Tongda; Wu, Pei; Pan, Defeng; Chen, Junhong; Chen, Jing; Zhang, Buchun; Zhu, Hong; Li, Dongye

    2017-01-01

    We previously found that luteolin (Lut) appeared to improve the contractility of cardiomyocytes during ischemia/reperfusion in rats. The enhancement was associated with the alteration in sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a). This finding prompted us to consider if the mechanism worked in heart failure (HF). We studied the regulation of SERCA2a by Lut in failing cardiomyocytes and intact heart of rats. Improvement of contractility and the mechanisms centered on SERCA2a were studied in isolated cardiomyocytes and intact heart. We found that Lut significantly improved contractility and Ca2+ transients, ameliorated expression, activity and stability of SERCA2a and upregulated expression of small ubiquitin-related modifier (SUMO) 1, which is a newfound SERCA2a regulator. Lut also increased phosphorylation of protein kinase B (Akt), phospholaban (PLB) and sumoylation of SERCA2a, specificity protein 1 (Sp1). Transcriptions of SUMO1 and SERCA2a were concurrently increased. Inhibition of posphatidylinositol 3 kinase/Akt (PI3K/Akt) pathway and SERCA2a activity both markedly abolished Lut-induced benefits in vitro and in vivo. Lut upregulated the expression ratio of Bcl-2/Bax, caspase-3/cleaved-Caspase3. Meanwhile, Lut ameliorated the myocardium fibrosis of HF. These discoveries provide an important potential therapeutic strategy that Lut targeted SERCA2a SUMOylation related to PI3K/Akt-mediated regulations on rescuing the dysfunction of HF.

  11. Modulation of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase activity and oxidative modification during the development of adjuvant arthritis.

    Science.gov (United States)

    Strosova, Miriam K; Karlovska, Janka; Zizkova, Petronela; Kwolek-Mirek, Magdalena; Ponist, Silvester; Spickett, Corinne M; Horakova, Lubica

    2011-07-01

    Adjuvant arthritis (AA) was induced by intradermal administration of Mycobacterium butyricum to the tail of Lewis rats. In sarcoplasmic reticulum (SR) of skeletal muscles, we investigated the development of AA. SR Ca(2+)-ATPase (SERCA) activity decreased on day 21, suggesting possible conformational changes in the transmembrane part of the enzyme, especially at the site of the calcium binding transmembrane part. These events were associated with an increased level of protein carbonyls, a decrease in cysteine SH groups, and alterations in SR membrane fluidity. There was no alteration in the nucleotide binding site at any time point of AA, as detected by a FITC fluorescence marker. Some changes observed on day 21 appeared to be reversible, as indicated by SERCA activity, cysteine SH groups, SR membrane fluidity, protein carbonyl content and fluorescence of an NCD-4 marker specific for the calcium binding site. The reversibility may represent adaptive mechanisms of AA, induced by higher relative expression of SERCA, oxidation of cysteine, nitration of tyrosine and presence of acidic phospholipids such as phosphatidic acid. Nitric oxide may regulate cytoplasmic Ca(2+) level through conformational alterations of SERCA, and decreasing levels of calsequestrin in SR may also play regulatory role in SERCA activity and expression.

  12. Rapid T cell–based identification of human tumor tissue antigens by automated two-dimensional protein fractionation

    Science.gov (United States)

    Beckhove, Philipp; Warta, Rolf; Lemke, Britt; Stoycheva, Diana; Momburg, Frank; Schnölzer, Martina; Warnken, Uwe; Schmitz-Winnenthal, Hubertus; Ahmadi, Rezvan; Dyckhoff, Gerhard; Bucur, Mariana; Jünger, Simone; Schueler, Thomas; Lennerz, Volker; Woelfel, Thomas; Unterberg, Andreas; Herold-Mende, Christel

    2010-01-01

    Identifying the antigens that have the potential to trigger endogenous antitumor responses in an individual cancer patient is likely to enhance the efficacy of cancer immunotherapy, but current methodologies do not efficiently identify such antigens. This study describes what we believe to be a new method of comprehensively identifying candidate tissue antigens that spontaneously cause T cell responses in disease situations. We used the newly developed automated, two-dimensional chromatography system PF2D to fractionate the proteome of human tumor tissues and tested protein fractions for recognition by preexisting tumor-specific CD4+ Th cells and CTLs. Applying this method using mice transgenic for a TCR that recognizes an OVA peptide presented by MHC class I, we demonstrated efficient separation, processing, and cross-presentation to CD8+ T cells by DCs of OVA expressed by the OVA-transfected mouse lymphoma RMA-OVA. Applying this method to human tumor tissues, we identified MUC1 and EGFR as tumor-associated antigens selectively recognized by T cells in patients with head and neck cancer. Finally, in an exemplary patient with a malignant brain tumor, we detected CD4+ and CD8+ T cell responses against two novel antigens, transthyretin and calgranulin B/S100A9, which were expressed in tumor and endothelial cells. The immunogenicity of these antigens was confirmed in 4 of 10 other brain tumor patients. This fast and inexpensive method therefore appears suitable for identifying candidate T cell antigens in various disease situations, such as autoimmune and malignant diseases, without being restricted to expression by a certain cell type or HLA allele. PMID:20458140

  13. Calcium uptake by sarcoplasmic reticulum in the presence of organophosphorus insecticide methyl-parathion

    Energy Technology Data Exchange (ETDEWEB)

    Blasiak, J. [Lodz Univ. (Poland)

    1995-12-31

    Using an isotope labelling technique it has been shown that an organophosphorus insecticide methyl parathion (0,0-diethyl 0-4-nitrophenyl phosphorothionate) depressed calcium uptake by sarcoplasmic reticulum isolated from rabbit hind leg muscle. The effect was significant for insecticide concentrations of 50 and 100 {mu}M and was dose-dependent. The insecticide exerted a more pronounced effect on calcium uptake in the presence of ATP in the reticulum environment than in the absence of ATP. The inhibitory action of methyl parathion on Ca{sup 2+} accumulation by sarcoplasmic reticulum can cause a rise in myoplasmic free Ca{sup 2+}, the essential prerequisite for contracture activation. Because methyl parathion, as well as other organophosphorus insecticides, is primarily neurotoxic, evidence of non-specific effect could be important for assessing its environmental safety. (author). 20 refs, 2 figs.

  14. Cholesterol Removal from Adult Skeletal Muscle impairs Excitation-Contraction Coupling and Aging reduces Caveolin-3 and alters the Expression of other Triadic Proteins

    Directory of Open Access Journals (Sweden)

    Genaro eBarrientos

    2015-04-01

    Full Text Available Cholesterol and caveolin are integral membrane components that modulate the function/location of many cellular proteins. Skeletal muscle fibers, which have unusually high cholesterol levels in transverse tubules, express the caveolin-3 isoform but its association with transverse tubules remains contentious. Cholesterol removal impairs excitation-contraction coupling in amphibian and mammalian fetal skeletal muscle fibers. Here, we show that treating single muscle fibers from adult mice with the cholesterol removing agent methyl-β-cyclodextrin decreased fiber cholesterol by 26%, altered the location pattern of caveolin-3 and of the voltage dependent calcium channel Cav1.1, and suppressed or reduced electrically evoked Ca2+ transients without affecting membrane integrity or causing sarcoplasmic reticulum calcium depletion. We found that transverse tubules from adult muscle and triad fractions that contain ~10% attached transverse tubules, but not sarcoplasmic reticulum membranes, contained caveolin-3 and Cav1.1; both proteins partitioned into detergent-resistant membrane fractions highly enriched in cholesterol. Aging entails significant deterioration of skeletal muscle function. We found that triad fractions from aged rats had similar cholesterol and RyR1 protein levels compared to triads from young rats, but had lower caveolin-3 and glyceraldehyde 3-phosphate dehydrogenase and increased Na+/K+-ATPase protein levels. Both triad fractions had comparable NADPH oxidase (NOX activity and protein content of NOX2 subunits (p47phox and gp91phox, implying that NOX activity does not increase during aging. These findings show that partial cholesterol removal impairs excitation-contraction coupling and alters caveolin-3 and Cav1.1 location pattern, and that aging reduces caveolin-3 protein content and modifies the expression of other triadic proteins. We discuss the possible implications of these findings for skeletal muscle function in young and aged

  15. [Calcium transport in sarcoplasmic reticulum in the presence of AR-L 115 BS].

    Science.gov (United States)

    Hasselbach, W

    1981-01-01

    2-[(2-Methoxy-4-methylsulfinyl)phenyl]-1H-imidazo[4,5-b]pyridine (AR-L 115 BS) is a substance with positive inotropic activity which does not influence the activity of the sarcoplasmic calcium pump. It can, therefore, be expected that AR-L 115 BS does not interfere with the distribution and movement of calcium in the resting and active muscle.

  16. Immunochemical quantitation of 3-(cystein-S-yl)acetaminophen protein adducts in subcellular liver fractions following a hepatotoxic dose of acetaminophen.

    Science.gov (United States)

    Pumford, N R; Roberts, D W; Benson, R W; Hinson, J A

    1990-08-01

    The hepatotoxicity of acetaminophen correlates with the formation of 3-(cystein-S-yl)acetaminophen protein adducts. Using a sensitive and specific immunochemical assay, we quantitated the formation of these protein adducts in liver fractions and serum after administration of a hepatotoxic dose of acetaminophen (400 mg/kg) to B6C3F1 mice. Adducts in the cytosolic fraction increased to 3.6 nmol/mg protein at 2 hr and then decreased to 1.1 nmol/mg protein by 8 hr. Concomitant with the decrease in adducts in the cytosol, 3-(cystein-S-yl)acetaminophen protein adducts appeared in serum and their levels paralleled increases in serum alanine aminotransferase. Microsomal protein adducts peaked at 1 hr (0.7 nmol/mg protein) and subsequently decreased to 0.2 nmol/mg at 8 hr. The 4000 g pellet (nuclei, plasma membranes, and cell debris) had the highest level of adducts (3.5 nmol/mg protein), which remained constant from 1 to 8 hr. Evaluation of fractions purified from a 960 g pellet indicated that the highest concentration of 3-(cystein-S-yl)acetaminophen protein adducts was located in plasma membranes and mitochondria; peak levels were 10.3 and 5.1 nmol/mg respectively. 3-(Cystein-S-yl)acetaminophen protein adducts were detected in nuclei only after enzymatic hydrolysis of the proteins. The localization of high levels of 3-(cystein-S-yl)acetaminophen protein adducts in plasma membranes and mitochondria may play a critical role in acetaminophen toxicity.

  17. Transient kinetics of Ca2+ transport of sarcoplasmic reticulum. A comparison of cardiac and skeletal muscle.

    Science.gov (United States)

    Sumida, M; Wang, T; Mandel, F; Froehlich, J P; Schwartz, A

    1978-12-25

    Current evidence supports similar functions and mechanisms for cardiac sarcoplasmic reticulum (CSR) as for skeletal sarcoplasmic reticulum (SSR). It is thought that the slower relaxation rate of cardiac muscle compared to fast skeletal muscle reflects the lower ATPase activity and calcium transport of CSR. Possible quantitative differences is phosphorylation, dephosphorylation, and calcium transport of the isolated preparations are studied using a quench-flow apparatus. The results show that both CSR and SSR bind calcium tightly in the absence of ATP, and coupling of E approximately P formation and calcium transport occurs in the transient phase of ATP hydrolysis. The rate of phosphorylation (t-1/2 - 10 ms) of sarcoplasmic reticulum (SR) preloaded with calcium is the same for cardiac and skeletal preparations. However, the rates of dissociation of extra vesicular calcium (10 s-1 versus 15 s-1), phosphorylation of calcium-free SR, and dephosphorylation of E approximately P (8 s-1 versus 12 s-1) are lower for CSR than for SSR. By computer simulation, the apparent rate constants associated with the reduced rates of phosphorylation of calcium-free SR were: 12 s-1 for CSR and 63 s-1 for SSR in the presence of high Mg2+. The difference in the rates may be partly responsible for the lower levels of ATPase and calcium transport activity with characterize cardiac muscle preparations.

  18. Molecular transformations in sarcoplasmic reticulum of fast-twitch muscle by electro-stimulation.

    Science.gov (United States)

    Heilmann, C; Pette, D

    1979-02-01

    Chronic electro-stimulation of fast-twitch rabbit muscle with the frequency pattern received by a slow-twitch muscle induces a progressive transformation of the sarcoplasmic reticulum. After 2 days stimulation activities of Ca2+-dependent ATPase and of Ca2+ transport begin to decrease, and are paralleled by a progressive decrease in Ca2+-dependent and Ca2+, Mg2+-dependent phosphoprotein formation, reduced rate of dephosphorylation and a rearrangement of the electrophoretic polypeptide and phosphoprotein patterns. These findings suggest a transformation of the sarcoplasmic reticulum to resemble that of a slow-twitch muscle. This transformation is paralleled by increase in time-to-peak of twitch contraction and half relaxation time and occurs before conversion of the myosin light chain pattern is observed. The parallel time course of changes in contractile properties of stimulated muscle and the molecular and functional properties of the sarcoplasmic reticulum emphasizes the definitive role of the latter in determining the twitch characteristics of fast and slow twitch muscles.

  19. Fermentation characteristics, chemical composition and fractionation of carbohydrates and crude protein of silage of elephant grass wilted or with addition of castor bean meal

    Directory of Open Access Journals (Sweden)

    Leandro Sampaio Oliveira Ribeiro

    2014-06-01

    Full Text Available The experiment was conducted to evaluate the concentrations of ammonia nitrogen, hydrogen potential, the losses of deriving the fermentative process, nutritional value, the fractioning of carbohydrates and protein the elephant grass silage wilted or not containing castor bean meal. The experimental design was completely randomized, with five treatments and with four replications: elephant grass wilted; elephant not wilted; elephant grass more castor bean meal (6%; elephant grass more castor bean meal (12% and elephant grass more castor bean meal (18%, the coproduct was added with base on natural matter. We adopted a specific mass of 600 kg/m3. The silage containing 18% castor bean meal showed higher (P0.05 among the silages with additives for fractions A+B1, B2 and C. For the protein fractioning, the fractions A and C decreased (P<0.05 with increase of the inclusion of castor bean meal, differently, of the fraction B1+B2 which increased. The castor bean stands out as a good additive in silage of elephant grass to reduce moisture and improve the fermentation characteristics of silages also was effective in increasing the protein value of silages, especially when using the dose 18%.

  20. A nuclear fraction of turnip crinkle virus capsid protein is important for elicitation of the host resistance response.

    Science.gov (United States)

    Kang, Sung-Hwan; Qu, Feng; Morris, T Jack

    2015-12-01

    The N-terminal 25 amino acids (AAs) of turnip crinkle virus (TCV) capsid protein (CP) are recognized by the resistance protein HRT to trigger a hypersensitive response (HR) and systemic resistance to TCV infection. This same region of TCV CP also contains a motif that interacts with the transcription factor TIP, as well as a nuclear localization signal (NLS). However, it is not yet known whether nuclear localization of TCV CP is needed for the induction of HRT-mediated HR and resistance. Here we present new evidence suggesting a tight correlation between nuclear inclusions formed by CP and the manifestation of HR. We show that a fraction of TCV CP localized to cell nuclei to form discrete inclusion-like structures, and a mutated CP (R6A) known to abolish HR failed to form nuclear inclusions. Notably, TIP-CP interaction augments the inclusion-forming activity of CP by tethering inclusions to the nuclear membrane. This TIP-mediated augmentation is also critical for HR resistance, as another CP mutant (R8A) known to elicit a less restrictive HR, though still self-associated into nuclear inclusions, failed to direct inclusions to the nuclear membrane due to its inability to interact with TIP. Finally, exclusion of CP from cell nuclei abolished induction of HR. Together, these results uncovered a strong correlation between nuclear localization and nuclear inclusion formation by TCV CP and induction of HR, and suggest that CP nuclear inclusions could be the key trigger of the HRT-dependent, yet TIP-reinforced, resistance to TCV.

  1. Synergistic skin heat shock protein expression in response to combined laser treatment with a diode laser and ablative fractional lasers.

    Science.gov (United States)

    Paasch, Uwe; Sonja, Grunewald; Haedersdal, Merete

    2014-06-01

    Diode laser-based skin heating has been shown to minimise scars by interfering with wound healing responses through the induction of heat shock proteins (HSP). HSP are also induced after ablative fractional laser (AFXL) wound healing. AFXL itself is highly recommended for scar treatment. Therefore, the sequential combination of both modalities may produce superior outcomes. The aim of this study was to examine the pretreatment effects of a diode laser before AFXL on wound healing responses in terms of HSP up-regulation in an in vitro model. Immediate responses and responses on days 1, 3 or 6 post-procedure were studied in an in vitro porcine skin model (n = 240). Untreated samples served as control. Immunohistochemical investigation (Hsp70) was performed in all untreated controls, diode laser-, AFXL-, and in diode laser + AFXL-treated samples. Hsp70 was shown to be up-regulated by all interventions between days 1 and 6 after interventions. The largest effect was caused by the combination of a diode laser and an AFXL procedure. Diode laser exposure induces a skin HSP response that can be further enhanced by sequential AFXL treatment. Clinical studies are necessary to investigate the dose response of HSP on scar formation and refine suitable laser exposure settings.

  2. Drug action of benzocaine on the sarcoplasmic reticulum Ca-ATPase from fast-twitch skeletal muscle.

    Science.gov (United States)

    Di Croce, D; Trinks, P W; Grifo, M B; Takara, D; Sánchez, G A

    2015-11-01

    The effect of the local anesthetic benzocaine on sarcoplasmic reticulum membranes isolated from fast-twitch muscles was tested. The effects on Ca-ATPase activity, calcium binding and uptake, phosphoenzyme accumulation and decomposition were assessed using radioisotopic methods. The calcium binding to the Ca-ATPase was noncompetitively inhibited, and the enzymatic activity decreased in a concentration-dependent manner (IC50 47.1 mM). The inhibition of the activity depended on the presence of the calcium ionophore calcimycin and the membrane protein concentration. The pre-exposure of the membranes to benzocaine enhanced the enzymatic activity in the absence of calcimycin, supporting the benzocaine permeabilizing effect, which was prevented by calcium. Benzocaine also interfered with the calcium transport capability by decreasing the maximal uptake (IC50 40.3 mM) without modification of the calcium affinity for the ATPase. It inhibited the phosphorylation of the enzyme, and at high benzocaine concentration, the dephosphorylation step became rate-limiting as suggested by the biphasic profile of phosphoenzyme accumulation at different benzocaine concentrations. The data reported in this paper revealed a complex pattern of inhibition involving two sites for interaction with low and high benzocaine concentrations. It is concluded that benzocaine not only exerts an indirect action on the membrane permeability to calcium but also affects key steps of the Ca-ATPase enzymatic cycle.

  3. Luteolin Exerts Cardioprotective Effects through Improving Sarcoplasmic Reticulum Ca2+-ATPase Activity in Rats during Ischemia/Reperfusion In Vivo

    Directory of Open Access Journals (Sweden)

    Changsheng Nai

    2015-01-01

    Full Text Available The flavonoid luteolin exists in many types of fruits, vegetables, and medicinal herbs. Our previous studies have demonstrated that luteolin reduced ischemia/reperfusion (I/R injury in vitro, which was related with sarcoplasmic reticulum Ca2+-ATPase (SERCA2a activity. However, the effects of luteolin on SERCA2a activity during I/R in vivo remain unclear. To investigate whether luteolin exerts cardioprotective effects and to monitor changes in SERCA2a expression and activity levels in vivo during I/R, we created a myocardial I/R rat model by ligating the coronary artery. We demonstrated that luteolin could reduce the myocardial infarct size, lactate dehydrogenase release, and apoptosis during I/R injury in vivo. Furthermore, we found that luteolin inhibited the I/R-induced decrease in SERCA2a activity in vivo. However, neither I/R nor luteolin altered SERCA2a expression levels in myocardiocytes. Moreover, the PI3K/Akt signaling pathway played a vital role in this mechanism. In conclusion, the present study has confirmed for the first time that luteolin yields cardioprotective effects against I/R injury by inhibiting the I/R-induced decrease in SERCA2a activity partially via the PI3K/Akt signaling pathway in vivo, independent of SERCA2a protein level regulation. SERCA2a activity presents a novel biomarker to assess the progress of I/R injury in experimental research and clinical applications.

  4. Proteolysis by sourdough lactic acid bacteria: effects on wheat flour protein fractions and gliadin peptides involved in human cereal intolerance.

    Science.gov (United States)

    Di Cagno, Raffaella; De Angelis, Maria; Lavermicocca, Paola; De Vincenzi, Massimo; Giovannini, Claudio; Faccia, Michele; Gobbetti, Marco

    2002-02-01

    Sourdough lactic acid bacteria were preliminarily screened for proteolytic activity by using a digest of albumin and globulin polypeptides as a substrate. Based on their hydrolysis profile patterns, Lactobacillus alimentarius 15M, Lactobacillus brevis 14G, Lactobacillus sanfranciscensis 7A, and Lactobacillus hilgardii 51B were selected and used in sourdough fermentation. A fractionated method of protein extraction and subsequent two-dimensional electrophoresis were used to estimate proteolysis in sourdoughs. Compared to a chemically acidified (pH 4.4) dough, 37 to 42 polypeptides, distributed over a wide range of pIs and molecular masses, were hydrolyzed by L. alimentarius 15M, L. brevis 14G, and L. sanfranciscensis 7A. Albumin, globulin, and gliadin fractions were hydrolyzed, while glutenins were not degraded. The concentrations of free amino acids, especially proline and glutamic and aspartic acids, also increased in sourdoughs. Compared to the chemically acidified dough, proteolysis by lactobacilli positively influenced the softening of the dough during fermentation, as determined by rheological analyses. Enzyme preparations of the selected lactobacilli which contained proteinase or peptidase enzymes showed hydrolysis of the 31-43 fragment of A-gliadin, a toxic peptide for celiac patients. A toxic peptic-tryptic (PT) digest of gliadins was used for in vitro agglutination tests on K 562 (S) subclone cells of human myelagenous leukemia origin. The lowest concentration of PT digest that agglutinated 100% of the total cells was 0.218 g/liter. Hydrolysis of the PT digest by proteolytic enzymes of L. alimentarius 15M and L. brevis 14G completely prevented agglutination of the K 562 (S) cells by the PT digest at a concentration of 0.875 g/liter. Considerable inhibitory effects by other strains and at higher concentrations of the PT digest were also found. The mixture of peptides produced by enzyme preparations of selected lactobacilli showed a decreased agglutination

  5. Sarcoplasmic reticulum calcium ATPase interactions with decaniobate, decavanadate, vanadate, tungstate and molybdate.

    Science.gov (United States)

    Fraqueza, Gil; Ohlin, C André; Casey, William H; Aureliano, Manuel

    2012-02-01

    Over the last few decades there has been increasing interest in oxometalate and polyoxometalate applications to medicine and pharmacology. This interest arose, at least in part, due to the properties of these classes of compounds as anti-cancer, anti-diabetic agents, and also for treatment of neurodegenerative diseases, among others. However, our understanding of the mechanism of action would be improved if biological models could be used to clarify potential toxicological effects in main cellular processes. Sarcoplasmic reticulum (SR) vesicles, containing a large amount of Ca(2+)-ATPase, an enzyme that accumulates calcium by active transport using ATP, have been suggested as a useful model to study the effects of oxometalates on calcium homeostasis. In the present article, it is shown that decavanadate, decaniobate, vanadate, tungstate and molybdate, all inhibited SR Ca(2+)-ATPase, with the following IC(50) values: 15, 35, 50, 400 μM and 45 mM, respectively. Decaniobate (Nb(10)), is the strongest P-type enzyme inhibitor, after decavanadate (V(10)). Atomic-absorption spectroscopy (AAS) analysis, indicates that decavanadate binds to the protein with a 1:1 decavanadate:Ca(2+)-ATPase stoichiometry. Furthermore, V(10) binds with similar extension to all the protein conformations, which occur during calcium translocation by active transport, namely E1, E1P, E2 and E2P, as analysed by AAS. In contrast, it was confirmed that the binding of monomeric vanadate (H(2)VO(4)(2-); V(1)) to the calcium pump is favoured only for the E2 and E2P conformations of the ATPase, whereas no significant amount of vanadate is bound to the E1 and E1P conformations. Scatchard plot analysis, confirmed a 1:1 ratio for decavanadate-Ca(2+)-ATPase, with a dissociation constant, k(d) of 1 μM(-1). The interaction of decavanadate V(10)O(28)(6-) (V(10)) with Ca(2+)-ATPase is prevented by the isostructural and isoelectronic decaniobate Nb(10)O(28)(6-) (Nb(10)), whereas no significant effects were

  6. Studies on Single Cell Culture in vitro in Wheat--The variation of grain protein content and its fractions from regenerated plants

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    On the basis of previous studies dealing with the variation of major agronomic and yield characteristics of regenerated plants derived from single cell culture in vitro of common wheat (Triticum aestivum L.Cultivar NE 7742), the grain protein content and its fractions from regenerated plants with stable agronomic characteristics were studied from 1992 to 1995. The results showed that the variation of grain protein content and its fractions in somaclones from single cell culture in vitro were very significant and the range was very wide (11.53~17.70%). Several types of variation were found in the studies, especially the type with higher protein content than that of cultivar NE 7742 (non-culture parent). Among them, -20.69% of lines the grain protein content was significantly higher than that of NE 7742 and combined with high yielding potential. The tendency of variation of the four protein fractions showed that the variation of albumin was not obvious and maintained the same level as NE7742, the content of gliadin increased in some somaclones and decreased in others. However, the percentages both globulin and glutenin tended to increase. The variation of total amount of structural protein and the ratio between globulin and glutenin tended to increase. The variation of total amount of structural protein and the ratio between globulin and albumm was mainly influenced by globulin under the condition of culture in vitro. The variation of total amount of storage protein and the ratio between gliadin and glutenin was mainly affected by glutenin. The results mentioned above demonstrated that the induction and screening of somaclonal variation could be an effective way in wheat improvement in combining high protein content with high yield.

  7. Nitrogen 15 abundance in protein fractions of beans fertilized with ({sup 15}NH{sub 4}){sub 2}SO{sub 4}

    Energy Technology Data Exchange (ETDEWEB)

    Chaud, Saula Goulart; Oliveira, Admar Costa de [Universidade Estadual de Campinas, SP (Brazil). Faculdade de Estudos Agricolas. Dept. de Planejamento Alimentar e Nutricao; Trivelin, Paulo Cesar Ocheuze [Centro de Energia Nuclear na Agricultura (CENA), Piracicaba, SP (Brazil). Lab. de Isotopos Estaveis]. E-mail: admarco@fea.unicamp.br

    2002-12-01

    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{sup -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)

  8. Short communication: Potential of Fresco-style cheese whey as a source of protein fractions with antioxidant and angiotensin-I-converting enzyme inhibitory activities.

    Science.gov (United States)

    Tarango-Hernández, S; Alarcón-Rojo, A D; Robles-Sánchez, M; Gutiérrez-Méndez, N; Rodríguez-Figueroa, J C

    2015-11-01

    Recently, traditional Mexican Fresco-style cheese production has been increasing, and the volume of cheese whey generated represents a problem. In this study, we investigated the chemical composition of Fresco-style cheese wheys and their potential as a source of protein fractions with antioxidant and angiotensin-I-converting enzyme (ACE)-inhibitory activities. Three samples from Fresco, Panela, and Ranchero cheeses whey were physicochemically characterized. Water-soluble extracts were fractionated to obtain whey fractions with different molecular weights: 10-5, 5-3, 3-1 and Panela and Fresco cheese whey fractions showed the highest ACE-inhibitory activity (0.57 ± 0.02 and 0.59 ± 0.04 μg/mL 50%-inhibitory concentration values, respectively). These results suggest that Fresco-style cheese wheys may be a source of protein fractions with bioactivity, and thus could be useful ingredients in the manufacture of functional foods with increased nutritional value.

  9. 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.

  10. Sarcoplasmic reticulum calcium mobilization in right ventricular pressure-overload hypertrophy in the ferret: relationships to diastolic dysfunction and a negative treppe.

    Science.gov (United States)

    Gwathmey, J K; Morgan, J P

    1993-03-01

    In a model of right-ventricular pressure-overload hypertrophy (POH) in the ferret, action potential duration (to 90% repolarization) was found to be significantly longer (228 +/- 11 vs 314 +/- 12 ms) with no change in amplitude (85 +/- 3 vs 85 +/- 2 mV) or resting membrane potential (-79 +/- 1.5 vs -79 +/- 1 mV) for control and POH, respectively. Peak sarcoplasmic reticulum Ca2+ release (expressed as the logarithm of the fractional luminescence, -4.2 +/- 0.1 vs -4.4 +/- 0.3) and resting calcium concentrations (-5.5 +/- 0.1 vs -5.7 +/- 0.1) were not different between the two groups (control vs POH respectively). Muscles from control and POH animals demonstrated a positive force/interval relationship in the presence of physiological extracellular [Ca2+]. However, unlike muscles from control animals, muscles from animals with POH subjected to increasing frequencies of contraction in the presence of increased extracellular [Ca2+] demonstrated further impairment of diastolic relaxation and a negative treppe. Exposure of muscles from POH animals to isoproterenol returned the slowed Ca2+ uptake by the sarcoplasmic reticulum as detected with aequorin to control values, although the relaxation phase of the isometric twitch remained prolonged compared to non-hypertrophied muscles. Exposure to milrinone also abbreviated the time course of the intracellular Ca2+ transient, but did not return it to that seen in normal myocardium. The exposure of non-hypertrophied isolated muscles to caffeine resulted in similar prolongation of the isometric twitch duration to that seen in hypertrophied myocardium. Results of these experiments suggest that impaired muscle relaxation in POH reflects changes at the level of the myofilaments. Thus, although slowed intracellular calcium mobilization contributes to diastolic relaxation abnormalities, it can not be the sole factor responsible for the slowed relaxation as has been suggested.

  11. Decolorization of direct dyes by salt fractionated turnip proteins enhanced in the presence of hydrogen peroxide and redox mediators.

    Science.gov (United States)

    Matto, Mahreen; Husain, Qayyum

    2007-09-01

    The present paper demonstrates the effect of salt fractionated turnip (Brassica rapa) proteins on the decolorization of direct dyes, used in textile industry, in the presence of various redox mediators. The rate and extent of decolorization of dyes was significantly enhanced by the presence of different types of redox mediators. Six out of 10 investigated compounds have shown their potential in enhancing the decolorization of direct dyes. The performance was evaluated at different concentrations of mediator and enzyme. The efficiency of each natural mediator depends on the type of dye treated. The decolorization of all tested direct dyes was maximum in the presence of 0.6mM redox mediator at pH 5.5 and 30 degrees C. Complex mixtures of dyes were also maximally decolorized in the presence of 0.6mM redox mediator (1-hydroxybenzotriazole/violuric acid). In order to examine the operational stability of the enzyme preparation, the enzyme was exploited for the decolorization of mixtures of dyes for different times in a stirred batch process. There was no further change in decolorization of an individual dye or their mixtures after 60 min; the enzyme caused more than 80% decolorization of all dyes in the presence of 1-hydroxybenzotriazole/violuric acid. However, there was no desirable increase in dye decolorization of the mixtures on overnight stay. Total organic carbon analysis of treated dyes or their mixtures showed that these results were quite comparable to the loss of color from solutions. However, the treatment of such polluted water in the presence of redox mediators caused the formation of insoluble precipitate, which could be removed by the process of centrifugation. The results suggested that catalyzed oxidative coupling reactions might be important for natural transformation pathways for dyes and indicate their potential use as an efficient means for removal of dyes color from waters and wastewaters.

  12. Phospholamban Modulates the Functional Coupling between Nucleotide Domains in Ca-ATPase Oligomeric Complexes in Cardiac Sarcoplasmic Reticulum

    Energy Technology Data Exchange (ETDEWEB)

    Chen, L.; Yao, Qing; Soares, Thereza A.; Squier, Thomas C.; Bigelow, Diana J.

    2009-03-24

    Oligomeric interactions between Ca-ATPase polypeptide chains and their modulation by phospholamban (PLB) were measured in native cardiac sarcoplasmic reticulum (SR) microsomes. Progressive modification of Lys514 with fluorescein-5-isothiocyanate (FITC), which physically blocks access to the nucleotide binding site by ATP, demonstrates that Ca-ATPase active sites function independently of one another prior to the phosphorylation of PLB. However, upon PKA-dependent phosphorylation of PLB, a second-order dependence between enzyme activity and the fraction of active sites is observed, consistent with a dimeric functional complex. Complementary distance measurements were made using FITC or 5-iodoacetamido-fluorescein (IAF) bound to Cys674 within the N- or P-domains respectively, to detect structural coupling within oligomeric complexes. Accompanying the phosphorylation of PLB, neighboring Ca-ATPase polypeptide chains exhibit a 4 ± 2 Å decrease in the proximity between FITC sites within the N-domain and a 9 ± 3 Å increase in the proximity between IAF sites within P-domains. Thus, the phosphorylation of PLB induces spatial rearrangements between the N- and P-domain elements of proximal Ca-ATPase polypeptide chains which restore functional interactions between neighboring polypeptide chains and, in turn, result in increased rates of catalytic turnover. These results are interpreted in terms of a structural model, calculated through optimization of shape complementarity, desolvation, and electrostatic energies, which suggests a dimeric arrangement of Ca-ATPase polypeptide chains through the proximal association of N-domains. We suggest that the phosphorylation of PLB acts to release constraints involving interdomain subunit interactions that enhance catalytically important N-domain motions.

  13. Combining Phytate/Ca2+Fractionation with Trichloroacetic Acid/Acetone Precipitation Improved Separation of Low-Abundant Proteins of Wheat (Triticum aestivum L.) Leaf for Proteomic Analysis

    Institute of Scientific and Technical Information of China (English)

    Muhammad A R F Sultan; LIU Hui; CHENG Yu-Feng; ZHANG Pei-pei; ZHAO Hui-xian

    2013-01-01

    Proteomic assessment of low-abundance leaf proteins is hindered by the large quantity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) present within plant leaf tissues. In the present study, total proteins were extracted from wheat (Triticum aestivum L.) leaves by a conventional trichloroacetic acid (TCA)/acetone method and a protocol first developed in this work. Phytate/Ca2+fractionation and TCA/acetone precipitation were combined to design an improved TCA/acetone method. The extracted proteins were analysed by two-dimensional gel electrophoresis (2-DE). The resulting 2-DE images were compared to reveal major differences. The results showed that large quantities of Rubisco were deleted from wheat leaf proteins prepared by the improved method. As many as (758±4) protein spots were detected from 2-DE images of protein extracts obtained by the improved method, 130 more than those detected by the TCA/acetone method. Further analysis indicated that more protein spots could be detected at regions of pI 4.00-4.99 and 6.50-7.00 in the improved method-based 2-DE images. Our findings indicated that the improved method is an efficient protein preparation protocol for separating low-abundance proteins in wheat leaf tissues by 2-DE analysis. The proposed protocol is simple, fast, inexpensive and also applicable to protein preparations of other plants.

  14. Digoxin activates sarcoplasmic reticulum Ca(2+)-release channels: a possible role in cardiac inotropy.

    OpenAIRE

    1993-01-01

    1. The effect of digoxin on rapid 45Ca2+ efflux from cardiac and skeletal sarcoplasmic reticulum (SR) vesicles was investigated. Additionally the interaction of digoxin with single cardiac and skeletal muscle SR Ca(2+)-release channels incorporated into planar phospholipid bilayers and held under voltage clamp was determined. 2. Digoxin (1 nM) increased the initial rate and amount of Ca(2+)-induced release of 45Ca2+ from cardiac SR vesicles, passively loaded with 45CaCl2, at an extravesicular...

  15. A fractionation method to identify qauntitative changes in protein expression mediated by IGF-1 on the proteome of murine C2C12 myoblasts

    Directory of Open Access Journals (Sweden)

    Friedmann Theodore

    2009-08-01

    Full Text Available Abstract Although much is known about signal transduction downstream of insulin-like growth factor-1 (IGF-1, relatively little is known about the global changes in protein expression induced by this hormone. In this study, the acute effects of IGF-1 on the proteome of murine C2C12 cells were examined. Cells were treated with IGF-1 for up to 24 hours, lysed, and fractionated into cytosolic, nuclear, and insoluble portions. Proteins from the cytosolic fraction were further separated using a new batch ion-exchange chromatography method to reduce sample complexity, followed by two-dimensional (2D electrophoresis, and identification of selected proteins by mass spectrometry. PDQuest software was utilized to identify and catalogue temporal changes in protein expression during IGF-1 stimulation. In response to IGF-1 stimulation, expression of 23 proteins increased at least three-fold and expression of 17 proteins decreased at least three-fold compared with control un-stimulated C2C12 cells. Changes in expression of selected proteins from each group, including Rho-GDI, cofillin, RAD50, enolase, IκB kinase b (IκBKb and Hsp70 were confirmed by Western blotting. Additionally, the position of 136 'landmark' proteins whose expression levels and physicochemical properties did not change appreciably or consistently during IGF-1 treatment were mapped and identified. This characterization of large-scale changes in protein expression in response to growth factor stimulation of C2C12 cells will further help to establish a comprehensive understanding of the networks and pathways involved in the action of IGF-1.

  16. Concentration and selective fractionation of an antihypertensive peptide from an alfalfa white proteins hydrolysate by mixed ion-exchange centrifugal partition chromatography.

    Science.gov (United States)

    Boudesocque, Leslie; Kapel, Romain; Paris, Cedric; Dhulster, Pascal; Marc, Ivan; Renault, Jean-Hugues

    2012-09-15

    This article reports a promising use of the mixed ion-exchange centrifugal partition chromatography (MIXCPC) technique in the field of downstream processes. A complex alfalfa white protein concentrate hydrolysate (AWPC hydrolysate) showing anti-hypertensive properties was successfully fractionated by MIXCPC to yield a L-valyl-L-tryptophan (VW) enriched fraction in one run. This dipeptide shows an interesting anti-angiotensin converting enzyme (anti-ACE) activity. An analytical method based on RP-LC/MS-MS was developed to quantify the target VW peptide in both the starting material and the enriched fractions. The best results for the MIXCPC fractionation were obtained by the combined use of the quaternary biphasic solvent system, methyl-tert-butylether/acetonitrile/n-butanol/water (2:1:2:5, v/v) in the descending mode, of the lipophilic di(2-ethylhexyl)phosphoric acid (DEHPA) cation-exchanger with an exchanger (DEHPA)/peptides ratio of 15, and of two displacers: calcium chloride and hydrochloric acid. The complexity of the starting material involved the selectivity optimization by splitting the stationary phase into two sections that differed by their triethylamine concentration. From 1g of AWPC hydrolysate containing 0.26% of VW, 30.7 mg of a VW enriched fraction were recovered with a purity of 10.9%, corresponding to a purification factor of 41 and a recovery of 97%.

  17. Determination of the Mercury Fraction Linked to Protein of Muscle and Liver Tissue of Tucunaré (Cichla spp.) from the Amazon Region of Brazil.

    Science.gov (United States)

    Vieira, José C S; Cavecci, Bruna; Queiroz, João V; Braga, Camila P; Padilha, Cilene C F; Leite, Aline L; Figueiredo, Wllyane S; Buzalaf, Marília A R; Zara, Luiz F; Padilha, Pedro M

    2015-11-01

    This study used metalloproteomic techniques to characterize mercury (Hg)-bound proteins in the muscle and liver tissue of Tucunaré (Cichla spp.) collected at the Jirau Hydroelectric Power Plant in Madeira River Basin, Brazil. The proteome of the muscle and liver tissue was obtained after two steps of fractional precipitation and separating the proteins by 2-D polyacrylamide gel electrophoresis. Hg was identified and quantified in the protein spots by graphite furnace atomic absorption spectrometry after acid mineralization in an ultrasound bath. Hg with a molecular weight <20 kDa and a concentration between 13.30 and 33.40 mg g(-1) was found in the protein spots. These protein spots were characterized by electrospray ionization tandem mass spectrometry after trypsin digestion. From a total of 12 analyzed spots, seven proteins showing Hg biomarker characteristics were identified: parvalbumin and its isoforms, ubiquitin-40S ribosomal protein S27a, zinc (Zn) finger and BTB domain-containing protein 24, and dual-specificity protein phosphatase 22-B.

  18. Vanadate oligomer inhibition of passive and active Ca2+ translocation by the Ca2+ pump of sarcoplasmic reticulum.

    Science.gov (United States)

    Aureliano, M

    2000-05-30

    'Monovanadate' containing mainly monomeric, dimeric and tetrameric vanadate species or 'decavanadate', containing mainly decameric vanadate species inhibits the passive and the active efflux of Ca2+ through the sarcoplasmic reticulum calcium pump. When the efflux of Ca2+ by sarcoplasmic reticulum vesicles is not associated with ATP synthesis both vanadate solutions inhibit the passive efflux of Ca2+. However, only 'decavanadate' exerts noticeable effects when the efflux of Ca2+ is associated with ATP synthesis being the active efflux of Ca2+ almost completely inhibited by decameric species concentration as low as 40 microM.

  19. Down-regulation of the cardiac sarcoplasmic reticulum ryanodine channel in severely food-restricted rats

    Directory of Open Access Journals (Sweden)

    V.A. Vizotto

    2007-01-01

    Full Text Available We have shown that myocardial dysfunction induced by food restriction is related to calcium handling. Although cardiac function is depressed in food-restricted animals, there is limited information about the molecular mechanisms that lead to this abnormality. The present study evaluated the effects of food restriction on calcium cycling, focusing on sarcoplasmic Ca2+-ATPase (SERCA2, phospholamban (PLB, and ryanodine channel (RYR2 mRNA expressions in rat myocardium. Male Wistar-Kyoto rats, 60 days old, were submitted to ad libitum feeding (control rats or 50% diet restriction for 90 days. The levels of left ventricle SERCA2, PLB, and RYR2 were measured using semi-quantitative RT-PCR. Body and ventricular weights were reduced in 50% food-restricted animals. RYR2 mRNA was significantly decreased in the left ventricle of the food-restricted group (control = 5.92 ± 0.48 vs food-restricted group = 4.84 ± 0.33, P < 0.01. The levels of SERCA2 and PLB mRNA were similar between groups (control = 8.38 ± 0.44 vs food-restricted group = 7.96 ± 0.45, and control = 1.52 ± 0.06 vs food-restricted group = 1.53 ± 0.10, respectively. Down-regulation of RYR2 mRNA expressions suggests that chronic food restriction promotes abnormalities in sarcoplasmic reticulum Ca2+ release.

  20. Expression of P-glycoprotein and multidrug resistance associated protein in Ehrlich ascites tumor cells after fractionated irradiation

    DEFF Research Database (Denmark)

    Nielsen, D; Maare, C; Eriksen, J

    2001-01-01

    AND MATERIALS: Sensitive Ehrlich ascites tumor cells (EHR2) were in vitro exposed to fractionated irradiation (60 Gy). Western blot analysis was performed for determination of PGP and Mrp1, reverse transcriptase-polymerase chain reaction (RT-PCR) for determination of mdr1a + b mRNA, and semiquantitative RT...

  1. Quantitative proteomics of fractionated membrane and lumen exosome proteins from isogenic metastatic and nonmetastatic bladder cancer cells reveal differential expression of EMT factors.

    Science.gov (United States)

    Jeppesen, Dennis Kjølhede; Nawrocki, Arkadiusz; Jensen, Steffen Grann; Thorsen, Kasper; Whitehead, Bradley; Howard, Kenneth A; Dyrskjøt, Lars; Ørntoft, Torben Falck; Larsen, Martin R; Ostenfeld, Marie Stampe

    2014-03-01

    Cancer cells secrete soluble factors and various extracellular vesicles, including exosomes, into their tissue microenvironment. The secretion of exosomes is speculated to facilitate local invasion and metastatic spread. Here, we used an in vivo metastasis model of human bladder carcinoma cell line T24 without metastatic capacity and its two isogenic derivate cell lines SLT4 and FL3, which form metastases in the lungs and liver of mice, respectively. Cultivation in CLAD1000 bioreactors rather than conventional culture flasks resulted in a 13- to 16-fold increased exosome yield and facilitated quantitative proteomics of fractionated exosomes. Exosomes from T24, SLT4, and FL3 cells were partitioned into membrane and luminal fractions and changes in protein abundance related to the gain of metastatic capacity were identified by quantitative iTRAQ proteomics. We identified several proteins linked to epithelial-mesenchymal transition, including increased abundance of vimentin and hepatoma-derived growth factor in the membrane, and casein kinase II α and annexin A2 in the lumen of exosomes, respectively, from metastatic cells. The change in exosome protein abundance correlated little, although significant for FL3 versus T24, with changes in cellular mRNA expression. Our proteomic approach may help identification of proteins in the membrane and lumen of exosomes potentially involved in the metastatic process.

  2. Retention behaviour of proteins on poly(vinylimidazole)-copper(II) complexes supported on silica: application to the fractionation of desialylated human alpha 1-acid glycoprotein variants.

    Science.gov (United States)

    Millot, M C; Hervé, F; Sébille, B

    1995-02-03

    The retention behaviour of various amino acids, peptides and proteins on poly(vinylimidazole)-Cu(II) complexes supported on silica was investigated. Free amino acids and peptides containing one histidine and in some instances one additional tryptophan residue in their primary structure were found to elute from the supports only after addition of a competing complexing agent to the mobile phase. However, the results obtained the proteins containing metal binding groups suggested that, in addition to the presence of donor-acceptor interactions between the macromolecules and the immobilized metal, other additional (essentially ionic and/or hydrophobic) interactions took place between the proteins and the surrounding of the metal. When donor-acceptor interactions were predominant, proteins were strongly adsorbed on the stationary phase and their elution required the addition of a competing complexing agent in the mobile phase. However, when the binding between the proteins and the supports via donor-acceptor interactions was less favourable, proteins were eluted from the columns without the addition of a competing agent in the mobile phase. With respect to the binding of these proteins, ionic and/or hydrophobic interactions were no longer negligible during the chromatographic process and the retention of the macromolecules by the stationary phase depended on the elution conditions (ionic strength, pH, etc.). These supports were used in the fractionation of the three main genetic variants of desialylated alpha 1-acid glycoprotein.

  3. Whole-body valine and cysteine kinetics and tissue fractional protein synthesis rates in lambs fed Sulla (Hedysarum coronarium) and infected or not infected with adult Trichostrongylus colubriformis.

    Science.gov (United States)

    Bermingham, Emma N; McNabb, Warren C; Sutherland, Ian A; Sinclair, Bruce R; Treloar, Bryan P; Roy, Nicole C

    2006-07-01

    Poor growth during parasitic infection may be due to a redistribution of amino acids away from skeletal muscle protein synthesis to the intestinal site of infection. The effect of a Trichostrongylus colubriformis infection on whole-body amino acid kinetics and tissue fractional protein synthesis rates were determined in lambs fed fresh Sulla (Hedysarum coronarium; 800 g DM/d). Lambs were dosed with 6000 L3 Trichostrongylus colubriformis larvae daily for 6 d (n 6) or kept as parasite-free controls (n 6). On day 45 post-infection, the lambs received an intravenous injection of 2H2O and infusions (8 h) of [35S]sulphate to measure the size of the whole-body water and sulphate pools, respectively. On day 48, the lambs were continuously infused for 8 h with [3,4-3H]valine into the jugular vein as well as with [1-13C]valine and [35S]cysteine into the abomasum. After the 8 h infusions, the lambs were killed and tissue samples collected from the duodenum, ileum, mesenteric lymph nodes, liver, spleen, thymus, muscle and skin. Feed intake (769 v. 689 (sd 47) g DM/d) was not affected by infection, whereas liveweight gains (50 v. -50 (sd 70) g/d) were lower and intestinal worm burdens (240 v. 18,000 (sd 7000) worms) higher in the infected lambs. Parasitic infection increased the fractional protein synthesis rates in the small intestine, mesenteric lymph nodes and liver but did not affect skin and skeletal muscle fractional protein synthesis rates during the established parasitic infection.

  4. The influence of protein fractions and electrolyte imbalance on refractive index of serum in patients with multiple myeloma

    Science.gov (United States)

    Plotnikova, L.; Polyanichko, A.; Kobeleva, M.; Uspenskaya, M.; Garifullin, A.; Voloshin, S.

    2017-01-01

    Refractometric analysis is very rapid, accurate and simple method of analysis measuring refractive index of biological liquids such as serum, plasma, spinal fluid, urine. This method can be used for definition total protein and solids concentrations in serum. The value of refractive index depends on all substances in serum including proteins, lipids as well as low molecular weight compounds, for example ions of different metals. Refractometric analysis shows strong correlations between protein concentrations in serum of patients with multiple myeloma and its(serum) refractive index which depends on protein concentration and doesn’t depends on electrolyte disbalance.

  5. Functional inactivation of a fraction of excitatory synapses in mice deficient for the active zone protein bassoon

    DEFF Research Database (Denmark)

    Altrock, Wilko D; tom Dieck, Susanne; Sokolov, Maxim;

    2003-01-01

    Mutant mice lacking the central region of the presynaptic active zone protein Bassoon were generated to establish the role of this protein in the assembly and function of active zones as sites of synaptic vesicle docking and fusion. Our data show that the loss of Bassoon causes a reduction in nor...

  6. Fractional Echoes

    CERN Document Server

    Karras, G; Billard, F; Lavorel, B; Siour, G; Hartmann, J -M; Faucher, O; Gershnabel, Erez; Prior, Yehiam; Averbukh, Ilya Sh

    2016-01-01

    We report the observation of fractional echoes in a double-pulse excited nonlinear system. Unlike standard echoes which appear periodically at delays which are integer multiple of the delay between the two exciting pulses, the fractional echoes appear at rational fractions of this delay. We discuss the mechanism leading to this phenomenon, and provide the first experimental demonstration of fractional echoes by measuring third harmonic generation in a thermal gas of CO2 molecules excited by a pair of femtosecond laser pulses.

  7. FRACTIONAL BANKING

    OpenAIRE

    Maria Klimikova

    2010-01-01

    Understanding the reasons of the present financial problems lies In understanding the substance of fractional reserve banking. The substance of fractional banking is in lending more money than the bankers have. Banking of partial reserves is an alternative form which links deposit banking and credit banking. Fractional banking is causing many unfavorable economic impacts in the worldwide system, specifically an inflation.

  8. FRACTIONAL BANKING

    OpenAIRE

    Maria Klimikova

    2010-01-01

    Understanding the reasons of the present financial problems lies In understanding the substance of fractional reserve banking. The substance of fractional banking is in lending more money than the bankers have. Banking of partial reserves is an alternative form which links deposit banking and credit banking. Fractional banking is causing many unfavorable economic impacts in the worldwide system, specifically an inflation.

  9. Fractional randomness

    Science.gov (United States)

    Tapiero, Charles S.; Vallois, Pierre

    2016-11-01

    The premise of this paper is that a fractional probability distribution is based on fractional operators and the fractional (Hurst) index used that alters the classical setting of random variables. For example, a random variable defined by its density function might not have a fractional density function defined in its conventional sense. Practically, it implies that a distribution's granularity defined by a fractional kernel may have properties that differ due to the fractional index used and the fractional calculus applied to define it. The purpose of this paper is to consider an application of fractional calculus to define the fractional density function of a random variable. In addition, we provide and prove a number of results, defining the functional forms of these distributions as well as their existence. In particular, we define fractional probability distributions for increasing and decreasing functions that are right continuous. Examples are used to motivate the usefulness of a statistical approach to fractional calculus and its application to economic and financial problems. In conclusion, this paper is a preliminary attempt to construct statistical fractional models. Due to the breadth and the extent of such problems, this paper may be considered as an initial attempt to do so.

  10. Fractional enrichment of proteins using [2-{sup 13}C]-glycerol as the carbon source facilitates measurement of excited state {sup 13}Cα chemical shifts with improved sensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Ahlner, Alexandra; Andresen, Cecilia; Khan, Shahid N. [Linköping University, Division of Chemistry, Department of Physics, Chemistry and Biology (Sweden); Kay, Lewis E. [The University of Toronto, Departments of Molecular Genetics, Biochemistry and Chemistry, One King’s College Circle (Canada); Lundström, Patrik, E-mail: patlu@ifm.liu.se [Linköping University, Division of Chemistry, Department of Physics, Chemistry and Biology (Sweden)

    2015-07-15

    A selective isotope labeling scheme based on the utilization of [2-{sup 13}C]-glycerol as the carbon source during protein overexpression has been evaluated for the measurement of excited state {sup 13}Cα chemical shifts using Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion (RD) experiments. As expected, the fractional incorporation of label at the Cα positions is increased two-fold relative to labeling schemes based on [2-{sup 13}C]-glucose, effectively doubling the sensitivity of NMR experiments. Applications to a binding reaction involving an SH3 domain from the protein Abp1p and a peptide from the protein Ark1p establish that accurate excited state {sup 13}Cα chemical shifts can be obtained from RD experiments, with errors on the order of 0.06 ppm for exchange rates ranging from 100 to 1000 s{sup −1}, despite the small fraction of {sup 13}Cα–{sup 13}Cβ spin-pairs that are present for many residue types. The labeling approach described here should thus be attractive for studies of exchanging systems using {sup 13}Cα spin probes.

  11. FRACIONAMENTO DE PROTEÍNAS DE SILAGEM DE CAPIM-ELEFANTE EMURCHECIDO OU COM FARELO DE CACAU PROTEIN FRACTIONING OF SILAGE OF ELEPHANTGRASS WILTED OR WITH COCOA MEAL

    Directory of Open Access Journals (Sweden)

    Gleidson Giordano Pinto de Carvalho

    2008-10-01

    Full Text Available

    Desenvolveu-se o experimento para determinar as frações que compõem as proteínas da silagem de capim-elefante (Pennisetum purpureum Schum. cv. Camaroon submetido ao emurchecimento ou à adição de diferentes níveis de farelo de cacau. O capim-elefante utilizado foi colhido aos 50 dias de rebrota após o corte de uniformização e submetido aos seguintes tratamentos: capim-elefante emurchecido ao sol por oito horas, e capim-elefante sem emurchecimento com níveis de 0 %, 7 %, 14%, 21 % e 28 % de farelo de cacau (FC (% da matéria natural. Acondicionou-se o material em silos de PVC com capacidade para 5,3 litros, que foram abertos após 45 dias. Para todas as frações de proteínas estimadas, o tratamento emurchecido apresentou valores semelhantes (P>0,05 ao do tratamento sem emurchecimento. As frações protéicas foram influenciadas pelas adições de FC, verificando-se redução dos teores das frações A e B1+B2 e aumentos das frações B3 e C, para os níveis crescentes de FC.

    PALAVRAS-CHAVES: Conservação de forragens, forrageira, Pennisetum purpureum Schum. cv. Cameroon, subproduto, Theobroma cacao L.

    The experiment was conducted to determine the fractions that compose the protein of silage on the submitted elephant grass forage to wilting under the sun light for eight hours. Other treatments involved the same elephant grass without exposing to sun light but with addition of 0, 7, 14, 21, and 28% of cocoa meal (CM at the ensilage processing. The PVC silos used in the experiment were 5.3 liters in capacity, and were opened in 45 days. To all protein-estimated fractions, the wilted treatment showed similar values (P>.05 to the treatment without wilting. The protein fractions were influenced by CM addictions, verifying reduction in contents of A and B1+B2 fractions and increase in B3 and C fractions, with CM increasing levels

  12. Alterations in mitochondria and sarcoplasmic reticulum from heart and skeletal muscle of horizontally casted primates

    Science.gov (United States)

    Sordahl, L. A.; Stone, H. L.

    1982-01-01

    Horizontally body-casted rhesus monkeys are used as an animal model in order to study the physiological changes known as cardiovascular deconditioning which occur during weightless conditions. No difference was found between the experimental and control animals in heart mitochondrial oxidative phosphorylation which indicates that no apparent changes occurred in the primary energy-producing system of the heart. A marked increase in cytochrome oxidase activity was observed in the casted primate heart mitochondria compared to controls, while a 25% decrease in respiratory substrate-supported calcium uptake was found in casted primate heart mitochondria compared to controls. Sacroplasmic reticulum isolated from the primate hearts revealed marked changes in calcium transport activities. It is concluded that the marked depression in cardiac sarcoplasmic reticulum functions indicates altered calcium homeostasis in the casted-primate heart which could be a factor in cardiovascular deconditioning.

  13. Taurine protects cardiac contractility in killifish, Fundulus heteroclitus, by enhancing sarcoplasmic reticular Ca(2+) cycling.

    Science.gov (United States)

    Henry, Elenor F; MacCormack, Tyson J

    2017-05-23

    Intracellular taurine is abundant in many animals and it influences an array of physiological processes, including osmoregulation, metabolism, and cardiac contractility. Taurine is an important osmolyte in teleost hearts, but its role in stress tolerance, cardiac metabolism, and contractility has not been assessed. The goal of this study was to determine if ventricular taurine concentration changes in response to environmental stress and to characterize its influence on contractility. Cardiac taurine concentrations varied in killifish (Fundulus heteroclitus) but were generally maintained following acute environmental challenges. In isometrically contracting ventricular strips, supplemental taurine (40 mmol L(-1)) protected peak tension development (F max) at high stimulation frequencies, an effect abolished by treatment with ryanodine, a blocker of sarcoplasmic reticulum Ca(2+) release. In the presence of ryanodine, taurine-treated preparations were also better able to maintain F max at supraphysiological extracellular Ca(2+) levels, but a prior anoxia exposure abolished this effect. Taurine had no impact on basal F max during or after anoxia, but it provided additive protection to high-frequency contractility post-anoxia. Tissue oxygen consumption and extracellular glucose utilization were unaffected by taurine in non-contracting preparations, indicating that it does not impact energy metabolism. Overall, the results suggest that cardiac taurine levels are well maintained on acute time scales in this highly stress-tolerant species. Supplemental taurine has no effect on aerobic metabolism in vitro, but it significantly improved cardiac contractility in a manner dependent upon sarcoplasmic reticulum Ca(2+) cycling. The data indicate that taurine likely plays an important role in the regulation of cardiac performance in teleosts.

  14. Formation of mono(ethylhexyl)phthalate from di(ethylhexyl)phthalate in human plasma stored in PVC bags and its presence in fractionated plasma proteins.

    Science.gov (United States)

    Vessman, J; Rietz, G

    1978-01-01

    The formation of mono(ethylhexyl)phthalate (MEHP) from di(ethylhexyl)phthalate in human plasma stored in bags of polyvinylchloride has been studied. Substantial amounts were formed and in ten bags from 4 to 56microgram/ml were found. After 2 weeks at room temperature the concentration of MEHP had increased to values between 27 and 79 microgram/ml. However, MEHP was also disappearing as shown in a recovery experiment. Of the fractionated proteins albumin contained MEHP in amounts from less than 3 to 290 microgram/g.

  15. Sarcoplasmic reticulum calcium release compared in slow-twitch and fast-twitch fibres of mouse muscle.

    Science.gov (United States)

    Baylor, S M; Hollingworth, S

    2003-08-15

    Experiments were carried out to compare the amplitude and time course of Ca2+ release from the sarcoplasmic reticulum (SR) in intact slow-twitch and fast-twitch mouse fibres. Individual fibres within small bundles were injected with furaptra, a low-affinity, rapidly responding Ca2+ indicator. In response to a single action potential at 16 degrees C, the peak amplitude and half-duration of the change in myoplasmic free [Ca2+] (Delta[Ca2+]) differed significantly between fibre types (slow-twitch: peak amplitude, 9.4 +/- 1.0 microM (mean +/- S.E.M.); half-duration, 7.7 +/- 0.6 ms; fast-twitch: peak amplitude 18.5 +/- 0.5 microM; half-duration, 4.9 +/- 0.3 ms). SR Ca2+ release was estimated from Delta[Ca2+] with a computational model that calculated Ca2+ binding to the major myoplasmic Ca2+ buffers (troponin, ATP and parvalbumin); buffer concentrations and reaction rate constants were adjusted to reflect fibre-type differences. In response to an action potential, the total concentration of released Ca2+ (Delta[CaT]) and the peak rate of Ca2+ release ((d/dt)Delta[CaT]) differed about 3-fold between the fibre types (slow-twitch: Delta[CaT], 127 +/- 7 microM; (d/dt)Delta[CaT], 70 +/- 6 microM ms-1; fast-twitch: Delta[CaT], 346 +/- 6 microM; (d/dt)Delta[CaT], 212 +/- 4 microM ms-1). In contrast, the half-duration of (d/dt)Delta[CaT] was very similar in the two fibre types (slow-twitch, 1.8 +/- 0.1 ms; fast-twitch, 1.6 +/- 0.0 ms). When fibres were stimulated with a 5-shock train at 67 Hz, the peaks of (d/dt)Delta[CaT] in response to the second and subsequent shocks were much smaller than that due to the first shock; the later peaks, expressed as a fraction of the amplitude of the first peak, were similar in the two fibre types (slow-twitch, 0.2-0.3; fast-twitch, 0.1-0.3). The results support the conclusion that individual SR Ca2+ release units function similarly in slow-twitch and fast-twitch mammalian fibres.

  16. Fractional thermoelasticity

    CERN Document Server

    Povstenko, Yuriy

    2015-01-01

    This book is devoted to fractional thermoelasticity, i.e. thermoelasticity based on the heat conduction equation with differential operators of fractional order. Readers will discover how time-fractional differential operators describe memory effects and space-fractional differential operators deal with the long-range interaction. Fractional calculus, generalized Fourier law, axisymmetric and central symmetric problems and many relevant equations are featured in the book. The latest developments in the field are included and the reader is brought up to date with current research.  The book contains a large number of figures, to show the characteristic features of temperature and stress distributions and to represent the whole spectrum of order of fractional operators.  This work presents a picture of the state-of-the-art of fractional thermoelasticity and is suitable for specialists in applied mathematics, physics, geophysics, elasticity, thermoelasticity and engineering sciences. Corresponding sections of ...

  17. Proteome-wide muscle protein fractional synthesis rates predict muscle mass gain in response to a selective androgen receptor modulator in rats.

    Science.gov (United States)

    Shankaran, Mahalakshmi; Shearer, Todd W; Stimpson, Stephen A; Turner, Scott M; King, Chelsea; Wong, Po-Yin Anne; Shen, Ying; Turnbull, Philip S; Kramer, Fritz; Clifton, Lisa; Russell, Alan; Hellerstein, Marc K; Evans, William J

    2016-03-15

    Biomarkers of muscle protein synthesis rate could provide early data demonstrating anabolic efficacy for treating muscle-wasting conditions. Androgenic therapies have been shown to increase muscle mass primarily by increasing the rate of muscle protein synthesis. We hypothesized that the synthesis rate of large numbers of individual muscle proteins could serve as early response biomarkers and potentially treatment-specific signaling for predicting the effect of anabolic treatments on muscle mass. Utilizing selective androgen receptor modulator (SARM) treatment in the ovariectomized (OVX) rat, we applied an unbiased, dynamic proteomics approach to measure the fractional synthesis rates (FSR) of 167-201 individual skeletal muscle proteins in triceps, EDL, and soleus. OVX rats treated with a SARM molecule (GSK212A at 0.1, 0.3, or 1 mg/kg) for 10 or 28 days showed significant, dose-related increases in body weight, lean body mass, and individual triceps but not EDL or soleus weights. Thirty-four out of the 94 proteins measured from the triceps of all rats exhibited a significant, dose-related increase in FSR after 10 days of SARM treatment. For several cytoplasmic proteins, including carbonic anhydrase 3, creatine kinase M-type (CK-M), pyruvate kinase, and aldolase-A, a change in 10-day FSR was strongly correlated (r(2) = 0.90-0.99) to the 28-day change in lean body mass and triceps weight gains, suggesting a noninvasive measurement of SARM effects. In summary, FSR of multiple muscle proteins measured by dynamics of moderate- to high-abundance proteins provides early biomarkers of the anabolic response of skeletal muscle to SARM.

  18. Validation of the flooding dose technique to determine fractional rates of protein synthesis in a model bivalve species, the blue mussel (Mytilus edulis L.).

    Science.gov (United States)

    McCarthy, Ian D; Nicholls, Ruth; Malham, Shelagh K; Whiteley, Nia M

    2016-01-01

    For the first time, use of the flooding dose technique using (3)H-Phenylalanine is validated for measuring whole-animal and tissue-specific rates of protein synthesis in the blue mussel Mytilus edulis (61mm shell length; 4.0g fresh body mass). Following injection, the phenylalanine-specific radioactivities in the gill, mantle and whole-animal free pools were elevated within one hour and remained elevated and stable for up to 6h following injection of (3)H-phenylalanine into the posterior adductor muscle. Incorporation of (3)H-phenylalanine into body protein was linear over time following injection and the non-significant intercepts for the regressions suggested incorporation into body protein occurred rapidly after injection. These results validate the technique for measuring rates of protein synthesis in mussels. There were no differences in the calculated rates following 1-6h incubation in gill, mantle or whole-animal and fractional rates of protein synthesis from the combined time course data were 9.5±0.8%d(-1) for the gill, 2.5±0.3%d(-1) for the mantle and 2.6±0.3%d(-1) for the whole-animal, respectively (mean values±SEM). The whole-animal absolute rate of protein synthesis was calculated as 18.9±0.6mg protein day(-1). The use of this technique in measuring one of the major components of maintenance metabolism and growth will provide a valuable and convenient tool in furthering our understanding of the protein metabolism and energetics of this keystone marine invertebrate and its ability to adjust and respond to fluctuations, such as that expected as a result of climate change.

  19. Short-term effects of β2-AR blocker ICI 118,551 on sarcoplasmic reticulum SERCA2a and cardiac function of rats with heart failure.

    Science.gov (United States)

    Gong, Haibin; Li, Yanfei; Wang, Lei; Lv, Qian; Wang, Xiuli

    2016-09-01

    The study was conducted to examine the effects of ICI 118,551 on the systolic function of cardiac muscle cells of rats in heart failure and determine the molecular mechanism of selective β2-adrenergic receptor (β2-AR) antagonist on these cells. The chronic heart failure model for rats was prepared through abdominal aortic constriction and separate cardiac muscle cells using the collagenase digestion method. The rats were then divided into Sham, HF and HF+ICI 50 nM goups and cultivated for 48 h. β2-AR, Gi/Gs and sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) protein expression levels in the cardiac muscle cells were evaluated by western blotting and changes in the systolic function of cardiac muscle cells based on the boundary detection system of contraction dynamics for individual cells was measured. The results showed that compared with the Sham group, the survival rate, percentage of basic contraction and maximum contraction amplitude percentage of cardiac muscle cells with heart failure decreased, Gi protein expression increased while Gs and SERCA2a protein expression decreased. Compared with the HF group, the maximum contraction amplitude percentage of cardiac muscle cells in group HF+ICI 50 nM decreased, the Gi protein expression level increased while the SERCA2a protein expression level decreased. Following the stimulation of Ca(2+) and ISO, the maximum contraction amplitude percentage of cardiac muscle cells in the HF+ICI 50 nM group was lower than that in group HF. This indicated that ICI 118,551 has negative inotropic effects on cardiac muscle cells with heart failure, which may be related to Gi protein. Systolic function of cardiac muscle cells with heart failure can therefore be reduced by increasing Gi protein expression and lowering SERCA2a protein expression.

  20. Adult Opisthorchis felineus major protein fractions deduced from transcripts: comparison with liver flukes Opisthorchis viverrini and Clonorchis sinensis.

    Science.gov (United States)

    Pomaznoy, Mikhail; Tatkov, Sergey; Katokhin, Alexey; Afonnikov, Dmitry; Babenko, Vladimir; Furman, Dagmara; Brusentsov, Ilya; Belavin, Pavel; Najakshin, Alexandr; Guselnikov, Sergey; Vasiliev, Gennady; Sivkov, Anton; Prokhortchouk, Egor; Skryabin, Konstantin; Mordvinov, Viatcheslav

    2013-10-01

    The epidemiologically important liver flukes Opisthorchis felineus, Opisthorchis viverrini, and Clonorchis sinensis are of interest to health professionals, epidemiologists, pharmacologists, and molecular biologists. Recently the transcriptomes of the latter two species were intensively investigated. However our knowledge on molecular biology of O. felineus is scarce. We report the first results of the O. felineus transcriptome analysis. We isolated and annotated a total of 2560 expressed sequence tag (EST) sequences from adult O. felineus (deposited within the database of expressed sequence tags (dbEST), under accession numbers GenBank: JK624271-JK626790, JK006511-JK006547, JK649790-JK649792). Clustering and analysis resulted in the detection of 267 contigs. Of the protein sequences deduced from these, 82% had homologs in the NCBI (nr) protein database and 63% contained conserved domains, allowing the functions to be interpreted using the Gene Ontology terms. Comprehensive analysis of Opisthorchiidae- and Trematoda-specific substitutions within amino acid sequences deduced for the proteins myoglobin, vitelline precursor protein, cathepsin F, and 28kDa glutathione transferase was carried out. The gene set of the 32 ribosomal proteins for the three Opisthorchiidae species with the addition of available Schistosoma and Fasciola orthologs was created and is provided in the supplementary. The orthologous gene set created was used for inferring phylogeny within the Trematoda with special attention to interrelations within the Opisthorchiidae. The phylogenetic analysis revealed a closer relationship between C. sinensis and O. viverrini and some divergence of O. felineus from either O. viverrini or C. sinensis.

  1. Membrane fractionation of herring marinade for separation and recovery of fats, proteins, amino acids, salt, acetic acid and water

    DEFF Research Database (Denmark)

    Fjerbæk Søtoft, Lene; Lizarazu, Juncal Martin; Razi Parjikolaei, Behnaz;

    2015-01-01

    containing sugars, amino acids and smaller peptides and a NF permeate containing salt and acetic acid ready for reuse. 42% of the spent marinade is recovered to substitute fresh water and chemicals. The Waste water amount is reduced 62.5%. Proteins are concentrated 30 times, while amino acids and smaller......In the production of marinated herring, nearly one ton of acidic saline marinade is produced per 1.5 tons herring fillet. This spent marinade contains highly valuable compounds such as proteins and amino acids. Membranes are suited to recover these substances. In this work, six membrane stages...... are employed: microfiltration (MF) (0.2 lm), ultrafiltration (UF) (50, 20, 10 and 1 kDa) and nanofiltration (NF). The most promising stages are 50 kDa UF and NF based on SDS–PAGE analyses and total amino acid concentration. The 50 kDa stage produces a protein concentrate (>17 kDa). NF produces a retentate...

  2. Comparison of the kinetics of calcium transport in vesicular dispersions and oriented multilayers of isolated sarcoplasmic reticulum membranes.

    Science.gov (United States)

    Pierce, D H; Scarpa, A; Trentham, D R; Topp, M R; Blasie, J K

    1983-12-01

    Knowledge of the functional properties of the protein in oriented multilayers, in addition to vesicular dispersions, of membranes such as the isolated sarcoplasmic reticulum (SR), extends the variety of techniques that can be effectively used in studies of the membrane protein's structure or structural changes associated with its function. One technique requiring the use of oriented multilayers to provide more direct time-averaged and time-resolved structural investigations of the SR membrane is x-ray diffraction. Therefore, the kinetics of ATP-induced calcium uptake by isolated SR vesicles in dispersions and hydrated, oriented multilayers were compared. Ca2+ uptake was necessarily initiated by the addition of ATP through flash photolysis of caged ATP, P3-1-(2-nitro)phenylethyl adenosine 5'-triphosphate, with either a frequency-doubled ruby laser or a 200 W Hg arc lamp, and measured with two different detector systems that followed the absorbance changes of the metallochromic indicator arsenazo III, which is sensitive to changes in the extravesicular [Ca2+]. The temperature range investigated was -2 degrees to 26 degrees C. The Ca2+ uptake kinetics of SR membranes in both the vesicular dispersions and oriented multilayers consist of at least two phases, an initial fast phase and a subsequent slow phase. The fast phase, generally believed to be associated with the formation of the phosphorylated enzyme, E approximately P, is kinetically comparable in both SR dispersions and multilayers. The slow phase mathematically follows first-order kinetics with specific rate constants of approximately 0.6 s-1 and approximately 1.2 s-1 for the dispersions at 26 degrees C and multilayers at 21 degrees C, respectively, with the given experimental conditions. The slow phase, generally believed to be associated with the translocation of Ca+2, across the membrane profile, appears to be the same process in SR dispersions and multilayers through their virtually identical rate constants

  3. Influence of black gram (Vigna mungo) trypsin inhibitory fraction on the hepatic protein catabolism in male albino mice.

    Science.gov (United States)

    Kamalakannan, V; Sathyamoorthy, A V; Motlag, D B

    1984-01-01

    The effect of black gram and black gram trypsin inhibitor on the protein catabolism of male albino mice has been investigated. Group 1 was given autoclaved black gram (control), Group II raw black gram and Group III the autoclaved black gram incorporated with 1% black gram trypsin inhibitor. Blood as well as urinary urea and creatine were found to be elevated in Groups II and III. Increased levels of arginase, ornithine transcarbamylase and transaminases were noted in Groups II and III. The results suggested an enhanced catabolism of proteins evoked by the native black gram trypsin inhibitor.

  4. Immunological evaluation of the alcohol-soluble protein fraction from gluten-free grains in relation to celiac disease.

    Science.gov (United States)

    Bergamo, Paolo; Maurano, Francesco; Mazzarella, Giuseppe; Iaquinto, Gaetano; Vocca, Immacolata; Rivelli, Anna Rita; De Falco, Enrica; Gianfrani, Carmen; Rossi, Mauro

    2011-08-01

    Celiac disease (CD) is a gluten-sensitive enteropathy with an immune basis. We established the immune reactivity of the alcohol-soluble fraction from two minor cereals (tef and millet) and two pseudocereals (amaranth and quinoa) which are believed to be nontoxic based on taxonomy. Grains were examined in intestinal T-cell lines (iTCLs), cultures of duodenal explants from HLA-DQ2(+) CD patients and HLA-DQ8 transgenic mice for signs of activation. Our data indicated that tef, millet, amaranth, and quinoa did not show any immune cross-reactivity toward wheat gliadin, and therefore confirming their safety in the diet of CD patients. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. In vitro availability of iron and zinc: Effects of the type, concentration and fractions of digestion products of the protein

    NARCIS (Netherlands)

    Pérez-Llamas, F.; Diepenmaat-Wolters, M.G.E.; Zamora, S.

    1996-01-01

    An in vitro dialysis method was employed to determine the effect on the Fe and Zn absorption of the type (beef, pork and soyabean) and the amount (10 and 30 g/kg) of protein present. In addition, the effects of low- and high-molecular-weight (LMW and HMW respectively) digestion products were investi

  6. Fracionamento dos carboidratos e proteínas de silagens de milho, sorgo e girassol Fractionation of carbohydrate and protein of corn, sorghum and sunflower silages

    Directory of Open Access Journals (Sweden)

    Renius Mello

    2004-10-01

    Full Text Available O trabalho caracteriza e quantifica as frações dos carboidratos e proteínas de silagens de milho, sorgo e girassol, com a finalidade de disponibilizar dados bromatológicos que possibilitem maximizar o aproveitamento desses alimentos e otimizar o desempenho animal. Foram avaliados dois híbridos de milho (Zea mays, DKB-215 e DKB-344, dois híbridos de sorgo (Sorghum bicolor, Ambar e AG-2005 e dois híbridos de girassol (Helianthus annuus, Rumbosol e M-734. A silagem de girassol apresentou menor valor de carboidratos totais (CT e B2 (celulose e hemicelulose e maior de C (lignina e fibra associada à lignina. A silagem de milho apresentou maior valor de carboidratos não-fibrosos (CNF, A+B1 (açúcares solúveis + amido e pectina e de CT juntamente com a silagem de sorgo, enquanto a de sorgo apresentou maior valor de B2 em função da maior contribuição de colmo. Houve diferença entre híbridos dentro da cultura do girassol para CT, sendo que o Rumbosol obteve maior valor que o M-734, em razão da aptidão dos mesmos, forrageiro e granífero respectivamente. A silagem de girassol apresentou maior valor de proteína bruta (PB e de suas frações. Não foi observada diferença entre híbridos nos valores de PB e de suas frações.This work evaluates and characterizes the carbohydrates and proteins fractions of corn, sorghum and sunflower silages. The purpose was to supply composition data that make it possible to maximize use of foods and optimize animal performance. Two corn (Zea mays hybrids, DKB-215 and DKB-344; sorghum (Sorghum bicolor hybrids, Ambar and AG-2005; and sunflower (Helianthus annuus hybrids, Rumbosol and M-734; were evaluated. Sunflower silage showed the lowest total carbohydrates (TC and B2 (cellulose and hemicelluloses values and the highest content of C (lignin and fiber associated lignin. Corn silages showed higher non-fiber carbohydrates (NFC, A+B1 (soluble sugars + starch and pectin and TC together sorghum silage while

  7. Determination of the ATP Affinity of the Sarcoplasmic Reticulum Ca(2+)-ATPase by Competitive Inhibition of [γ-(32)P]TNP-8N3-ATP Photolabeling.

    Science.gov (United States)

    Clausen, Johannes D; McIntosh, David B; Woolley, David G; Andersen, Jens Peter

    2016-01-01

    The photoactivation of aryl azides is commonly employed as a means to covalently attach cross-linking and labeling reagents to proteins, facilitated by the high reactivity of the resultant aryl nitrenes with amino groups present in the protein side chains. We have developed a simple and reliable assay for the determination of the ATP binding affinity of native or recombinant sarcoplasmic reticulum Ca(2+)-ATPase, taking advantage of the specific photolabeling of Lys(492) in the Ca(2+)-ATPase by [γ-(32)P]2',3'-O-(2,4,6-trinitrophenyl)-8-azido-adenosine 5'-triphosphate ([γ-(32)P]TNP-8N3-ATP) and the competitive inhibition by ATP of the photolabeling reaction. The method allows determination of the ATP affinity of Ca(2+)-ATPase mutants expressed in mammalian cell culture in amounts too minute for conventional equilibrium binding studies. Here, we describe the synthesis and purification of the [γ-(32)P]TNP-8N3-ATP photolabel, as well as its application in ATP affinity measurements.

  8. Milk digesta and milk protein fractions influence the adherence of Lactobacillus gasseri R and Lactobacillus casei FMP to human cultured cells.

    Science.gov (United States)

    Volstatova, Tereza; Havlik, Jaroslav; Potuckova, Miroslava; Geigerova, Martina

    2016-08-10

    Adhesion to the intestinal epithelium is considered an important feature of probiotic bacteria, which may increase their persistence in the intestine, allowing them to exert their beneficial health effect or promote the colonisation process. However, this feature might be largely dependent on the host specificity or diet. In the present study, we investigated the effect of selected milks and milk protein fractions on the ability of selected lactobacilli to adhere to the cells of an intestinal model based on co-culture Caco-2/HT29-MTX cell lines. Most milk digesta did not significantly affect bacterial adhesion except for UHT-treated milk and sheep milk. The presence of UHT-treated milk digesta reduced the adhesion of Lactobacillus gasseri R by 61% but not that of Lactobacillus casei FMP. However, sheep milk significantly increased the adherence of L. casei FMP (P < 0.05) but not of L. gasseri R. Among the protein fractions, rennet casein (RCN) and bovine serum albumin (BSA) showed reproducible patterns and strain-specific effects on bacterial adherence. While RCN reduced the adherence of L. gasseri R to <50% compared to the control, it did not have a significant effect on L. casei FMP. In contrast, BSA reduced L. casei FMP adherence to a higher extent than that of L. gasseri R. Whey protein (WH) tended to increase the adherence of both strains by 130%-180%. Recently, interactions between the host diet and its microbiota have attracted considerable interest. Our results may explain one of the aspects of the role of milk in the development of microbiota or support of probiotic supplements. Based on our data, we conclude that the persistence of probiotic strains supplemented as part of dairy food or constitutional microbiota in the gut might be affected negatively or positively by the food matrix through complex strain or concentration dependent effects.

  9. Disturbances of the sarcoplasmic reticulum and transverse tubular system in 24-h electrostimulated fast-twitch skeletal muscle

    DEFF Research Database (Denmark)

    Frías, J A; Cadefau, J A; Prats, C;

    2005-01-01

    -migration of terminal cisternae and t-tubules from R3 to R4, indicating the presence of triads. This density change may be associated with calcium overload of the sarcoplasmic reticulum, since total calcium rose three- to fourfold in stimulated muscle homogenates. These changes correlate well with ultrastructural...... damage to longitudinal sarcoplasmic reticulum and swelling of t-tubules revealed by electron microscopy. The ultrastructural changes observed here reflect exercise-induced damage of membrane systems that might severely compromise muscle function. Since this process is reversible, we suggest that it may......Chronic low-frequency stimulation of rabbit tibialis anterior muscle over a 24-h period induces a conspicuous loss of isometric tension that is unrelated to muscle energy metabolism (J.A. Cadefau, J. Parra, R. Cusso, G. Heine, D. Pette, Responses of fatigable and fatigue-resistant fibres of rabbit...

  10. Biphasic decay of the Ca transient results from increased sarcoplasmic reticulum Ca leak

    Science.gov (United States)

    Sankaranarayanan, Rajiv; Li, Yatong; Greensmith, David J.; Eisner, David A.

    2016-01-01

    Key points Ca leak from the sarcoplasmic reticulum through the ryanodine receptor (RyR) reduces the amplitude of the Ca transient and slows its rate of decay.In the presence of β‐adrenergic stimulation, RyR‐mediated Ca leak produces a biphasic decay of the Ca transient with a fast early phase and a slow late phase.Two forms of Ca leak have been studied, Ca‐sensitising (induced by caffeine) and non‐sensitising (induced by ryanodine) and both induce biphasic decay of the Ca transient.Only Ca‐sensitising leak can be reversed by traditional RyR inhibitors such as tetracaine.Ca leak can also induce Ca waves. At low levels of leak, waves occur. As leak is increased, first biphasic decay and then slowed monophasic decay is seen. The level of leak has major effects on the shape of the Ca transient. Abstract In heart failure, a reduction in Ca transient amplitude and contractile dysfunction can by caused by Ca leak through the sarcoplasmic reticulum (SR) Ca channel (ryanodine receptor, RyR) and/or decreased activity of the SR Ca ATPase (SERCA). We have characterised the effects of two forms of Ca leak (Ca‐sensitising and non‐sensitising) on calcium cycling and compared with those of SERCA inhibition. We measured [Ca2+]i with fluo‐3 in voltage‐clamped rat ventricular myocytes. Increasing SR leak with either caffeine (to sensitise the RyR to Ca activation) or ryanodine (non‐sensitising) had similar effects to SERCA inhibition: decreased systolic [Ca2+]i, increased diastolic [Ca2+]i and slowed decay. However, in the presence of isoproterenol, leak produced a biphasic decay of the Ca transient in the majority of cells while SERCA inhibition produced monophasic decay. Tetracaine reversed the effects of caffeine but not of ryanodine. When caffeine (1 mmol l−1) was added to a cell which displayed Ca waves, the wave frequency initially increased before waves disappeared and biphasic decay developed. Eventually (at higher caffeine concentrations), the

  11. Immunoblot analysis of protein containing 3-(cystein-S-yl)acetaminophen adducts in serum and subcellular liver fractions from acetaminophen-treated mice.

    Science.gov (United States)

    Pumford, N R; Hinson, J A; Benson, R W; Roberts, D W

    1990-07-01

    The hepatotoxicity of acetaminophen is believed to be mediated by the metabolic activation of acetaminophen to N-acetyl-p-benzoquinone imine which covalently binds to cysteinyl residues on proteins as 3-(cystein-S-yl)acetaminophen adducts. The formation of these adducts in hepatic protein correlates with the hepatotoxicity. In this study, the formation of 3-(cystein-S-yl)acetaminophen adducts in specific cellular proteins was investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detected using affinity-purified antisera specific for 3-(cystein-S-yl)acetaminophen adducts on immunoblots. These techniques were used to investigate the liver 10,000g supernatant and serum from B6C3F1 mice that received hepatotoxic doses of acetaminophen. More than 15 proteins containing 3-(cystein-S-yl)acetaminophen adducts were detected in the liver 10,000g supernatant. The most prominent protein containing 3-(cystein-S-yl)acetaminophen adducts in the hepatic 10,000g supernatant had a relative molecular mass of 55 kDa. Serum proteins containing 3-(cystein-S-yl)acetaminophen adducts had molecular masses similar to those found in the liver 10,000g supernatant (55, 87, and approximately 102 kDa). These data, combined with our previous findings describing the temporal relationship between the appearance of 3-(cystein-S-yl)acetaminophen adducts in protein in the serum and the decrease in the levels of 3-(cystein-S-yl)acetaminophen adducts in protein in the liver, suggested that liver adducts were released into the serum following lysis of hepatocytes. The temporal relationship between the formation of specific adducts and hepatotoxicity in mice following a hepatotoxic dose of acetaminophen was examined using immunoblots of mitochondria, microsomes, cytosol, and plasma membranes. Hepatotoxicity indicated by serum alanine aminotransferase levels was increased at 2 and 4 hr after dosing. The cytosolic fraction contained numerous proteins with 3-(cystein

  12. Fractional motions

    Energy Technology Data Exchange (ETDEWEB)

    Eliazar, Iddo I., E-mail: eliazar@post.tau.ac.il [Holon Institute of Technology, P.O. Box 305, Holon 58102 (Israel); Shlesinger, Michael F., E-mail: mike.shlesinger@navy.mil [Office of Naval Research, Code 30, 875 N. Randolph St., Arlington, VA 22203 (United States)

    2013-06-10

    Brownian motion is the archetypal model for random transport processes in science and engineering. Brownian motion displays neither wild fluctuations (the “Noah effect”), nor long-range correlations (the “Joseph effect”). The quintessential model for processes displaying the Noah effect is Lévy motion, the quintessential model for processes displaying the Joseph effect is fractional Brownian motion, and the prototypical model for processes displaying both the Noah and Joseph effects is fractional Lévy motion. In this paper we review these four random-motion models–henceforth termed “fractional motions” –via a unified physical setting that is based on Langevin’s equation, the Einstein–Smoluchowski paradigm, and stochastic scaling limits. The unified setting explains the universal macroscopic emergence of fractional motions, and predicts–according to microscopic-level details–which of the four fractional motions will emerge on the macroscopic level. The statistical properties of fractional motions are classified and parametrized by two exponents—a “Noah exponent” governing their fluctuations, and a “Joseph exponent” governing their dispersions and correlations. This self-contained review provides a concise and cohesive introduction to fractional motions.

  13. ON-COLUMN ENRICHMENT OF HYDROPHOBIC CYP450 PROTEINS IN HPLC FRACTIONATION OF MOUSE MICROSOMES PRIOR TO PROTEIN DIGESTION AND NANOSPRAY-LC/MSMS ANALYSIS

    Science.gov (United States)

    IntroductionMembrane proteins play crucial role in many cellular processes and are promising candidates for biomarker discovery but are under-represented in the field of proteomics due to their hydrophobic nature. Although standard reversed-phase LC methods often exhibit ...

  14. Antihepatitis B Virus Activity of a Protein-Enriched Fraction from Housefly (Musca domestica in a Stable HBV-Producing Cell Line

    Directory of Open Access Journals (Sweden)

    Xuemei Lu

    2014-01-01

    Full Text Available Hepatitis B virus (HBV infection remains a major public health problem. Although several vaccines and therapeutic strategies are currently being implemented to combat HBV virus, effective antiviral therapy against HBV infection has not been fully developed. Alternative strategies and new drugs to combat this disease are urged. Insects and insect derivatives are a large and unexploited source of potentially useful compounds for modern medicine. In the present study, we investigated the first anti-HBV activity of a protein-enriched fraction (PE from the larvae of the housefly (Musca domestica in a stable HBV-producing cell line. HBsAg and HBeAg in the culture medium were measured by enzyme-linked immunosorbent assay. HBV-DNA was quantified by fluorescent quantification PCR. HBV core protein was assayed by immunofluorescent staining. Results indicate PE treatment inhibited both HBsAg, HBeAg secretion, and HBV-DNA replication. Furthermore, PE could also suppress HBV core protein expression. PE could be a potential candidate for the development of a novel and effective drug for the treatment of HBV infection.

  15. Antihepatitis B virus activity of a protein-enriched fraction from housefly (Musca domestica) in a stable HBV-producing cell line.

    Science.gov (United States)

    Lu, Xuemei; Jin, Xiaobao; Wang, Jie; Chu, Fujiang; Zhu, Jiayong

    2014-01-01

    Hepatitis B virus (HBV) infection remains a major public health problem. Although several vaccines and therapeutic strategies are currently being implemented to combat HBV virus, effective antiviral therapy against HBV infection has not been fully developed. Alternative strategies and new drugs to combat this disease are urged. Insects and insect derivatives are a large and unexploited source of potentially useful compounds for modern medicine. In the present study, we investigated the first anti-HBV activity of a protein-enriched fraction (PE) from the larvae of the housefly (Musca domestica) in a stable HBV-producing cell line. HBsAg and HBeAg in the culture medium were measured by enzyme-linked immunosorbent assay. HBV-DNA was quantified by fluorescent quantification PCR. HBV core protein was assayed by immunofluorescent staining. Results indicate PE treatment inhibited both HBsAg, HBeAg secretion, and HBV-DNA replication. Furthermore, PE could also suppress HBV core protein expression. PE could be a potential candidate for the development of a novel and effective drug for the treatment of HBV infection.

  16. 姜黄素对心力衰竭兔肌浆网钙泵表达的影响%Effects of curcumin on sarcoplasmic reticulum Ca~(2+) -ATPase in rabbits with heart failure

    Institute of Scientific and Technical Information of China (English)

    张艳; 林国生; 包明威; 武欣迎; 王澈; 杨波

    2010-01-01

    目的 探讨姜黄素对心力衰竭(心衰)兔肌浆网钙泵表达的影响.方法 采用主动脉瓣反流联合腹主动脉缩窄制作慢性心衰家兔模型.随机分为心衰姜黄素组、心衰安慰剂组、对照姜黄素组、对照安慰剂组.8周后计算心脏重量与体重比值,观察超微结构,检测肌浆网钙泵mRNA和蛋白的表达水平及活性.结果 心衰姜黄素组和心衰安慰剂组心脏重量与体重比值均大于对照组(P<0.05);且心衰姜黄素组比值小于心衰安慰剂组(P<0.05).电子显微镜显示心衰姜黄素组的心脏超微结构有所改善.心衰姜黄素组和心衰安慰剂组肌浆网钙泵mRNA、蛋白表达及活性均小于对照组(P<0.05),但心衰姜黄素组均显著高于心衰安慰剂组(P<0.05).结论 姜黄素能在mRNA水平和蛋白水平提高心衰家兔肌浆网钙泵的表达,提高肌浆网钙泵的活性,这可能是姜黄素改善心衰的机制之一.%Objective To investigate the effects of curcumin on sarcoplasmic reticulum Ca~(2+)-ATPase in heart failure rabbits.Methods Rabbit heart failure model was made with aortic regurgitation and abdominal aorta constriction and 40 rabbits were randomly divided into 4 groups including:(1) heart failure treated with curcumin;(2) heart failure treated with placebo;(3) healthy control treated with curcumin and (4) healthy control treated with placebo.All rabbits were administrated with curcumin capsules or placebo capsules 100 mg·kg~(-1)·d~(-1),respectively.All groups were sacrificed after eight weeks.Myocardial ultrastructural organization was detected by transmission electron microscope.RT-PCR and Western blot were used to measure the expression of sarcoplasmic reticulum Ca~(2+)-ATPase in mRNA and protein levels,respectively.Malachite green colorimetric assay was used to evaluate the activity of sarcoplasmic reticulum Ca~(2+) -ATPase.Results All detected parameters were similar between control curcumin group and control placebo

  17. Delineation of pulmonary airway fluid protein fractions with HRPO binding-avidity by far-Western ligand blot and mass spectrometry analyses: a model methodology for detecting mannose-binding protein expression profiles.

    Science.gov (United States)

    Coyne, Cody P; Rashmir-Raven, Ann; Jones, Toni; Mochal, Cathleen; Linford, Robert L; Brashier, Michael; Eddy, Alison

    2009-01-01

    Limited research to date has characterized the potential for HRPO to function as a primary molecular probe. Pulmonary airway fluid was developed by non-reducing far-Western (ligand) blot analyses utilizing conjugated HRPO-strepavidin or non-conjugated HRPO without the presence of primary immunoglobulin. Endogenous esterase-like biochemical activity of fractions within pulmonary airway fluid was inactivated to determine if they were capable of biochemically converting HRPO chemiluminescent substrate. Complementary analyses modified pulmonary fluid and HRPO with beta-galactosidase and alpha-mannosidase respectively, in addition to determining the influence of mannose and maltose competitive binding on HRPO far-Western (ligand) blot analyses. Identification of pulmonary fluid fractions detected by HRPO far-Western blot analyses was determined by mass spectrometry. Modification of pulmonary fluid with beta-galactosidase, and HRPO with alpha-mannosidase in concert with maltose and mannose competitive binding analyses altered the intensity and spectrum of pulmonary fluid fractions detected by HRPO far-Western blot analysis. Identity of pulmonary airway fluid fractions detected by HRPO far-Western (ligand) blot analysis were transferrin, dynein, albumin precursor, and two 156 kDa equine peptide fragments. HRPO can function as a partially-selective primary molecular probe when applied in either a conjugated or non-conjugated form. Some protein fractions can form complexes with HRPO through molecular mechanisms that involve physical interactions at the terminal alpha-mannose-rich regions of HRPO glycan side-chains. Based on its known molecular composition and structure, HRPO provides an opportunity for the development of diagnostics methodologies relevant to disease biomarkers that possess mannose-binding avidity.

  18. 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.

  19. Quantitative Measurement of Ca2+ in the Sarcoplasmic Reticulum Lumen of Mammalian Skeletal Muscle

    Science.gov (United States)

    Ziman, Andrew P.; Ward, Christopher W.; Rodney, George G.; Lederer, W. Jonathan; Bloch, Robert J.

    2010-01-01

    Skeletal muscle stores Ca2+ in the sarcoplasmic reticulum (SR) and releases it to initiate contraction, but the concentration of luminal Ca2+ in the SR ([Ca2+]SR) and the amount that is released by physiological or pharmacological stimulation has been difficult to measure. Here we present a novel, yet simple and direct, method that provides the first quantitative estimates of static content and dynamic changes in [Ca2+]SR in mammalian skeletal muscle, to our knowledge. The method uses fluo-5N loaded into the SR of single, mammalian skeletal muscle cells (murine flexor digitorum brevis myofibers) and confocal imaging to detect and calibrate the signals. Using this method, we have determined that [Ca2+]SR, free is 390 μM. 4-Chloro-m-cresol, an activator of the skeletal muscle ryanodine receptor, reduces [Ca2+]SR, free to ∼8 μM, when values are corrected for background fluorescence from cytoplasmic pools of dye. Prolonged electrical stimulation (10 s) at 50 Hz releases 88% of the SR Ca2+ content, whereas stimulation at 1 Hz (10 s) releases only 20%. Our results lay the foundation for molecular modeling of the dynamics of luminal SR Ca2+ and for future studies of the role of SR Ca2+ in healthy and diseased mammalian muscle. PMID:20959112

  20. Calcium handling by the sarcoplasmic reticulum during oscillatory contractions of skinned skeletal muscle fibres.

    Science.gov (United States)

    Szentesi, P; Zaremba, R; Stienen, G J

    1998-08-01

    Isometric ATP consumption and force were investigated in mechanically skinned fibres from iliofibularis muscle of Xenopus laevis. Measurements were performed at different [Ca2+], in the presence and absence of caffeine (5 nM). In weakly Ca2+-buffered solutions without caffeine, spontaneous oscillations in force and ATPase activity occurred. The repetition frequency was [Ca2+]-and temperature-dependent. The Ca2+ threshold (+/- SEM) for the oscillations corresponded to a pCa of 6.5 +/- 0.1. The maximum ATP consumption associated with calcium uptake by the sarcoplasmic reticulum (SR) reached during the oscillations was similar to the activity under steady-state conditions at saturating calcium concentrations in the presence of caffeine. Maximum activity was reached when the force relaxation was almost complete. The calculated amount of Ca2+ taken up by the SR during a complete cycle corresponded to 5.4 +/ 0.4 mmol per litre cell volume. In strongly Ca2+-buffered solutions, caffeine enhanced the calcium sensitivity of the contractile apparatus and, at low calcium concentrations, SR Ca uptake. These results suggest that when the SR is heavily loaded by net Ca uptake, there is a massive calcium-induced calcium release. Subsequent net Ca uptake by the SR then gives rise to the periodic nature of the calcium transient.

  1. Impaired sarcoplasmic reticulum Ca(2+) release rate after fatiguing stimulation in rat skeletal muscle

    DEFF Research Database (Denmark)

    Ørtenblad, Niels; Sjøgaard, G; Madsen, Klavs

    2000-01-01

    to 66% that persisted for 1 h, followed by a gradual recovery to 87% of prefatigue release rate at 3 h recovery. Tetanic force and rate of force development (+dF/dt) and relaxation (-dF/dt) were depressed by approximately 80% after stimulation. Recovery occurred in two phases: an initial phase, in which......The purpose of the study was to characterize the sarcoplasmic reticulum (SR) function and contractile properties before and during recovery from fatigue in the rat extensor digitorum longus muscle. Fatiguing contractions (60 Hz, 150 ms/s for 4 min) induced a reduction of the SR Ca(2+) release rate...... during the first 0.5-1 h the metabolic state recovered to resting levels, and a slow phase from 1-3 h characterized by a rather slow recovery of the mechanical properties. The recovery of SR Ca(2+) release rate was closely correlated to +dF/dt during the slow phase of recovery (r(2) = 0.51; P

  2. Vanadate oligoanions interact with the proton ejection by the Ca2+ pump of sarcoplasmic reticulum.

    Science.gov (United States)

    Aureliano, M; Madeira, V M

    1994-11-30

    Decameric vanadate differs from other oligomeric vanadate species in inhibiting Ca2+ uptake and H+ ejection promoted by sarcoplasmic reticulum ATPase. A decavanadate solution, 2 mM in total vanadium, containing about 200 microM decameric species, inhibits by about 50% the uptake of Ca2+ and by 75% the H+ ejection, whereas 2 mM nominal monovanadate slightly increases the uptake of Ca2+ and inhibits the ejection of H+ by 25%. Moreover, decavanadate linearly increases the Ca2+/H+ ratio, whereas monovanadate mimicks decavanadate behavior only at concentrations up to 1.2 mM. For higher concentrations of monovanadate, this effect is reversed probably due to the formation of metavanadates, namely tetravandate. It is concluded that Ca2+ uptake is tightly coupled to proton ejection through molecular events that are sensitive to the interaction of vanadate species. Apparently, the stoichiometry is variable and modulated by molecular events involved in vanadate interaction suggesting alterations in the energetic coupling associated with Ca2+ translocation.

  3. Identification of a novel small cysteine-rich protein in the fraction from the biocontrol Fusarium oxysporum strain CS-20 that mitigates Fusarium wilt symptoms and triggers defense responses in tomato

    Directory of Open Access Journals (Sweden)

    Larisa A. Shcherbakova

    2016-01-01

    Full Text Available The biocontrol effect of the nonpathogenic F. oxysporum strain CS-20 against the tomato wilt pathogen F. oxysporum f. sp. lycopersici (FOL has been previously reported to be primarily plant-mediated. This study shows that CS-20 produces proteins, which elicit defense responses in tomato plants. Three protein-containing fractions were isolated from CS-20 biomass using size exclusion chromatography. Exposure of seedling roots to one of these fractions prior to inoculation with pathogenic FOL strains significantly reduced wilt severity. This fraction initiated an ion exchange response in cultured tomato cells resulting in a reversible alteration of extracellular pH; increased tomato chitinase activity, and induced systemic resistance by enhancing PR-1 expression in tomato leaves. Two other protein fractions were inactive in seedling protection. The main polypeptide (designated CS20EP, which was specifically present in the defense-inducing fraction and was not detected in inactive protein fractions, was identified. The nucleotide sequence encoding this protein was determined, and its complete amino acid sequence was deduced from direct Edman degradation (25 N-terminal amino acid residues and DNA sequencing. The CS20EP was found to be a small basic cysteine-rich protein with a pI of 9.87 and 23.43% of hydrophobic amino acid residues. BLAST search in the NCBI database showed that the protein is new; however, it displays 48% sequence similarity with a hypothetical protein FGSG_10784 from F. graminearum strain PH-1. The contribution of CS20EP to elicitation of tomato defense responses resulting in wilt mitigating is discussed.

  4. Identification of a Novel Small Cysteine-Rich Protein in the Fraction from the Biocontrol Fusarium oxysporum Strain CS-20 that Mitigates Fusarium Wilt Symptoms and Triggers Defense Responses in Tomato

    Science.gov (United States)

    Shcherbakova, Larisa A.; Odintsova, Tatyana I.; Stakheev, Alexander A.; Fravel, Deborah R.; Zavriev, Sergey K.

    2016-01-01

    The biocontrol effect of the non-pathogenic Fusarium oxysporum strain CS-20 against the tomato wilt pathogen F. oxysporum f. sp. lycopersici (FOL) has been previously reported to be primarily plant-mediated. This study shows that CS-20 produces proteins, which elicit defense responses in tomato plants. Three protein-containing fractions were isolated from CS-20 biomass using size exclusion chromatography. Exposure of seedling roots to one of these fractions prior to inoculation with pathogenic FOL strains significantly reduced wilt severity. This fraction initiated an ion exchange response in cultured tomato cells resulting in a reversible alteration of extracellular pH; increased tomato chitinase activity, and induced systemic resistance by enhancing PR-1 expression in tomato leaves. Two other protein fractions were inactive in seedling protection. The main polypeptide (designated CS20EP), which was specifically present in the defense-inducing fraction and was not detected in inactive protein fractions, was identified. The nucleotide sequence encoding this protein was determined, and its complete amino acid sequence was deduced from direct Edman degradation (25 N-terminal amino acid residues) and DNA sequencing. The CS20EP was found to be a small basic cysteine-rich protein with a pI of 9.87 and 23.43% of hydrophobic amino acid residues. BLAST search in the NCBI database showed that the protein is new; however, it displays 48% sequence similarity with a hypothetical protein FGSG_10784 from F. graminearum strain PH-1. The contribution of CS20EP to elicitation of tomato defense responses resulting in wilt mitigating is discussed. PMID:26779237

  5. Polymer fractionation

    Energy Technology Data Exchange (ETDEWEB)

    Hadermann, A. F.

    1985-04-09

    Soluble polymers are fractionated according to molecular weight by cryogenically comminuting the polymer and introducing the polymer particles, while still in the active state induced by cryogenic grinding, into a liquid having a solvent power selected to produce a coacervate fraction containing high molecular weight polymer species and a dilute polymer solution containing lower molecular weight polymer species. The coacervate may be physically separated from the solution and finds use in the production of antimisting jet fuels and the like.

  6. In vitro versus in vivo protein digestibility techniques for calculating PDCAAS (protein digestibility-corrected amino acid score) applied to chickpea fractions.

    Science.gov (United States)

    Tavano, Olga Luisa; Neves, Valdir Augusto; da Silva Júnior, Sinézio Inácio

    2016-11-01

    Seven different in vitro methods to determine the protein digestibility for chickpea proteins were considered and also the application of these methodologies for calculating PDCAAS (protein digestibility-corrected amino acid score), seeking their correlations with the in vivo methodology. In vitro digestibility of raw and heated samples were determined using pepsin-pancreatin hydrolysis, considering soluble nitrogen via Kjeldahl (ppKJ) and hydrolysed peptide linkages using trinitrobenzenesulfonic acid and o-phthaldialdehyde. In vitro digestibility was also determined using trypsin, chymotrypsin and peptidase (3-Enz) or trypsin, chymotrypsin, peptidase and pronase solution (4-Enz). None of the correlations between in vitro and in vivo digestibilities were significant (at pvitro and in vivo results. PDCAAS-ppKJ, PDCAAS-3-Enz and PDCAAS-4-Enz presented the highest correlations with in vivo method, r=0.9316, 0.9442 and 0.9649 (pvitro methods for calculating PDCAAS may be promising and deserves more discussions. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Interaction between endoplasmic/sarcoplasmic reticulum stress (ER/SR stress), mitochondrial signaling and Ca(2+) regulation in airway smooth muscle (ASM).

    Science.gov (United States)

    Delmotte, Philippe; Sieck, Gary C

    2015-02-01

    Airway inflammation is a key aspect of diseases such as asthma. Several inflammatory cytokines (e.g., TNFα and IL-13) increase cytosolic Ca(2+) ([Ca(2+)]cyt) responses to agonist stimulation and Ca(2+) sensitivity of force generation, thereby enhancing airway smooth muscle (ASM) contractility (hyper-reactive state). Inflammation also induces ASM proliferation and remodeling (synthetic state). In normal ASM, the transient elevation of [Ca(2+)]cyt induced by agonists leads to a transient increase in mitochondrial Ca(2+) ([Ca(2+)]mito) that may be important in matching ATP production with ATP consumption. In human ASM (hASM) exposed to TNFα and IL-13, the transient increase in [Ca(2+)]mito is blunted despite enhanced [Ca(2+)]cyt responses. We also found that TNFα and IL-13 induce reactive oxidant species (ROS) formation and endoplasmic/sarcoplasmic reticulum (ER/SR) stress (unfolded protein response) in hASM. ER/SR stress in hASM is associated with disruption of mitochondrial coupling with the ER/SR membrane, which relates to reduced mitofusin 2 (Mfn2) expression. Thus, in hASM it appears that TNFα and IL-13 result in ROS formation leading to ER/SR stress, reduced Mfn2 expression, disruption of mitochondrion-ER/SR coupling, decreased mitochondrial Ca(2+) buffering, mitochondrial fragmentation, and increased cell proliferation.

  8. Altered calcium pump and secondary deficiency of gamma-sarcoglycan and microspan in sarcoplasmic reticulum membranes isolated from delta-sarcoglycan knockout mice.

    Science.gov (United States)

    Solares-Pérez, Alhondra; Alvarez, Rocío; Crosbie, Rachelle H; Vega-Moreno, Jesús; Medina-Monares, Joel; Estrada, Francisco J; Ortega, Alicia; Coral-Vazquez, Ramón

    2010-07-01

    Sarcoglycans (SGs) and sarcospan (SSPN) are transmembrane proteins of the dystrophin-glycoprotein complex. Mutations in the genes encoding SGs cause many inherited forms of muscular dystrophy. In this study, using purified membranes of wild-type (WT) and delta-SG knockout (KO) mice, we found the specific localization of the SG-SSPN isoforms in transverse tubules (TT) and sarcoplasmic reticulum (SR) membranes. Immunoblotting revealed that the absence of delta-SG isoforms in TT and SR results in a secondary deficiency of gamma-SG and microSPN. Our results showed augmented ATP hydrolytic activity, ATP-dependent calcium uptake and passive calcium efflux, probably through SERCA1 in KO compared to WT mice. Furthermore, we found a conformational change in SERCA1 isolated from KO muscle as demonstrated by calorimetric analysis. Following these alterations with mechanical properties, we found an increase in force in KO muscle with the same rate of fatigue but with a decreased fatigue recovery compared to WT. Together our observations suggest, for the first time, that the delta-SG isoforms may stabilize the expression of gamma-SG and microSPN in the TT and SR membranes and that this possible complex may play a role in the maintenance of a stable level of resting cytosolic calcium concentration in skeletal muscle. Copyright 2010 Elsevier Ltd. All rights reserved.

  9. 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 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.

  10. Understanding Multiplication of Fractions.

    Science.gov (United States)

    Sweetland, Robert D.

    1984-01-01

    Discussed the use of Cuisenaire rods in teaching the multiplication of fractions. Considers whole number times proper fraction, proper fraction multiplied by proper fraction, mixed number times proper fraction, and mixed fraction multiplied by mixed fractions. (JN)

  11. Herbal adaptogens combined with protein fractions from bovine colostrum and hen egg yolk reduce liver TNF-α expression and protein carbonylation in Western diet feeding in rats.

    Science.gov (United States)

    Mobley, C Brooks; Toedebusch, Ryan G; Lockwood, Christopher M; Heese, Alexander J; Zhu, Conan; Krieger, Anna E; Cruthirds, Clayton L; Hofheins, John C; Company, Joseph M; Wiedmeyer, Charles E; Kim, Dae Y; Booth, Frank W; Roberts, Michael D

    2014-01-01

    We examined if a purported anti-inflammatory supplement (AF) abrogated Western-diet (WD)-induced liver pathology in rats. AF contained: 1) protein concentrates from bovine colostrum and avian egg yolk; 2) herbal adaptogens and antioxidants; and 3) acetyl-L-carnitine. Nine month-old male Brown Norway rats were allowed ad libitum access to WD for 41-43 days and randomly assigned to WD + AF feeding twice daily for the last 31-33 days (n = 8), or WD and water-placebo feeding twice daily for the last 31-33 days (n = 8). Rats fed a low-fat/low-sucrose diet (CTL, n = 6) for 41-43 days and administered a water-placebo twice daily for the last 31-33 days were also studied. Twenty-four hours following the last gavage-feed, liver samples were analyzed for: a) select mRNAs (via RT-PCR) as well as genome-wide mRNA expression patterns (via RNA-seq); b) lipid deposition; and, c) protein carbonyl and total antioxidant capacity (TAC). Serum was also examined for TAC, 8-isoprostane and clinical chemistry markers. WD + AF rats experienced a reduction in liver Tnf-α mRNA (-2.8-fold, p < 0.01). Serum and liver TAC was lower in WD + AF versus WD and CTL rats (p < 0.05), likely due to exogenous antioxidant ingredients provided through AF as evidenced by a tendency for mitochondrial SOD2 mRNA to increase in WD + AF versus CTL rats (p = 0.07). Liver fat deposition nor liver protein carbonyl content differed between WD + AF versus WD rats, although liver protein carbonyls tended to be lower in WD + AF versus CTL rats (p = 0.08). RNA-seq revealed that 19 liver mRNAs differed between WD + AF versus WD when both groups were compared with CTL rats (+/- 1.5-fold, p < 0.01). Bioinformatics suggest that AF prevented WD-induced alterations in select genes related to the transport and metabolism of carbohydrates in favor of select genes related to lipid transport and metabolism. Finally, serum clinical safety markers and liver pathology

  12. Polyethylene glycol method is superior to ammonium sulfate and protein-A sepharose-4B method in fractionating thyroid stimulating immunoglobulin in Graves' disease.

    Science.gov (United States)

    Jap, T S; Jenq, S F; Wong, M C; Chiang, H

    1995-03-01

    The purpose of this study is to compare the performance of three methods, i.e., ammonium sulfate, polyethylene glycol (PEG) and protein A sepharose 4B in fractionation of thyroid stimulating immunoglobulin. Twelve patients with Graves' disease and twelve age-, sex-matched normal controls were recruited. 0.9 ml of saturated ammonium sulfate was added slowly to 1.1 ml of sera of all subjects and was stirred until reaching 45% saturation. The precipitation was allowed to form at 4 degrees C overnight. The solution was centrifuged at 10,000 x g for 5 minutes. The precipitate was washed twice with 45% ammonium sulfate, dissolved in 0.55 ml of distilled water equal to half of the initial volume of the serum and dialyzed against 200 volumes of phosphate-buffered saline (PBS) for 48 hours. Another 0.5 ml of serum sample from the same patients was mixed with 1.5 ml of 20% polyethylene, followed by centrifugation at 2,800 g for 20 minutes. The pellet was dissolved in 0.6 ml of Hanks' medium without NaCl containing 1.5% bovine serum albumin, 20mM HEPES: 1 ml of sera was applied to 0.75 g of protein A sepharose-4B in PBS buffer and eluted with glycine, pH 2.3. By adding immunoglobulin (Ig) from the different methods to the FRTL-5 tissue culture, we determined the cAMP generation. Patients with Graves' disease were found to have higher serum thyroxine and triiodothyronine concentrations (p TSI) while with ammonium sulfate precipitation, nine out 12 patients (75%) showed positive TSI. On the other hand, all patients showed positive TSI with PEG precipitation. When the degree of thyrotoxicosis was classified in terms of thyroid hormone concentrations, there was no correlation between TSI titers and thyroid hormone concentration. Polyethylene glycol is superior to both ammonium sulfate and protein A sepharose-4B in fractionating TSI in terms of sensitivity and convenience in operation.

  13. FRAKSINASI PROTEIN KAPANG LAUT Xylaria psidii KT30 DAN SITOTOKSISITASNYA TERHADAP SEL HeLa [Fractionation of Proteins of Marine Fungus Xylaria psidii KT30 and their Cytotoxicity against HeLa Cells

    Directory of Open Access Journals (Sweden)

    Mita Gebriella Inthe

    2014-06-01

    Full Text Available Cervical cancer is the most common cause of death for Indonesian women after human breast cancer. One of the efforts of cancer treatment is the utilization of natural compounds. One of the microorganisms having the potential as anticancer agent is endophytic fungi. Endophytic fungi from the marine habitat can be isolated from sea weeds, sea grasses, sponges, and mangroves. Xylaria psidii KT30, a marine fungus used in this study was isolated from red seaweed Kappaphycus alvarezii. Xylaria psidii KT30 was cultivated in potato dextrose broth medium for nine days at room temperature 27-29°C in shaking condition. This study aimed to obtain protein fractions from X. psidii KT30 and determine their toxicity againt Chang and HeLa cells. The fractionation process was conducted using DEAE Sephadex A-50 column chromatography and the toxicity was determined by Brine Shrimp Lethality Test (BSLT. The metabolites excreted in the culture broth was extracted using 90% of ammonium sulphate. The extract was then tested for their toxicity against HeLa and Chang cells by Microculture Tetrazolium Technique (MTT assay.The results revealed that LC50 of the protein extract of X. psidii KT30 was 104.95 ppm and IC50 was 69.9 ppm. Based on the National Cancer Institute (NCI, this value showed moderate cytotoxicity against HeLa cells.

  14. Fatty acid profile and composition of milk protein fraction in dairy cows fed long-chain unsaturated fatty acids during the transition period

    Directory of Open Access Journals (Sweden)

    Francisco Palma Rennó

    2013-11-01

    Full Text Available The objective of this study was to evaluate the utilization of different sources of unsaturated long-chain fatty acids in diets for dairy cows during the transition period and early lactation on the milk fatty acid profile and composition of the protein fraction. Thirty-six Holstein cows were divided into three groups, fed the following diets: control (C; soybean oil (SO; and calcium salts of long-chain unsaturated fatty acids (CS. The milk samples utilized for analysis were obtained weekly from parturition to twelve weeks of lactation; each one of the samples originated from two daily milkings. Milk composition and total nitrogen, non-protein nitrogen and non-casein nitrogen levels were analyzed. The cows receiving the diet with calcium salts had lower concentrations of non-protein nitrogen (%CP in milk compared with the animals fed the diet with soybean oil. There was a decrease in concentration of medium-chain fatty acids C12-C16, and a concomitant increase in concentrations of long-chain fatty acids >C18 in milk fat for the animals fed the diets CS and SO when compared with diet C. Soybean oil and CS diets increased milk-fat concentrations of the acids C18: 1 trans-11, C18: 2 cis-9, trans-11 and C18: 2 trans-10 cis-12 in relation to diet C. The utilization of sources of long-chain fatty acids in the diet of dairy cows increases the biological value of milk in early lactation due to higher concentrations of specific fatty acids such as CLA C18: 2cis-9, trans-11.

  15. Optimization of heterologous expression of the phytase (PPHY) of Pichia anomala in P. pastoris and its applicability in fractionating allergenic glycinin from soy protein.

    Science.gov (United States)

    Joshi, Swati; Satyanarayana, T

    2014-06-01

    The phytase (PPHY) of Pichia anomala has the requisite properties of thermostability and acidstability, broad substrate spectrum, and protease insensitivity, which make it a suitable candidate as a feed and food additive. The 1,389-bp PPHY gene was amplified from P. anomala genomic DNA, cloned in pPICZαA, and expressed extracellularly in P. pastoris X33. Three copies of PPHY have been detected integrated into the chromosomal DNA of the recombinant P. pastoris. The size exclusion chromatography followed by electrophoresis of the pure rPPHY confirmed that this is a homohexameric glycoprotein of ~420 kDa with a 24.3 % portion as N-linked glycans. The temperature and pH optima of rPPHY are 60 °C and 4.0, similar to the endogenous enzyme. The kinetic characteristics K(m), V(max), K(cat), and K(cat)/K(m) of rPPHY are 0.2 ± 0.03 mM, 78.2 ± 1.43 nmol mg(-1) s(-1), 65,655 ± 10.92 s(-1), and 328.3 ± 3.12 μM(-1) s(-1), respectively. The optimization of medium components led to a 21.8-fold improvement in rPPHY production over the endogenous yeast. The rPPHY titer attained in shake flasks could also be sustained in the laboratory fermenter. The rPPHY accounts for 57.1 % of the total secreted protein into the medium. The enzyme has been found useful in fractionating allergenic protein glycinin from soya protein besides dephytinization.

  16. Renoprotective Effects, Protein Thiols and Liver Glycogen Content of Alloxan-induced Diabetic Rats Treated with Different Fractions of Heartwood of Pterocarpus marsupium.

    Science.gov (United States)

    Bhata, Vinutha; Nayak, B Shivananda

    2015-11-01

    Oxidative stress is believed to be a pathogenic factor in the development of diabetic complications. In the present study, we aimed to evaluate the effects of different fractions of heart wood of Pterocarpus marsupium on antioxidant enzyme like protein thiols and also check the efficacy of the extract for the protection of the renal function in alloxan induced diabetic rats. The present study also investigates the levels of liver glycogen which are considered as the best biomarker for assaying the hypoglycemic activity of any drug. Diabetes was induced by administering alloxan dissolved in saline, while the normal control group was given propylene glycol. Diabetes induced animals were randomly assigned into different groups. Blood samples were collected from all the experimental and control groups. Estimation of urea, uric acid and creatinine along with protein thiols was made on day 30 only. At the end, all the animals were sacrificed to collect liver tissue to analyze glycogen content. The 30 days treatment with various extracts (75 mg/kg body wt) significantly lowered protein thiol levels, which probably represents increased utilization for neutralizing free radicals. There was no significant increase in the levels of renal parameters in the extract treated groups which revealed that the employed dose of the extract is nontoxic to the kidney. There was also a significant decrease in the glycogen content in insulin and alcohol-extract treated groups and should be encouraging for the treatment of diabetes mellitus. The extract showed a promising antioxidant effect, as well as hypoglycemic activity, and should be encouraged for the treatment of diabetes.

  17. Matching of sarcoplasmic reticulum and contractile properties in rat fast- and slow-twitch muscle fibres.

    Science.gov (United States)

    Trinh, Huong H; Lamb, Graham D

    2006-07-01

    1. The twitch characteristics (fast-twitch or slow-twitch) of skeletal muscle fibres are determined not only by the contractile apparatus properties of the fibre, but also by the time-course of Ca2+ release and re-uptake by the sarcoplasmic reticulum (SR). The present study examined, in individual fibres from non-transforming muscle of the rat, whether particular SR properties are matched to the contractile apparatus properties of the fibre, in particular in the case of fibres with fast-twitch contractile apparatus located in a slow-twitch muscle, namely the soleus. 2. Force was recorded in single, mechanically skinned fibres from extensor digitorum longus (EDL), gastrocnemius, peroneus longus and soleus muscles. Using repeated cycles in which the SR was emptied of all releasable Ca2+ and then reloaded, it was possible to determine the relative amount of Ca2+ present in the SR endogenously, the maximum SR capacity and the rate of Ca2+ loading. The sensitivity of the contractile apparatus to Ca2+ and Sr2+ was used to classify the fibres as fast-twitch (FT), slow-twitch (ST) or mixed (< 3% of the fibres examined) and thereby identify the likely troponin C and myosin heavy chain types present. 3. There was no significant difference in SR properties between the groups of FT fibres obtained from the four different muscles, including soleus. Despite some overlap in the SR properties of individual fibres between the FT and ST groups, the properties of the FT fibres in all four muscles studied were significantly different from those of the ST and mixed fibres. 4. In general, in FT fibres the SR had a larger capacity and the endogenous Ca2+ content was a relatively lower percentage of maximum compared with ST fibres. Importantly, in terms of their SR properties, FT fibres from soleus muscle more closely resembled FT fibres from other muscles than they did ST fibres from soleus muscle.

  18. Crosstalk between mitochondrial and sarcoplasmic reticulum Ca2+ cycling modulates cardiac pacemaker cell automaticity.

    Directory of Open Access Journals (Sweden)

    Yael Yaniv

    Full Text Available BACKGROUND: Mitochondria dynamically buffer cytosolic Ca(2+ in cardiac ventricular cells and this affects the Ca(2+ load of the sarcoplasmic reticulum (SR. In sinoatrial-node cells (SANC the SR generates periodic local, subsarcolemmal Ca(2+ releases (LCRs that depend upon the SR load and are involved in SANC automaticity: LCRs activate an inward Na(+-Ca(2+ exchange current to accelerate the diastolic depolarization, prompting the ensemble of surface membrane ion channels to generate the next action potential (AP. OBJECTIVE: To determine if mitochondrial Ca(2+ (Ca(2+ (m, cytosolic Ca(2+ (Ca(2+ (c-SR-Ca(2+ crosstalk occurs in single rabbit SANC, and how this may relate to SANC normal automaticity. RESULTS: Inhibition of mitochondrial Ca(2+ influx into (Ru360 or Ca(2+ efflux from (CGP-37157 decreased [Ca(2+](m to 80 ± 8% control or increased [Ca(2+](m to 119 ± 7% control, respectively. Concurrent with inhibition of mitochondrial Ca(2+ influx or efflux, the SR Ca(2+ load, and LCR size, duration, amplitude and period (imaged via confocal linescan significantly increased or decreased, respectively. Changes in total ensemble LCR Ca(2+ signal were highly correlated with the change in the SR Ca(2+ load (r(2 = 0.97. Changes in the spontaneous AP cycle length (Ru360, 111 ± 1% control; CGP-37157, 89 ± 2% control in response to changes in [Ca(2+](m were predicted by concurrent changes in LCR period (r(2 = 0.84. CONCLUSION: A change in SANC Ca(2+ (m flux translates into a change in the AP firing rate by effecting changes in Ca(2+ (c and SR Ca(2+ loading, which affects the characteristics of spontaneous SR Ca(2+ release.

  19. Prognostic usefulness of insulin-like growth factor-binding protein 7 in heart failure with reduced ejection fraction: a novel biomarker of myocardial diastolic function?

    Science.gov (United States)

    Gandhi, Parul U; Gaggin, Hanna K; Sheftel, Alex D; Belcher, Arianna M; Weiner, Rory B; Baggish, Aaron L; Motiwala, Shweta R; Liu, Peter P; Januzzi, James L

    2014-11-15

    Insulin-like growth factor-binding protein 7 (IGFBP7) is a biomarker that has recently been associated with heart failure and cardiac hypertrophy. The aim of this study was to examine IGFBP7 relative to echocardiographic abnormalities reflecting diastolic dysfunction. One hundred twenty-four patients with ambulatory heart failure with reduced ejection fraction and baseline detailed 2-dimensional echocardiograms were followed for a mean of 10 months. IGFBP7 was measured serially at each office visit; 108 patients underwent follow-up echocardiography. Echocardiographic parameters of diastolic function were compared at baseline and over time. IGFBP7 concentrations were not linked to left ventricular size or systolic function. In contrast, those with elevated baseline IGFBP7 concentrations were more likely to have abnormalities of parameters describing diastolic function, such as higher left atrial volume index, transmitral E/A ratio, E/E' ratio, and right ventricular systolic pressure. IGFBP7 was correlated with left atrial volume index (ρ = 0.237, p = 0.008), transmitral E/A ratio (ρ = 0.304, p = 0.001), E/E' ratio (ρ = 0.257, p = 0.005), and right ventricular systolic pressure (ρ = 0.316, p = 0.001). Furthermore, each was found to be independently predictive of IGFBP7 in adjusted analysis. In subjects with baseline and final echocardiograms, more time spent with elevated IGFBP7 concentrations in serial measurement was associated with worsening diastolic function and increasing left atrial volume index or right ventricular systolic pressure. IGFBP7 concentrations were predictive of an increased risk for cardiovascular events independent of echocardiographic measures of diastolic function (p = 0.006). In conclusion, IGFBP7 is a novel prognostic biomarker for heart failure with reduced ejection fraction and shows significant links to the presence and severity of echocardiographic parameters of abnormal diastolic function.

  20. Mystery Fractions

    Science.gov (United States)

    Bhattacharyya, Sonalee; Namakshi, Nama; Zunker, Christina; Warshauer, Hiroko K.; Warshauer, Max

    2016-01-01

    Making math more engaging for students is a challenge that every teacher faces on a daily basis. These authors write that they are constantly searching for rich problem-solving tasks that cover the necessary content, develop critical-thinking skills, and engage student interest. The Mystery Fraction activity provided here focuses on a key number…

  1. Mystery Fractions

    Science.gov (United States)

    Bhattacharyya, Sonalee; Namakshi, Nama; Zunker, Christina; Warshauer, Hiroko K.; Warshauer, Max

    2016-01-01

    Making math more engaging for students is a challenge that every teacher faces on a daily basis. These authors write that they are constantly searching for rich problem-solving tasks that cover the necessary content, develop critical-thinking skills, and engage student interest. The Mystery Fraction activity provided here focuses on a key number…

  2. Immunogenicity and allergenicity of 2S, 7S and 11S soy protein fractions Imunogenicidade e alergenicidade das frações protéicas 2S, 7S and 11S da soja

    Directory of Open Access Journals (Sweden)

    Alvorita Leite Bittencourt

    2007-12-01

    Full Text Available It is known that in a part of the population, mainly among children, some are hypersensitive to soybean protein, although it is not yet completely elucidated which protein fraction is more immunogenic/allergenic. The objective of the study was to compare the immunogenicity and allergenicity of the soy protein fractions. The 2S (conglycinin, 7S (beta conglycinin and 11S (glycinin fractions were isolated from soy protein by affinity chromatography. These purified soy protein fractions were used as antigens for immunizing BALB/c mice to evaluate their immunogenicity by following the appearance of specific IgM and IgG antibodies in blood serum by ELISA. The allergenicity of these soy protein fractions was evaluated by the following approaches: i the production of IgE antibodies against 2S, 7S and 11S soy protein fractions by BALBc mice in the anaphylactic cutaneous passive test (PCA, and ii the production of IgG1 specific antibodies against the 7S fraction in BALB/c mice. The 7S and 11S fractions induced the formation of IgM and IgG antibodies. The PCA test showed that only the 7S fraction was allergenic leading to the production of IgE antibodies. Another evidence that reinforces the allergenicity of the 7S soy protein fraction is the presence of IgG1 specific antibodies reactive to this protein fraction in immunized mice. Our study shows that the 7S soy protein fraction is important to elicit allergic reactions in mice and may contribute to elucidate the allergenicity of soy-derived products.Sabe-se que uma parte da população, principalmente crianças, são hipersensíveis à proteína de soja, embora não esteja completamente esclarecido qual a fração protéica mais alergênica. O objetivo do estudo foi comparar a imunogenicidade e alergenicidade das frações protéicas 2S (conglicinina, 7S (beta conglicinina e 11S (glicinina da soja, isoladas por cromatografia de afinidade. Essas frações protéicas foram usadas como antígenos para

  3. Antibody reactivity in patients with IgE-mediated wheat allergy to various subunits and fractions of gluten and non-gluten proteins from ω-gliadin-free wheat genotypes.

    Science.gov (United States)

    Skoczowski, Andrzej; Obtułowicz, Krystyna; Czarnobilska, Ewa; Dyga, Wojciech; Mazur, Marcel; Stawoska, Iwona; Waga, Jacek

    2017-05-11

    [b]Abstract Introduction and objective[/b]. Gluten proteins (gliadins and glutenins) are polymorphic wheat storage proteins of allergenic properties. Significant differences in chemical composition between both protein groups allow to expect highly specific immunological response of individual subunits and fractions in reactions with IgE sera of people allergic to wheat. The aim of these studies was to identify and characterize the most allergenic gluten proteins (GP) and nongluten proteins (NGP) occurred in two closely related wheat hybrid genotypes. [b]Materials and method.[/b] 3xC and 3xN wheat hybrids, which differ strongly in regard of gliadin composition, were analyzed. Seven people manifesting different symptoms of wheat allergy donated sera for the experiment. The technique of immunoblotting after SDS-PAGE was used for identification of allergenic subunits and fractions among GP and NGP. Immunologically active protein bands were visualized by chemiluminescence. [b]Results[/b]. Great variation of immunodetection spectra was observed. Results of immunoblotting showed LMW glutenins to be of highest, gliadins of medium, while NGP of lowest allergenicity for selected patients. The 43-kDa and 47-kDa LMW glutenin subunits, 40-kDa and 43-kDa γ-gliadin fractions and 49-kDa NGP can be considered as the most immunoreactive among all protein bands [b]separated by SDS-PAGE. [/b] The observed differentiation of immunodetection spectra allows to model highly specific IgE-binding profiles of allergenic wheat proteins attributed to individual patients with symptoms of gluten intolerance. Highly immunoreactive subunits and fractions among GP and NGP were identified. The observed immunoreactivity of 49 kDa NGP is worth to emphasize, as it has never been reported as wheat allergenic protein before.

  4. Fraction Reduction through Continued Fractions

    Science.gov (United States)

    Carley, Holly

    2011-01-01

    This article presents a method of reducing fractions without factoring. The ideas presented may be useful as a project for motivated students in an undergraduate number theory course. The discussion is related to the Euclidean Algorithm and its variations may lead to projects or early examples involving efficiency of an algorithm.

  5. Selective 'unlabeling' of amino acids in fractionally 13C labeled proteins: An approach for stereospecific NMR assignments of CH3 groups in Val and Leu residues

    Energy Technology Data Exchange (ETDEWEB)

    Atreya, H.S.; Chary, K.V.R. [Tata Institute of Fundamental Research, Department of Chemical Sciences (India)

    2001-03-15

    A novel methodology for stereospecific NMR assignments of methyl (CH{sub 3}) groups of Val and Leu residues in fractionally {sup 13}C-labeled proteins is presented. The approach is based on selective 'unlabeling' of specific amino acids in proteins while fractionally {sup 13}C-labeling the rest. A 2D [{sup 13}C-{sup 1}H] HSQC spectrum recorded on such a sample is devoid of peaks belonging to the 'unlabeled' amino acid residues. Such spectral simplification aids in unambiguous stereospecific assignment of diastereotopic CH{sub 3} groups in Val and Leu residues in large proteins. This methodology has been demonstrated on a 15 kDa calcium binding protein from Entamoeba histolytica (Eh-CaBP)

  6. Characteristics of the effect of insecticide karate on the metabolism of the nucleic acids and proteins in the subcellular fractions of the liver and intestine

    Directory of Open Access Journals (Sweden)

    Mukaddas Khamrakulova

    2012-12-01

    Full Text Available Karate is contact insecticide from the group of pyrethroids, its chemical formulae C23H19ClF3NO3, molecular mass 449.9. (1-RS-cis-2.2-dimethyl-3-(3.3.3-triphtoro-2chloropropanyl-cyclopropan of the carbonic acid-1 (RS-α-cyano-3-phenoxybenzolic ether. Purpose of this work was to study effect of insecticide Karate on the contents of DNA and RNA and total protein in the subcellular elements of the liver and intestinal mucosa in white rats.Experiments were performed on the outbred white rats of mass 120-250 g in 2 stages. Analysis of the results obtained showed that in acute poisoning with pesticide Karate the acute decrease in RNA and DNA occurred in the subcellular elements of liver and intestine mucosa during the first 7 days of experiment with subsequent normalization of the levels during the following 7 and 15 days. In chronic poisoning the number of RNA in the studied hepatic biomedium decreased in the homogenate to 66.47 and 55%, in the nucleic fractions to 64-62.5%, and in the supernatant fluid - to 52.6-48%; and in the small intestine - from 55.6% to 64.6%, respectively, according to the time of experiment. The DNA level reduced from 20% to 55%. In acute and chronic poisoning with pesticides of group pyrethroids, concentration of nucleic acids sharply reduced in the elements of liver and intestinal mucosa, that indicated about possibility of the inhibition of the DNA and RNA synthesis, which effect on the synthesis of protein in the cells.

  7. The effect of high and low levels of supplementation on milk production, nitrogen utilization efficiency, and milk protein fractions in late-lactation dairy cows.

    Science.gov (United States)

    Reid, M; O'Donovan, M; Murphy, J P; Fleming, C; Kennedy, E; Lewis, E

    2015-08-01

    To fill the feed deficit in the autumn/late lactation period in a seasonal grazing system, supplementation is required. This study aimed to investigate the use of baled grass silage or concentrate as supplementation to grazing dairy cows in late lactation. Eighty-four grass-based spring-calving dairy cows, averaging 212d in milk, were allocated to 1 of 6 treatments [high grass allowance (HG), low grass allowance (LG), grass with a low concentrate allocation (GCL), grass with a low grass silage allocation (GSL), grass with a high concentrate allocation (GCH), and grass with a high grass silage allocation (GSH)] to measure the effects of using baled grass silage or concentrate as supplements to grazed grass. Effects on intake, milk yield, milk composition and N fractions, and N utilization efficiency were measured. Treatments HG and LG received 17 and 14kg of dry matter (DM) grass/cow per d, respectively. Treatments GCL and GSL were offered 14kg of DM grass/cow per d and 3kg of DM of supplementation/cow per d. Treatments GCH and GSH were offered 11kg of DM grass/cow per d and 6kg of DM of supplementation/cow per d. Milk yield was greatest in the GCH treatment and milk solids yield was greatest in both concentrate-supplemented treatments. The HG and LG treatments excreted a greater quantity of N as a proportion of N intake than the supplemented treatments. The HG treatment also excreted the greatest total quantity of N. This indicates an improvement in N utilization efficiency when supplementation is offered compared with grazing only. Offering 6kg of DM of either grass silage or concentrate as supplementation decreased milk true protein concentration compared with offering a grass-only diet. This suggests that increasing the proportion of supplementation relative to grass may negatively affect milk processability, which is associated with milk true protein concentration.

  8. Decavanadate, decaniobate, tungstate and molybdate interactions with sarcoplasmic reticulum Ca(2+)-ATPase: quercetin prevents cysteine oxidation by vanadate but does not reverse ATPase inhibition.

    Science.gov (United States)

    Fraqueza, Gil; Batista de Carvalho, Luís A E; Marques, M Paula M; Maia, Luisa; Ohlin, C André; Casey, William H; Aureliano, Manuel

    2012-11-07

    Recently we demonstrated that the decavanadate (V(10)) ion is a stronger Ca(2+)-ATPase inhibitor than other oxometalates, such as the isoelectronic and isostructural decaniobate ion, and the tungstate and molybdate monomer ions, and that it binds to this protein with a 1 : 1 stoichiometry. The V(10) interaction is not affected by any of the protein conformations that occur during the process of calcium translocation (i.e. E1, E1P, E2 and E2P) (Fraqueza et al., J. Inorg. Biochem., 2012). In the present study, we further explore this subject, and we can now show that the decaniobate ion, [Nb(10) = Nb(10)O(28)](6-), is a useful tool in deducing the interaction and the non-competitive Ca(2+)-ATPase inhibition by the decavanadate ion [V(10) = V(10)O(28)](6-). Moreover, decavanadate and vanadate induce protein cysteine oxidation whereas no effects were detected for the decaniobate, tungstate or molybdate ions. The presence of the antioxidant quercetin prevents cysteine oxidation, but not ATPase inhibition, by vanadate or decavanadate. Definitive V(IV) EPR spectra were observed for decavanadate in the presence of sarcoplasmic reticulum Ca(2+)-ATPase, indicating a vanadate reduction at some stage of the protein interaction. Raman spectroscopy clearly shows that the protein conformation changes that are induced by V(10), Nb(10) and vanadate are different from the ones induced by molybdate and tungstate monomer ions. Here, Mo and W cause changes similar to those by phosphate, yielding changes similar to the E1P protein conformation. The putative reduction of vanadium(V) to vanadium(IV) and the non-competitive binding of the V(10) and Nb(10) decametalates may explain the differences in the Raman spectra compared to those seen in the presence of molybdate or tungstate. Putting it all together, we suggest that the ability of V(10) to inhibit the Ca(2+)-ATPase may be at least in part due to the process of vanadate reduction and associated protein cysteine oxidation. These

  9. Levels and Age Dependency of Neurofilament Light and Glial Fibrillary Acidic Protein in Healthy Individuals and Their Relation to the Brain Parenchymal Fraction.

    Science.gov (United States)

    Vågberg, Mattias; Norgren, Niklas; Dring, Ann; Lindqvist, Thomas; Birgander, Richard; Zetterberg, Henrik; Svenningsson, Anders

    2015-01-01

    Neurofilament light (NFL) and Glial Fibrillary Acidic Protein (GFAP) are integral parts of the axonal and astrocytal cytoskeletons respectively and are released into the cerebrospinal fluid (CSF) in cases of cellular damage. In order to interpret the levels of these biomarkers in disease states, knowledge on normal levels in the healthy is required. Another biomarker for neurodegeneration is brain atrophy, commonly measured as brain parenchymal fraction (BPF) using magnetic resonance imaging (MRI). Potential correlations between levels of NFL, GFAP and BPF in healthy individuals have not been investigated. To present levels of NFL and GFAP in healthy individuals stratified for age, and investigate the correlation between them as well as their correlation with BPF. The CSF was analysed in 53 healthy volunteers aged 21 to 70 (1 sample missing for GFAP analysis) and 48 of the volunteers underwent determination of BPF using MRI. Mean (±SD) NFL was 355 ng/L (±214), mean GFAP was 421 ng/L (±129) and mean BPF was 0.867 (±0.035). All three biomarkers correlated with age. NFL also correlated with both GFAP and BPF. When controlled for age, only the correlation between NFL and GFAP retained statistical significance. This study presents data on age-stratified levels of NFL and GFAP in the CSF of healthy individuals. There is a correlation between levels of NFL and GFAP and both increase with age. A correlation between NFL and BPF was also found, but did not retain statistical significance if controlled for age.

  10. High sensitive troponin T and heart fatty acid binding protein: Novel biomarker in heart failure with normal ejection fraction?: A cross-sectional study

    Directory of Open Access Journals (Sweden)

    Barroso Michael

    2011-07-01

    Full Text Available Abstract Background High sensitive troponin T (hsTnT and heart fatty acid binding protein (hFABP are both markers of myocardial injury and predict adverse outcome in patients with systolic heart failure (SHF. We tested whether hsTnT and hFABP plasma levels are elevated in patients with heart failure with normal ejection fraction (HFnEF. Methods We analyzed hsTnT, hFABP and N-terminal brain natriuretic peptide in 130 patients comprising 49 HFnEF patients, 51 patients with asymptomatic left ventricular diastolic dysfunction (LVDD, and 30 controls with normal diastolic function. Patients were classified to have HFnEF when the diagnostic criteria as recommended by the European Society of Cardiology were met. Results Levels of hs TnT and hFABP were significantly higher in patients with asymptomatic LVDD and HFnEF (both p Conclusion In HFnEF patients, hsTnT and hFABP are elevated independent of coronary artery disease, suggesting that ongoing myocardial damage plays a critical role in the pathophysiology. A combination of biomarkers and echocardiographic parameters might improve diagnostic accuracy and risk stratification of patients with HFnEF.

  11. Antiviral, immunomodulatory, and free radical scavenging activities of a protein-enriched fraction from the larvae of the housefly, Musca domestica.

    Science.gov (United States)

    Ai, Hui; Wang, Furong; Zhang, Na; Zhang, Lingyao; Lei, Chaoliang

    2013-01-01

    In our previous study, protein-enriched fraction (PEF) that was isolated from the larvae of the housefly, Musca domestica L. (Diptera: Muscidae), showed excellent hepatoprotective activity as well as the potential for clinical application in therapy for liver diseases. In this study, antiviral, immunomodulatory, and free radical scavenging activities of PEF were evaluated. The antiviral results demonstrated that PEF inhibited the infection of avian influenza virus H9N2 and had a virucidal effect against the multicapsid nucleopolyhedrovirus of the alfalfa looper, Autographa californica Speyer (Lepidoptera: Noctuidae) in vitro. The mortality of silkworm larve in a PEF treatment group decreased significantly compared with a negative control. PEF showed excellent scavenging activity for 1,1-diphenyl-2-picrylhydrazyl and superoxide anion radicals, which were similar to those of ascorbic acid. The imunomodulatory results suggested that PEF could effectively improve immune function in experimental mice. Our results indicated that PEF could possibly be used for the prophylaxis and treatment of diseases caused by avian influenza virus infection. In addition, PEF with virucidal activity against insect viruses might provide useful for the development of antimicrobial breeding technology for economically important insects. As a natural product from insects, PEF could be a potential source for the discovery of potent antioxidant and immunomodulatory agents.

  12. Insoluble fraction of buckwheat (Fagopyrum esculentum Moench) protein possessing cholesterol-binding properties that reduce micelle cholesterol solubility and uptake by Caco-2 cells.

    Science.gov (United States)

    Metzger, Brandon T; Barnes, David M; Reed, Jess D

    2007-07-25

    Buckwheat (Fagopyrum esculentum Moench) protein (BWP) exhibits hypocholesterolemic activity in several animal models by increasing fecal excretion of neutral and acidic sterols. In the current study, the ability of BWP to disrupt micelle cholesterol solubility by sequestration of cholesterol was investigated. When BWP (0.2%) was incubated with cholesterol and micelle lipid components prior to micelle formation, cholesterol solubility was reduced 40%. In contrast, cholesterol solubility was not decreased when BWP (0.2%) was incubated after micelle formation and incorporation of soluble cholesterol. Buckwheat flour, from which BWP was derived, had no significant effect on cholesterol solubility. Cholesterol uptake in Caco-2 cells from micelles made in the presence of BWP (0.2%) was reduced by 47, 36, 35, and 33% when compared with buckwheat flour, bovine serum albumin, casein, and gelatin, respectively. Reduction in cholesterol uptake in Caco-2 cells was dose-dependent, with maximum reductions at 0.1-0.4% BWP. In cholesterol-binding experiments, 83% of the cholesterol was associated with an insoluble BWP fraction, indicating strong cholesterol-binding capacity that disrupts solubility and uptake by Caco-2 cells.

  13. Ca2+ uptake, Ca2+-ATPase activity, phosphoprotein formation and phosphate turnover in a microsomal fraction of smooth muscle.

    Science.gov (United States)

    Raeymaekers, L; Hasselbach, W

    1981-05-15

    Vesicles capable of phosphate-stimulated calcium uptake were isolated from the microsomal fraction of the smooth muscle of the pig stomach according to a previously described procedure which consists in increasing the density of the vesicles by loading them with calcium phosphate and isolating them by centrifugation [Raeymaekers, L., Agostini, B., and Hasselbach, W. (1981) Histochemistry, 70, 139--150]. These vesicles, which contain calcium phosphate deposits, are able to accumulate an additional amount of calcium. This calcium uptake is accompanied by calcium-stimulated ATPase activity and by the formation of an acid-stable phosphoprotein. The acid-denatured phosphoprotein is dephosphorylated by hydroxylamine, which indicates that an acylphosphate is formed. This phosphoprotein probably represents a phosphorylated transport intermediate similar to that seen with the Ca2+-ATPase of sarcoplasmic reticulum of skeletal muscle. As with the Ca2+-ATPase of sarcoplasmic reticulum vesicles, this vesicular fraction catalyses an exchange between inorganic phosphate and the gamma-phosphate of ATP (ATP-Pi exchange) which is dependent on the presence of intravesicular calcium, and an exchange of phosphate between ATP and ADP (ATP-ADP exchange). The results further indicate that the turnover rate of the calcium pump, calculated from the ratio of calcium-stimulated ATPase activity to the steady-state level of phosphoprotein, is similar to that of Ca2+-ATPase of sarcoplasmic reticulum of skeletal muscle.

  14. Determination of low isotopic enrichment of L-[1-C-13]valine by gas chromatography combustion isotope ratio mass spectrometry : a robust method for measuring protein fractional synthetic rates in vivo

    NARCIS (Netherlands)

    Reijngoud, DJ; Hellstern, G; Elzinga, H; de Sain-van der Velden, MG; Okken, A; Stellaard, F

    1998-01-01

    A method was developed for measuring protein fractional synthetic rates using the N-methoxycarbonylmethyl ester (MCM) derivative of L-[1-C-13]valine and on-line gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). The derivatization procedure can be performed rapidly and GC sep

  15. Determination of low isotopic enrichment of L-[1-C-13]valine by gas chromatography combustion isotope ratio mass spectrometry : a robust method for measuring protein fractional synthetic rates in vivo

    NARCIS (Netherlands)

    Reijngoud, DJ; Hellstern, G; Elzinga, H; de Sain-van der Velden, MG; Okken, A; Stellaard, F

    A method was developed for measuring protein fractional synthetic rates using the N-methoxycarbonylmethyl ester (MCM) derivative of L-[1-C-13]valine and on-line gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). The derivatization procedure can be performed rapidly and GC

  16. Detergent resistant membrane fractions are involved in calcium signaling in Müller glial cells of retina.

    Science.gov (United States)

    Krishnan, Gopinath; Chatterjee, Nivedita

    2013-08-01

    Compartmentalization of the plasma membrane into lipid microdomains promotes efficient cellular processes by increasing local molecular concentrations. Calcium signaling, either as transients or propagating waves require integration of complex macromolecular machinery. Calcium waves represent a form of intercellular signaling in the central nervous system and the retina. We hypothesized that the mechanism for calcium waves would require effector proteins to aggregate at the plasma membrane in lipid microdomains. The current study shows that in Müller glia of the retina, proteins involved in calcium signaling aggregate in detergent resistant membranes identifying rafts and respond by redistributing on stimulation. We have investigated Purinoreceptor-1 (P2Y1), Ryanodine receptor (RyR), and Phospholipase C (PLC-β1). P2Y1, RyR and PLC-β1, redistribute from caveolin-1 and flotillin-1 positive fractions on stimulation with the agonists, ATP, 2MeS-ATP and Thapsigargin, an inhibitor of sarcoplasmic-endoplasmic reticulum Ca-ATPase (SERCA). Redistribution is absent on treatment with cyclopiazonic acid, another SERCA inhibitor. Disruption of rafts by removing cholesterol cause proteins involved in this machinery to redistribute and change agonist-induced calcium signaling. Cholesterol depletion from raft lead to increase in time to peak of calcium levels in agonist-evoked calcium signals in all instances, as seen by live imaging. This study emphasizes the necessity of a sub-population of proteins to cluster in specialized lipid domains. The requirement for such an organization at the raft-like microdomains may have implications on intercellular communication in the retina. Such concerted interaction at the rafts can regulate calcium dynamics and could add another layer of complexity to calcium signaling in cells.

  17. Effects of Sodium Tripolyphosphate, Microbial Transglutaminase and Enzyme-hydrolyzed Soy Protein Fraction on the Quality of Cooked Pork Batter by Response Surface Methodology

    Directory of Open Access Journals (Sweden)

    Xingjian Huang

    2014-11-01

    Full Text Available We investigated the compound effects of sodium tripolyphosphate (STPP, microbial transglutaminase (MTGase and enzyme-hydrolyzed soy protein fraction (denoted as TSF, molecular weight cut-off = 0.5 kDa to 10 kDa on the texture properties (hardness, springiness, cohesiveness and chewiness, cooking yield and sensory attributes (firmness, elasticity and juiciness of cooked pork batter. The hardness and springiness of the cooked pork batter were both significantly affected by the amount of MTGase and TSF added. In the presence of TSF, the textural characteristics of cooked pork batter were not significantly affected by STPP (p>0.05. The amount of TSF elicited negative linear (p<0.001 and positive quadratic effects (p<0.01 on the cohesiveness and chewiness of cooked pork batter. The interaction between MTGase and TSF positively affected (p<0.01 the cohesiveness of cooked pork batter. Furthermore, the amount of MTGase showed positive linear (p<0.01 effects on the chewiness of cooked pork batter. However, the interaction between STPP and TSF significantly weakened (p<0.05 the chewiness of cooked pork batter. Both TSF and MTGase positively affected (p<0.01 and p<0.05, respectively cooking yield. Both hardness versus firmness and springiness versus elasticity presented distinct correlations (p<0.01 and p<0.001, respectively. The cohesiveness and chewiness of cooked pork batter significantly affected cooking yield and sensory attributes (firmness, elasticity and juiciness. Overall acceptability poorly correlated with instrumental attributes and sensory partial attribute. Sensory analysis results indicated that the cooked pork batter with 0.4% MTGase, 4% TSF and 0.4% STPP was the most common sample, which presented the best synthetic mouth feeling.

  18. Levels and Age Dependency of Neurofilament Light and Glial Fibrillary Acidic Protein in Healthy Individuals and Their Relation to the Brain Parenchymal Fraction.

    Directory of Open Access Journals (Sweden)

    Mattias Vågberg

    Full Text Available Neurofilament light (NFL and Glial Fibrillary Acidic Protein (GFAP are integral parts of the axonal and astrocytal cytoskeletons respectively and are released into the cerebrospinal fluid (CSF in cases of cellular damage. In order to interpret the levels of these biomarkers in disease states, knowledge on normal levels in the healthy is required. Another biomarker for neurodegeneration is brain atrophy, commonly measured as brain parenchymal fraction (BPF using magnetic resonance imaging (MRI. Potential correlations between levels of NFL, GFAP and BPF in healthy individuals have not been investigated.To present levels of NFL and GFAP in healthy individuals stratified for age, and investigate the correlation between them as well as their correlation with BPF.The CSF was analysed in 53 healthy volunteers aged 21 to 70 (1 sample missing for GFAP analysis and 48 of the volunteers underwent determination of BPF using MRI.Mean (±SD NFL was 355 ng/L (±214, mean GFAP was 421 ng/L (±129 and mean BPF was 0.867 (±0.035. All three biomarkers correlated with age. NFL also correlated with both GFAP and BPF. When controlled for age, only the correlation between NFL and GFAP retained statistical significance.This study presents data on age-stratified levels of NFL and GFAP in the CSF of healthy individuals. There is a correlation between levels of NFL and GFAP and both increase with age. A correlation between NFL and BPF was also found, but did not retain statistical significance if controlled for age.

  19. Mercury distribution and lipid oxidation in fish muscle: Effects of washing and isoelectric protein precipitation

    Science.gov (United States)

    Gong, Y.; Krabbenhoft, D.P.; Ren, L.; Egelandsdal, B.; Richards, M.P.

    2011-01-01

    Nearly all the mercury (Hg) in whole muscle from whitefish (Coregonus clupeaformis) and walleye (Sander vitreus) was present as methyl mercury (MeHg). The Hg content in whole muscle from whitefish and walleye was 0.04-0.09 and 0.14-0.81 ppm, respectively. The myofibril fraction contained approximately three-fourths of the Hg in whitefish and walleye whole muscle. The sarcoplasmic protein fraction (e.g., press juice) was the next most abundant source of Hg. Isolated myosin, triacylglycerols, and cellular membranes contained the least Hg. Protein isolates prepared by pH shifting in the presence of citric acid did not decrease Hg levels. Addition of cysteine during washing decreased the Hg content in washed muscle probably through the interaction of the sulfhydryl group in cysteine with MeHg. Primary and secondary lipid oxidation products were lower during 2 ??C storage in isolates prepared by pH shifting compared to those of washed or unwashed mince from whole muscle. This was attributed to removing some of the cellular membranes by pH shifting. Washing the mince accelerated lipid peroxide formation but decreased secondary lipid oxidation products compared to that of the unwashed mince. This suggested that there was a lipid hydroperoxide generating system that was active upon dilution of aqueous antioxidants and pro-oxidants. ?? 2011 American Chemical Society.

  20. Proteomic analysis of lettuce seed germination and thermoinhibition by sampling of individual seeds at germination and removal of storage proteins by polyethylene glycol fractionation

    DEFF Research Database (Denmark)

    Wang, Wei-Qing; Song, Bin-Yan; Deng, Zhi-Jun;

    2015-01-01

    seeds at 25°C, a thermoinhibitory temperature. Before twodimensional gel electrophoresis analysis, storage proteins (greater than 50% of total extractable protein) were removed by polyethylene glycol precipitation, which significantly improved the detection of less abundant proteins on two...

  1. Role of the Calcium-Sensing Receptor in Cardiomyocyte Apoptosis via the Sarcoplasmic Reticulum and Mitochondrial Death Pathway in Cardiac Hypertrophy and Heart Failure

    Directory of Open Access Journals (Sweden)

    Fang-Hao Lu

    2013-05-01

    Full Text Available Aims: Alterations in calcium homeostasis in the intracellular endo/sarcoplasmic reticulum (ER/SR and mitochondria of cardiomyocytes cause cell death via the SR and mitochondrial apoptotic pathway, contributing to ventricular dysfunction. However, the role of the calcium-sensing receptor (CaR in cardiac hypertrophy and heart failure has not been studied. This study examined the possible involvement of CaR in the SR and mitochondrial apoptotic pathway in an experimental model of heart failure. Methods and Results: In Wistar rats, cardiac hypertrophy and heart failure were induced by subcutaneous injection of isoproterenol (Iso. Calindol, an activator of CaR, and calhex231, an inhibitor of CaR, were administered by caudal vein injection. Cardiac remodeling and left ventricular function were then analyzed in these rats. After 2, 4, 6 and 8 weeks after the administration of Iso, the rats developed cardiac hypertrophy and failure. The cardiac expression of ER chaperones and related apoptotic proteins was significantly increased in the failing hearts. Furthermore, the expression of ER chaperones and the apoptotic rate were also increased with the administration of calindol, whereas the expression of these proteins was reduced with the treatment of calhex231. We also induced cardiac hypertrophy and failure via thoracic aorta constriction (TAC in mice. After 2 and 4 weeks of TAC, the expression of ER chaperones and apoptotic proteins were increased in the mouse hearts. Furthermore, Iso induced ER stress and apoptosis in cultured cardiomyocytes, while pretreatment with calhex231 prevented ER stress and protected the myocytes against apoptosis. To further investigate the effect of CaR on the concentration of intracellular calcium, the calcium concentration in the SR and mitochondria was determined with Fluo-5N and x-rhod-1 and the mitochondrial membrane potential was examined with JC-1 using laser confocal microscopy. After treatment with Iso for 48 hours

  2. Role of the calcium-sensing receptor in cardiomyocyte apoptosis via the sarcoplasmic reticulum and mitochondrial death pathway in cardiac hypertrophy and heart failure.

    Science.gov (United States)

    Lu, Fang-Hao; Fu, Song-Bin; Leng, Xiaoning; Zhang, Xinying; Dong, Shiyun; Zhao, Ya-Jun; Ren, Huan; Li, Hulun; Zhong, Xin; Xu, Chang-Qing; Zhang, Wei-Hua

    2013-01-01

    Alterations in calcium homeostasis in the intracellular endo/sarcoplasmic reticulum (ER/SR) and mitochondria of cardiomyocytes cause cell death via the SR and mitochondrial apoptotic pathway, contributing to ventricular dysfunction. However, the role of the calcium-sensing receptor (CaR) in cardiac hypertrophy and heart failure has not been studied. This study examined the possible involvement of CaR in the SR and mitochondrial apoptotic pathway in an experimental model of heart failure. In Wistar rats, cardiac hypertrophy and heart failure were induced by subcutaneous injection of isoproterenol (Iso). Calindol, an activator of CaR, and calhex231, an inhibitor of CaR, were administered by caudal vein injection. Cardiac remodeling and left ventricular function were then analyzed in these rats. After 2, 4, 6 and 8 weeks after the administration of Iso, the rats developed cardiac hypertrophy and failure. The cardiac expression of ER chaperones and related apoptotic proteins was significantly increased in the failing hearts. Furthermore, the expression of ER chaperones and the apoptotic rate were also increased with the administration of calindol, whereas the expression of these proteins was reduced with the treatment of calhex231. We also induced cardiac hypertrophy and failure via thoracic aorta constriction (TAC) in mice. After 2 and 4 weeks of TAC, the expression of ER chaperones and apoptotic proteins were increased in the mouse hearts. Furthermore, Iso induced ER stress and apoptosis in cultured cardiomyocytes, while pretreatment with calhex231 prevented ER stress and protected the myocytes against apoptosis. To further investigate the effect of CaR on the concentration of intracellular calcium, the calcium concentration in the SR and mitochondria was determined with Fluo-5N and x-rhod-1 and the mitochondrial membrane potential was examined with JC-1 using laser confocal microscopy. After treatment with Iso for 48 hours, activation of CaR reduced [Ca(2+)]SR

  3. Digoxin activates sarcoplasmic reticulum Ca(2+)-release channels: a possible role in cardiac inotropy.

    Science.gov (United States)

    McGarry, S J; Williams, A J

    1993-04-01

    1. The effect of digoxin on rapid 45Ca2+ efflux from cardiac and skeletal sarcoplasmic reticulum (SR) vesicles was investigated. Additionally the interaction of digoxin with single cardiac and skeletal muscle SR Ca(2+)-release channels incorporated into planar phospholipid bilayers and held under voltage clamp was determined. 2. Digoxin (1 nM) increased the initial rate and amount of Ca(2+)-induced release of 45Ca2+ from cardiac SR vesicles, passively loaded with 45CaCl2, at an extravesicular [Ca2+] of 0.1 microM. The efflux in the presence and absence of digoxin was inhibited at pM extravesicular Ca2+ and blocked by 5 mM Mg2+. 3. To elucidate the mechanism of action of digoxin, single-channel recording was used. Digoxin (1-20 nM) increased single-channel open probability (Po) when added to the cytosolic but not the luminal face of the cardiac channel in the presence of sub-maximally activating Ca2+ (0.1 microM-10 microM) with an EC50 of 0.91 nM at 10 microM Ca2+. The mechanisms underlying the action of digoxin appear to be concentration-dependent. The activation observed at 1 nM digoxin appears to be consistent with the sensitization of the channel to the effects of Ca2+. At higher concentrations the drug appears to interact synergistically with Ca2+ to produce values of Po considerably greater than those seen with Ca2+ as the sole activating ligand. 4. Digoxin had no effect on single-channel conductance or the Ca2+/Tris permeability ratio. In channels activated by digoxin the Po was decreased by Mg2+. Single-channels were characteristically modified to along lasting open, but reduced, conductance state when 100 nM ryanodine was added to the cytosolic side of the channel.5. Activation of the cardiac SR Ca2+-release channel was observed with similar concentrations of digitoxin, however, higher concentrations of ouabain were required to increase PO. In contrast, a steroid which is not positively inotropic, chlormadinone acetate, had no effect on either cardiac or

  4. Sarcoplasmic reticulum calcium release in frog skeletal muscle fibres estimated from Arsenazo III calcium transients.

    Science.gov (United States)

    Baylor, S M; Chandler, W K; Marshall, M W

    1983-01-01

    Single twitch fibres, dissected from frog muscle, were injected with the metallochromic dye Arsenazo III. Changes in dye-related absorbance measured at 650 or 660 nm were used to estimate the time course of myoplasmic free [Ca2+] following either action potential stimulation or voltage-clamp depolarization (temperature, 15-17 degrees C). The amplitude of the Ca2+ transient decreased when fibres were stretched to sarcomere spacings approaching 4 microns. The effect appeared to be less marked in H2O Ringer than in D2O Ringer, where a reduction of about 40% was observed in going from 3.0 microns to 3.7-3.9 microns. In fibres heavily injected with dye (1.5-2.2 mM-dye) at least 0.1 mM-Ca2+ was complexed with Arsenazo III following a single action potential, implying that at least 0.1 mM-Ca2+ was released from the sarcoplasmic reticulum (s.r.) into the myoplasm. Computer simulations were carried out to estimate the flux of Ca2+ between the s.r. and myoplasm (in fibres containing no more that 0.8 mM-dye). The amounts and time courses of Ca2+ bound to the Ca2+-regulatory sites on troponin and to the Ca2+, Mg2+ sites on parvalbumin were estimated from the free [Ca2+] wave form and the law of mass action. In the computations the total myoplasmic [Ca2+] was taken as the total amount of Ca2+ existing either as free ion or as ion complexed with dye, troponin or parvalbumin. The time derivative of total myoplasmic [Ca2+] was used as an estimate of net Ca2+ flux (release minus uptake) from the s.r. into myoplasm. Rate constants for formation of cation: receptor complex were taken from published values. For the Ca2+-regulatory sites on troponin, three sets of rate constants, corresponding to two values of dissociation constant (0.2 and 2 microM) were used. Each set of three simulations was carried out both with and without parvalbumin. The simulations show that following action potential stimulation, 0.2-0.3 mM-Ca2+ enters the myoplasm from the s.r. The wave form of s.r. Ca2

  5. The changes of cardioelectrical activity of rat with myocardial infarction receiving sarcoplasmic reticulum Ca2+-ATPase gene modified bone marrow stem cell transplantation by microelectrode array technology

    Institute of Scientific and Technical Information of China (English)

    范平

    2012-01-01

    Objective Therapy effects and cardiac electrical activity comparison of bone marrow stem cells (BMSCs) transplantation and sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) gene modified BMSCs transplantation after acute myocardial infarction(AMI) in rats.Methods Rats with AMI were divided

  6. Fractional complex transforms for fractional differential equations

    National Research Council Canada - National Science Library

    Ibrahim, Rabha W

    2012-01-01

    The fractional complex transform is employed to convert fractional differential equations analytically in the sense of the Srivastava-Owa fractional operator and its generalization in the unit disk...

  7. Transitions of protein traffic from cardiac ER to junctional SR

    OpenAIRE

    Sleiman, Naama H.; McFarland, Timothy P.; Jones, Larry R.; Cala, Steven E.

    2015-01-01

    The junctional sarcoplasmic reticulum (jSR) is an important and unique ER subdomain in the adult myocyte that concentrates resident proteins to regulate Ca2+ release. To investigate cellular mechanisms for sorting and trafficking proteins to jSR, we overexpressed canine forms of junctin (JCT) or triadin (TRD) in adult rat cardiomyocytes. Protein accumulation over time was visualized by confocal fluorescence microscopy using species-specific antibodies. Newly synthesized JCTdog and TRDdog appe...

  8. Fractional complex transform for fractional differential equations

    National Research Council Canada - National Science Library

    Lİ, Zheng Biao; HE, Ji Huan

    2010-01-01

    Fractional complex transform is proposed to convert fractional differential equations into ordinary differential equations, so that all analytical methods devoted to advanced calculus can be easily...

  9. Effects of Preslaughter Stress Levels on the Post-mortem Sarcoplasmic Proteomic Profile of Gilthead Seabream Muscle

    DEFF Research Database (Denmark)

    Silva, Tomé Santos; Cordeiro, Odete D; Matos, Elisabete D.

    2012-01-01

    affects these post-mortem processes. For the experiment, two groups of gilthead seabream (n = 5) were subjected to distinct levels of preslaughter stress, with three muscle samples being taken from each fish. Proteins were extracted from the muscle samples, fractionated, and separated by 2DE. Protein......Fish welfare is an important concern in aquaculture, not only due to the ethical implications but also for productivity and quality-related reasons. The purpose of this study was to track soluble proteome expression in post-mortem gilthead seabream muscle and to observe how preslaughter stress...... been hastened by preslaughter stress, confirming that it induces clear post-mortem changes in the muscle proteome of gilthead seabream....

  10. Temperature and Ca2+-dependence of the sarcoplasmic reticulum Ca2(+)-ATPase in haddock, salmon, rainbow trout and zebra cichlid

    DEFF Research Database (Denmark)

    Godiksen, Helene; Jessen, Flemming

    2002-01-01

    Temperature dependence of Ca2+-ATPase from the sarcoplasmic reticulum (SR) in rabbit muscle has been widely studied, and it is generally accepted that a break point in Arrhenius plot exist at approximately 20 degreesC. Whether the break point arises as a result of temperature dependent changes...... nigrofasciatum). The Arrhenius plot of zebra cichlid showed a break point at 20 degreesC, and the haddock Arrhenius plot was non-linear with pronounced changes in slope in the. temperature area, 6-14 degreesC. In Arrhenius plot from both salmon and rainbow trout a plateau exists with an almost constant SR Ca2.......5) between 60 and 250 muM. Results indicated that interaction between SR Ca2+-ATPase and its lipid environment may play an important role for the different Arrhenius plot of the different types of fish species investigated. (C) 2002 Elsevier Science Inc. All rights reserved....

  11. Protein fraction and digestibility of marandu, xaraes and campo grande grasses in monocropping and intercropping systems under different sowing methods - doi: 10.4025/actascianimsci.v35i1.15134

    Directory of Open Access Journals (Sweden)

    Welma Santos Crunivel

    2013-01-01

    Full Text Available A study was carried out to evaluate the protein fraction and in vitro dry matter digestibility of marandu, xaraes grasses and campo grande in monocropping and intercropping systems under different planting methods, for a period of two years. The experimental design was a complete randomized block with four replications. The treatments consisted of the following crop systems: campo grande in monocropping; xaraés grass in monocropping; marandu grass in monocropping; xaraés intercropped with campo grande in rows; xaraés intercropped with campo grande, broadcast; marandu grass intercropped with campo grande in rows; and marandu intercropped with campo grand, broadcast. The evaluations were conducted for two years, consisting of seasonal evaluations (autumn, winter, spring and summer in the same plots, with repeated measurements over time. The results showed that xaraes and marandu grasses were similar between crop systems, indicating that both can be intercropped with campo grande. The intercropping of campo grande with Brachiaria brizantha cultivars improved the protein fraction and digestibility. The row method of planting provided better protein fractions and in vitro dry matter digestibility.

  12. Meadow based Fraction Theory

    OpenAIRE

    Bergstra, Jan A.

    2015-01-01

    In the context of an involutive meadow a precise definition of fractions is formulated and on that basis formal definitions of various classes of fractions are given. The definitions follow the fractions as terms paradigm. That paradigm is compared with two competing paradigms for storytelling on fractions: fractions as values and fractions as pairs.

  13. Pitavastatin-attenuated cardiac dysfunction in mice with dilated cardiomyopathy via regulation of myocardial calcium handling proteins

    Directory of Open Access Journals (Sweden)

    Hu Wei

    2014-03-01

    Full Text Available C57BL/6 mice with dilated cardiomyopathy (DCM were randomly divided to receive placebo or pitavastatin at a dose of 1 or 3 mg kg-1d-1. After 8 weeks treatment, mice with dilated cardiomyopathy developed serious cardiac dysfunction characterized by significantly enhanced left ventricular end-diastolic diameter (LVIDd, decreased left ventricular ejection fraction (LVEF as well as left ventricular short axis fractional shortening (LVFS, accompanied with enlarged cardiomyocytes, and increased plasma levels of N-terminal pro-B type natriuretic peptide (NT-proBNP and plasma angiotensin II (AngII concentration. Moreover, myocardium sarcoplasmic reticulum Ca2+ pump (SERCA-2 activity was decreased. The ratio of phosphorylated phospholamban (PLB to total PLB decreased significantly with the down-regulation of SERCA- -2a and ryanodine receptor (RyR2 expression. Pitavastatin was found to ameliorate the cardiac dysfunction in mice with dilated cardiomyopathy by reversing the changes in the ratios of phosphorylated PLB to total PLB, SERCA-2a and RyR2 via reducing the plasma AngII concentration and the expressions of myocardium angiotensin II type 1 receptor (AT1R and protein kinase C (PKCb2. The possible underlying mechanism might be the regulation of myocardial AT1R-PKCb2-Ca2+ handling proteins.

  14. Protein

    Science.gov (United States)

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

  15. 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

    2012-01-15

    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.

  16. Role of Mitochondrial Enzymes and Sarcoplasmic ATPase in Cardioprotection Mediated by Aqueous Extract of Desmodium gangeticum (L) DC Root on Ischemic Reperfusion Injury.

    Science.gov (United States)

    Kurian, G A; Paddikkala, J

    2010-11-01

    The present study investigate the protective effect of aqueous root extract of Desmodium gangeticum in preserving mitochondrial and sarcoplasmic ATPase during ischemia reperfusion injury. The isolated rat hearts in both drug and control group were subjected to warm ischemia (37°), followed by reperfusion with the Langendorff perfusion system. The aqueous root extract of Desmodium gangeticum (L) at a dose of 50 mg/kg body weight was found to be effective in the rat heart for the management of ischemic reperfusion injury. Physiological parameters were significantly (PDesmodium gangeticum treated rat heart. These results suggest that Desmodium gangeticum aqueous root extract can preserve the mitochondrial and sarcoplasmic ATPase in the myocardium, resulting in the improvement of cardiac function after ischemia reperfusion injury.

  17. Fracionamento de proteína e carboidratos em silagens de capim-elefante contendo subprodutos agrícolas Protein and carbohydrate fractioning in elephantgrass silage with agricultural by-products

    Directory of Open Access Journals (Sweden)

    Izabela Vieira Oliveira Andrade

    2010-11-01

    Full Text Available Um experimento foi conduzido para determinar o fracionamento de carboidratos e proteína da silagem de capim-elefante contendo farelo de mandioca, casca de café farelo de cacau. Utilizou-se o delineamento experimental inteiramente casualizado, em esquema fatorial 3 × 4, composto de três subprodutos adicionados ao capim-elefante em quatro níveis (0, 10, 20 e 30% da matéria natural, cada um com cinco repetições. O material foi ensilado em silos de PVC, que permaneceram fechados por 60 dias. O farelo de mandioca contribuiu para redução do teor de nitrogênio insolúvel em detergente ácido, apresentando os maiores valores para a fração A da proteína e para os teores das frações B1+B2. Em contrapartida, o farelo de cacau proporcionou acréscimo da fração C, aumentando significativamente o teor de proteína indisponível para os microrganismos ruminais. A adição do farelo de mandioca, seguido da casca de café, proporcionou os maiores teores de carboidratos totais em todos os níveis utilizados. As frações A+B1 dos carboidratos aumentaram de acordo com os níveis de subprodutos adicionados, e a casca de café foi responsável pelo menor teor dessa fração nas silagens. O farelo de cacau favoreceo aumento das frações nitrogenadas, porém a elevação do teor de nitrogênio insolúvel em detergente ácido, fração C, das silagens produzidas com este aditivo é um fator limitante. O farelo de mandioca aumenta o teor de carboidratos não-fibrosos da silagem, enquanto a casca de café e o farelo de cacau aumentam a fração não-digerível dos carboidratos.The experiment was conducted to determine fractioning of carbohydrate and protein of elephantgrass silage containing cassava meal, coffee hulls and cocoa meal. A completely randomized experimental design was used in a 3 × 4 factorial scheme, composed of three byproducts added to elephantgrass at four levels (0, 10, 20 and 30% of natural matter, each one with five repetitions

  18. A novel artificial microRNA expressing AAV vector for phospholamban silencing in cardiomyocytes improves Ca2+ uptake into the sarcoplasmic reticulum.

    Directory of Open Access Journals (Sweden)

    Tobias Gröβl

    Full Text Available In failing rat hearts, post-transcriptonal inhibition of phospholamban (PLB expression by AAV9 vector-mediated cardiac delivery of short hairpin RNAs directed against PLB (shPLBr improves both impaired SERCA2a controlled Ca2+ cycling and contractile dysfunction. Cardiac delivery of shPLB, however, was reported to cause cardiac toxicity in canines. Thus we developed a new AAV vector, scAAV6-amiR155-PLBr, expressing a novel engineered artificial microRNA (amiR155-PLBr directed against PLB under control of a heart-specific hybrid promoter. Its PLB silencing efficiency and safety were compared with those of an AAV vector expressing shPLBr (scAAV6-shPLBr from an ubiquitously active U6 promoter. Investigations were carried out in cultured neonatal rat cardiomyocytes (CM over a period of 14 days. Compared to shPLBr, amiR155-PLBr was expressed at a significantly lower level, resulting in delayed and less pronounced PLB silencing. Despite decreased knockdown efficiency of scAAV6-amiR155-PLBr, a similar increase of the SERCA2a-catalyzed Ca2+ uptake into sarcoplasmic reticulum (SR vesicles was observed for both the shPLBr and amiR155-PLBr vectors. Proteomic analysis confirmed PLB silencing of both therapeutic vectors and revealed that shPLBr, but not the amiR155-PLBr vector, increased the proinflammatory proteins STAT3, STAT1 and activated STAT1 phosphorylation at the key amino acid residue Tyr701. Quantitative RT-PCR analysis detected alterations in the expression of several cardiac microRNAs after treatment of CM with scAAV6-shPLBr and scAAV6-amiR155-PLBr, as well as after treatment with its related amiR155- and shRNAs-expressing control AAV vectors. The results demonstrate that scAAV6-amiR155-PLBr is capable of enhancing the Ca2+ transport function of the cardiac SR PLB/SERCA2a system as efficiently as scAAV6-shPLBr while offering a superior safety profile.

  19. Nitric oxide-dependent activation of CaMKII increases diastolic sarcoplasmic reticulum calcium release in cardiac myocytes in response to adrenergic stimulation.

    Directory of Open Access Journals (Sweden)

    Jerry Curran

    Full Text Available Spontaneous calcium waves in cardiac myocytes are caused by diastolic sarcoplasmic reticulum release (SR Ca(2+ leak through ryanodine receptors. Beta-adrenergic (β-AR tone is known to increase this leak through the activation of Ca-calmodulin-dependent protein kinase (CaMKII and the subsequent phosphorylation of the ryanodine receptor. When β-AR drive is chronic, as observed in heart failure, this CaMKII-dependent effect is exaggerated and becomes potentially arrhythmogenic. Recent evidence has indicated that CaMKII activation can be regulated by cellular oxidizing agents, such as reactive oxygen species. Here, we investigate how the cellular second messenger, nitric oxide, mediates CaMKII activity downstream of the adrenergic signaling cascade and promotes the generation of arrhythmogenic spontaneous Ca(2+ waves in intact cardiomyocytes. Both SCaWs and SR Ca(2+ leak were measured in intact rabbit and mouse ventricular myocytes loaded with the Ca-dependent fluorescent dye, fluo-4. CaMKII activity in vitro and immunoblotting for phosphorylated residues on CaMKII, nitric oxide synthase, and Akt were measured to confirm activity of these enzymes as part of the adrenergic cascade. We demonstrate that stimulation of the β-AR pathway by isoproterenol increased the CaMKII-dependent SR Ca(2+ leak. This increased leak was prevented by inhibition of nitric oxide synthase 1 but not nitric oxide synthase 3. In ventricular myocytes isolated from wild-type mice, isoproterenol stimulation also increased the CaMKII-dependent leak. Critically, in myocytes isolated from nitric oxide synthase 1 knock-out mice this effect is ablated. We show that isoproterenol stimulation leads to an increase in nitric oxide production, and nitric oxide alone is sufficient to activate CaMKII and increase SR Ca(2+ leak. Mechanistically, our data links Akt to nitric oxide synthase 1 activation downstream of β-AR stimulation. Collectively, this evidence supports the hypothesis

  20. A Novel Artificial MicroRNA Expressing AAV Vector for Phospholamban Silencing in Cardiomyocytes Improves Ca2+ Uptake into the Sarcoplasmic Reticulum

    Science.gov (United States)

    Größl, Tobias; Hammer, Elke; Bien-Möller, Sandra; Geisler, Anja; Pinkert, Sandra; Röger, Carsten; Poller, Wolfgang; Kurreck, Jens; Völker, Uwe

    2014-01-01

    In failing rat hearts, post-transcriptonal inhibition of phospholamban (PLB) expression by AAV9 vector-mediated cardiac delivery of short hairpin RNAs directed against PLB (shPLBr) improves both impaired SERCA2a controlled Ca2+ cycling and contractile dysfunction. Cardiac delivery of shPLB, however, was reported to cause cardiac toxicity in canines. Thus we developed a new AAV vector, scAAV6-amiR155-PLBr, expressing a novel engineered artificial microRNA (amiR155-PLBr) directed against PLB under control of a heart-specific hybrid promoter. Its PLB silencing efficiency and safety were compared with those of an AAV vector expressing shPLBr (scAAV6-shPLBr) from an ubiquitously active U6 promoter. Investigations were carried out in cultured neonatal rat cardiomyocytes (CM) over a period of 14 days. Compared to shPLBr, amiR155-PLBr was expressed at a significantly lower level, resulting in delayed and less pronounced PLB silencing. Despite decreased knockdown efficiency of scAAV6-amiR155-PLBr, a similar increase of the SERCA2a-catalyzed Ca2+ uptake into sarcoplasmic reticulum (SR) vesicles was observed for both the shPLBr and amiR155-PLBr vectors. Proteomic analysis confirmed PLB silencing of both therapeutic vectors and revealed that shPLBr, but not the amiR155-PLBr vector, increased the proinflammatory proteins STAT3, STAT1 and activated STAT1 phosphorylation at the key amino acid residue Tyr701. Quantitative RT-PCR analysis detected alterations in the expression of several cardiac microRNAs after treatment of CM with scAAV6-shPLBr and scAAV6-amiR155-PLBr, as well as after treatment with its related amiR155- and shRNAs-expressing control AAV vectors. The results demonstrate that scAAV6-amiR155-PLBr is capable of enhancing the Ca2+ transport function of the cardiac SR PLB/SERCA2a system as efficiently as scAAV6-shPLBr while offering a superior safety profile. PMID:24670775

  1. The Antioxidant Properties of Chickpea Protein Hydrolyzed Fractions%鹰嘴豆蛋白降解产物的抗氧化作用

    Institute of Scientific and Technical Information of China (English)

    陈晓飞; 孙玉飞; 冯菲; 王雪妍; 周伏忠

    2015-01-01

    旨在分析鹰嘴豆分离蛋白(CPI)及其不同分子量短肽的抗氧化活性。用碱性蛋白酶(Alcalase)处理CPI,得到其降解产物,该降解产物经超滤离心,得到分子量分别为10 kD的短肽。结果表明,与其它大分子肽段相比,10 kD, respectively, using membrane ultrafiltration. Results showed that, the <3 kD peptides exhibited better ferric reducing power and radicals scavenging activities when compared to peptide fractions of higher molecular weights. CPI and its peptide fractions had significant ability to chelate metal ions compared to glutathione(GSH), and also had some ferric reducing powers. The remarkable antioxidant properties indicate that CPI and its peptide fractions have the potential to be used in manufacturing antioxidant functional foods and healthy foods.

  2. Spontaneous Ca2+ release from the sarcoplasmic reticulum limits Ca2+- dependent twitch potentiation in individual cardiac myocytes. A mechanism for maximum inotropy in the myocardium

    OpenAIRE

    1988-01-01

    We hypothesized that the occurrence of spontaneous Ca2+ release from the sarcoplasmic reticulum (SR), in diastole, might be a mechanism for the saturation of twitch potentiation common to a variety of inotropic perturbations that increase the total cell Ca. We used a videomicroscopic technique in single cardiac myocytes to quantify the amplitude of electrically stimulated twitches and to monitor the occurrence of the mechanical manifestation of spontaneous SR Ca2+ release, i.e., the spontaneo...

  3. Proteomic analysis of lettuce seed germination and thermoinhibition by sampling of individual seeds at germination and removal of storage proteins by polyethylene glycol fractionation.

    Science.gov (United States)

    Wang, Wei-Qing; Song, Bin-Yan; Deng, Zhi-Jun; Wang, Yue; Liu, Shu-Jun; Møller, Ian Max; Song, Song-Quan

    2015-04-01

    Germination and thermoinhibition in lettuce (Lactuca sativa 'Jianyexianfeng No. 1') seeds were investigated by a proteomic comparison among dry seeds, germinated seeds at 15°C, at 15°C after imbibition at 25°C for 48 h, or at 25°C in KNO3 (all sampled individually at germination), and ungerminated seeds at 25°C, a thermoinhibitory temperature. Before two-dimensional gel electrophoresis analysis, storage proteins (greater than 50% of total extractable protein) were removed by polyethylene glycol precipitation, which significantly improved the detection of less abundant proteins on two-dimensional gels. A total of 108 protein spots were identified to change more than 2-fold (Pgermination treatment. Nineteen proteins increasing and one protein decreasing in abundance during germination had higher abundance in germinated 15°C, 15°C after imbibition at 25°C for 48 h, and 25°C in KNO3 seeds than in ungerminated 25°C seeds. Gene expression of 12 of those proteins correlated well with the protein accumulation. Methionine metabolism, ethylene production, lipid mobilization, cell elongation, and detoxification of aldehydes were revealed to be potentially related to lettuce seed germination and thermoinhibition. Accumulation of three proteins and expression of five genes participating in the mevalonate (MVA) pathway of isoprenoid biosynthesis correlated positively with seed germinability. Inhibition of this pathway by lovastatin delayed seed germination and increased the sensitivity of germination to abscisic acid. MVA pathway-derived products, cytokinins, partially reversed the lovastatin inhibition of germination and released seed thermoinhibition at 25°C. We conclude that the MVA pathway for isoprenoid biosynthesis is involved in lettuce seed germination and thermoinhibition.

  4. Coordinated collagen and muscle protein synthesis in human patella tendon and quadriceps muscle after exercise

    DEFF Research Database (Denmark)

    Miller, Benjamin F; Olesen, Jens L; Hansen, Mette

    2005-01-01

    We hypothesized that an acute bout of strenuous, non-damaging exercise would increase rates of protein synthesis of collagen in tendon and skeletal muscle but these would be less than those of muscle myofibrillar and sarcoplasmic proteins. Two groups (n = 8 and 6) of healthy young men were studied...... collagen (0.077% h(-1)), muscle collagen (0.054% h(-1)), myofibrillar protein (0.121% h(-1)), and sarcoplasmic protein (0.134% h(-1))). The rates decreased toward basal values by 72 h although rates of tendon collagen and myofibrillar protein synthesis remained elevated. There was no tissue damage...... of muscle visible on histological evaluation. Neither tissue microdialysate nor serum concentrations of IGF-I and IGF binding proteins (IGFBP-3 and IGFBP-4) or procollagen type I N-terminal propeptide changed from resting values. Thus, there is a rapid increase in collagen synthesis after strenuous exercise...

  5. Matrix fractional systems

    Science.gov (United States)

    Tenreiro Machado, J. A.

    2015-08-01

    This paper addresses the matrix representation of dynamical systems in the perspective of fractional calculus. Fractional elements and fractional systems are interpreted under the light of the classical Cole-Cole, Davidson-Cole, and Havriliak-Negami heuristic models. Numerical simulations for an electrical circuit enlighten the results for matrix based models and high fractional orders. The conclusions clarify the distinction between fractional elements and fractional systems.

  6. Toxic effects of copper sulfate and copper nanoparticles on minerals, enzymes, thyroid hormones and protein fractions of plasma and histopathology in common carp Cyprinus carpio.

    Science.gov (United States)

    Hoseini, Seyyed Morteza; Hedayati, Aliakbar; Taheri Mirghaed, Ali; Ghelichpour, Melika

    2016-10-01

    Differences in toxicological effects of dissolved copper and copper nanoparticles were studied in common carp, Cyprinus carpio. The fish were exposed to 0.25mgL(-1) copper as copper sulfate (0.25Cu), 0.25mgL(-1) copper as copper oxide nanoparticles (0.25NCu) and 25mgL(-1) copper as copper oxide nanoparticles (25NCu) over 14days. Plasma biochemical, enzymatic and hormonal characteristics, and liver and kidney histopathology were examined at the end of the experiment. The results showed that both forms of copper had no significant effects on plasma calcium levels, however, significantly increased plasma phosphorous levels, compared to control group (no added copper). Plasma alanine transaminase (ALT) activity increased in 0.25Cu and 25NCu treatments compared to the control and 0.25NCu treatments. Nanoparticle copper exposure significantly decreased plasma alkaline phosphatase (ALP) activity compared to the control and 0.25Cu treatments. Only copper sulfate exposure caused plasma aspartate transaminase (AST) elevation. Both copper forms increased plasma T4 and free T4 (FT4); however, copper sulfate effect was higher than nanoparticle copper. Copper sulfate exposure increased plasma albumin fraction, whereas, 25mgL(-1) copper nanoparticle exposure increased plasma α2-globulin fraction compared to the control. Both copper forms damaged the fish liver and kidney, however, copper sulfate caused more severe damages compared to nanoparticle copper. Overall, except for plasma ALP and α2-globulin fraction, dissolved copper seems to be more toxic than nanoparticle copper in common carp. Copyright © 2016 Elsevier GmbH. All rights reserved.

  7. Effect of Zn2+ ions on ryanodine binding to sarcoplasmic reticulum of striated muscles in the presence of pyrithione

    Institute of Scientific and Technical Information of China (English)

    Hong XIE; Ke-ying CHEN; Pei-hong ZHU

    2004-01-01

    AIM: To explore whether the differential effects of Zn2+ on ryanodine binding to the sarcoplasmic reticulum (SR)of skeletal and cardiac muscles resulted from different permeability of the SR to Zn2+. METHODS: [3H]ryanodine binding assays were performed to examine the effect of Zn2+ on ryanodine binding to the SR in the presence of pyrithione sodium (PyNa), a specific Zn2+ ionophore. RESULTS: As a control, PyNa up to 50 μmol/L did not induce any effect on ryanodine binding to the SR of cardiac muscle. But PyNa 1-100 μmol/L increased ryanodine binding in skeletal muscle with maximum binding (222.2 %+20.9 % of the control) and inhibited ryanodine binding to 50 % of the control at about 500 μrnol/L. In the presence of PyNa 10 and 50 μmol/L the dose-dependence of the effect of Zn2+ in cardiac muscle was still monophasic and not changed by PyNa, while the biphasic effect of Zn2+in skeletal muscle became monophasic. CONCLUSION: Different permeability of the SR to Zn2+ may account for the differential effects of Zn2+on ryanodine binding in skeletal and cardiac muscles. PyNa is not a strictly specific Zn2+ ionophore.

  8. Effect of chloride on Ca2+ release from the sarcoplasmic reticulum of mechanically skinned skeletal muscle fibres.

    Science.gov (United States)

    Coonan, J R; Lamb, G D

    1998-04-01

    The effect of intracellular Cl- on Ca2+ release in mechanically skinned fibres of rat extensor digitorum longus (EDL) and toad iliofibularis muscles was examined under physiological conditions of myoplasmic [Mg2+] and [ATP] and sarcoplasmic reticulum (SR) Ca2+ loading. Both in rat and toad fibres, the presence of 20 mM Cl- in the myoplasm increased Ca2+ leakage from the SR at pCa (i.e. -log10 [Ca2+]) 6.7, but not at pCa 8. Ca2+ uptake was not significantly affected by the presence of Cl-. This Ca2+-dependent effect of Cl- on Ca2+ leakage was most likely due to a direct action on the ryanodine receptor/Ca2+ release channel, and could influence channel sensitivity and the resting [Ca2+] in muscle fibres in vivo. In contrast to this effect, acute addition of 20 mM Cl- to the myoplasm caused a 40-50% reduction in Ca2+ release in response to a low caffeine concentration both in toad and rat fibres. One possible explanation for this latter effect is that the addition of Cl- induces a potential across the SR (lumen negative) which might reduce Ca2+ release via several different mechanisms.

  9. Effects of thapsigargin and cyclopiazonic acid on the sarcoplasmic reticulum Ca2+ pump of skinned fibres from frog skeletal muscle.

    Science.gov (United States)

    Du, G G; Ashley, C C; Lea, T J

    1994-12-01

    Thapsigargin has been reported to inhibit ATP-dependent Ca2+ uptake by isolated sarcoplasmic reticulum (SR) vesicles of vertebrate skeletal muscle fibres at nanomolar concentrations. There have been no reports confirming this effect in skinned muscle fibre preparations. We have examined the ability of thapsigargin to inhibit the uptake of Ca2+ by the SR in mechanically skinned fibres of frog iliofibularis muscles, using the size of the caffeine-induced contracture to assess the Ca2+ content of the SR. The SR was first depleted of Ca2+ and then reloaded for 1 min at pCa 6.2 in the presence and absence of thapsigargin. When 5 min were allowed for diffusion, a thapsigargin concentration of at least 131 microM was required to inhibit Ca2+ loading by 50%. In contrast, another SR Ca2+ uptake inhibitor, cyclopiazonic acid, was more effective, producing 50% inhibition at 7.0 microM and total inhibition at 50 microM. When cyclopiazonic acid (100 microM) was applied after, rather than during, Ca2+ loading, the caffeine-induced contracture was not changed. Thapsigargin (300 microM), on the other hand, caused some reduction in the peak amplitude of the caffeine-induced contracture when applied after Ca2+ loading. The poor effectiveness of thapsigargin in the skinned fibres, compared with in SR vesicles, is attributed to its slow diffusion into the skinned fibres, perhaps as a result of binding to myofibrillar components.

  10. Influence of inorganic phosphate and pH on sarcoplasmic reticular ATPase in skinned muscle fibres of Xenopus laevis.

    Science.gov (United States)

    Stienen, G J; Papp, Z; Zaremba, R

    1999-08-01

    1. The influence of 30 mM inorganic phosphate (Pi) and pH (6.2-7.4) on the rate of ATP utilization was determined in mechanically skinned bundles of myofibrils from the iliofibularis muscle of Xenopus laevis at approximately 5 C. 2. BDM (2,3-butanedione monoxime; 10 mM) depressed isometric force production and actomyosin (AM) ATPase activity equally. Therefore sarcoplasmic reticular (SR) ATPase activity could be determined by extrapolation of the total ATPase activity to zero force. 3. The SR ATPase activity without added Pi at pH 7.1 was 42 +/- 2 % of the total ATPase activity. Addition of 30 mM Pi reduced SR ATPase activity slightly, by 9 +/- 5 %, and depressed force by 62 +/- 2 % and AM ATPase activity by 21 +/- 6 %. 4. At pH 6.2, force, SR ATPase activity and AM ATPase activity were reduced by 21 +/- 5, 61 +/- 5 and 10 +/- 4 % of their respective values at pH 7.1. 5. The SR ATPase activity at 30 mM Pi and pH 6.2 was reduced markedly to 20 +/- 6 % of the value under control conditions, suggesting that the maximum rate of Ca2+ uptake during muscle fatigue was strongly depressed. This reduction was larger than expected on the basis of the effects of Pi and pH alone.

  11. Cellular mechanisms of reduced sarcoplasmic reticulum Ca2+ content in L-thyroxin-induced rat ventricular hypertrophy

    Institute of Scientific and Technical Information of China (English)

    Lai-jing SONG; Guan-lei WANG; Jie LIU; Qin-ying QIU; Jing-hua OU; Yong-yuan GUAN

    2008-01-01

    Aim:To examine how the sarcoplasmic reticulum (SR) Ca2+ content changes and the underlying mechanism in L-thyroxin-induced cardiac hypertrophy. Methods:Echocardiography was used to confirm the establishment of the cardiac hypertro-phy model. The confocal microscopy and fluorescent indicator Fluo-3 was ap-plied to examine the intracellular Ca2+ concentration ([Ca2+]I), the Ca2+ sparks, and the caffeine-induced Ca2+ transient in freshly isolated cardiac ventricular myocytes. The activity of sarcolemmal and SR Ca2+-ATPase 2a (SERCA2a) in the ventricular tissue was also measured, respectively. Results:L-thyroxin (1 mg/kg injection for 10 d) induces left ventricular cardiac hypertrophy with normal myocardial function. The decreased caffeine-induced Ca2+ transient in the Ca2+-free solution was detected. The spontaneous Ca2+ sparks in hypertrophied myocytes occurred more frequently than in normal cells, with similar duration and spatial spread, but smaller amplitude. Then the basal [Ca2+]I increase was observed in quiescent left ventricular myocytes from hyperthyroidism rats. The activity of sarcolemmal and SR Ca2+-ATPase was decreased in the hypertrophied ventricle tissue. Conclusion:The results suggested that the reduced SR Ca2+ content may be associated with an increased Ca2+ leak and reduced SERCA2a activity, contributing to abnormal intracellular Ca2+ handling during hypertrophy in hyperthyroidism rats.

  12. Effects of vacuum and modified atmosphere on textural parameters and structural proteins of cultured meagre (Argyrosomus regius) fillets.

    Science.gov (United States)

    Sáez, María I; Martínez, Tomás F; Cárdenas, Salvador; Suárez, María D

    2015-09-01

    The influence of two preservation strategies (vacuum package and modified atmosphere package) on the post-mortem changes of textural parameters, pH, water holding capacity, sarcoplasmic and myofibrillar proteins, and collagen content of meagre (Argyrosomus regius) fillets was studied. Fillets were stored in a cold room in aerobic (control, C), vacuum (V) and modified atmosphere (MA) package. Samples were withdrawn at six sampling points throughout 15-day storage, and post-mortem changes were assessed. The textural parameters were significantly enhanced in V and MA compared to C. Both V and MA treatments reduced the intensity of a group of myofibrillar protein fractions (140-195 kDa) and increased insoluble collagen compared to C. Consequently, the post-mortem flesh softening in C was attributed to increased proteolysis in both intracellular and extracellular structural proteins. The preservation of the textural and biochemical characteristics of meagre fillets subjected to V and MA treatments makes these two treatments highly recommendable for the commercialization of meagre fillets. © The Author(s) 2014.

  13. Characterization of flagellar cysteine-rich sperm proteins involved in motility, by the combination of cellular fractionation, fluorescence detection, and mass spectrometry analysis.

    Science.gov (United States)

    Cabrillana, María E; Monclus, María A; Sáez Lancellotti, Tania E; Boarelli, Paola V; Clementi, Marisa A; Vincenti, Amanda E; Yunes, Roberto F M; Fornés, Miguel W

    2011-09-01

    Mammalian sperm proteins undergo thiol group (SH) oxidation to form disulfides bonds (SS) as they travel through the epididymis during cell maturation. Disulfide bonds are involved in chromatin condensation and tail organelle stabilization. In this work, we used a fluorescent thiol-selective labeling agent, monobromobimane (mBBr), to study the protein thiol status of rat sperm during maturation. Fluorescence signal decrease along the epididymal trip, more evidently in the head, but also in the tail, indicates that both sub cellular regions participate in the thiol changes. The sources of the fluorescence signal are sulfhydryls sperm proteins labeled by mBBr (mBBr-spp). Initial attempts to identify the mBBr-spp labeled were detected in the initial-caput, but not in the distal cauda-segment of the epididymis in sodium dodecyl sulfate (SDS)-PAGE analysis. This phenomenon could be due to protein resistance to solubilization. For this reason, disulfide bond reduction was accomplished by sodium dodecyl sulfate plus dithiothreitol treatment to recover the mBBr signal in SDS-PAGE. Under this protocol, a major 27 kDa protein band displays a strong signal. Protein identification by mass spectrometry and sequence database searching correlated this protein with the outer dense fiber 1 (ODF1). The mBBr specifically bound to N-terminal domain cysteine of ODF1. The mBBr reduces rat sperm motility, quantitatively and qualitatively, and the effects are dose dependent, without significantly increasing the percentage of dead sperm. Thus, we found that ODF1 is highly responsible for mBBr fluorescence detection in the sperm tail, and the motility inhibition by the fluorescence marker indicates that ODF1 N-terminal domain are related to sperm motility. © 2011 Wiley-Liss, Inc.

  14. Testing the dependence of stabilizing effect of osmolytes on the fractional increase in the accessible surface area on thermal and chemical denaturations of proteins.

    Science.gov (United States)

    Rahman, Safikur; Ali, Syed Ausaf; Islam, Asimul; Hassan, Md Imtaiyaz; Ahmad, Faizan

    2016-02-01

    Here we have generated two different denatured states using heat- and guanidinium chloride (GdmCl)-induced denaturations of three disulfide bond free proteins (barstar, cytochrome-c and myoglobin). We have observed that these two denatured states of barstar and myoglobin are structurally and energetically different, for, heat-induced denatured state contains many un-melted residual structure that has a significant amount of secondary and tertiary interactions. We show that structural properties of the denatured state determine the magnitude of the protein stabilization in terms of Gibbs free energy change (ΔGD°) induced by an osmolyte, i.e., the greater the exposed surface area, the greater is the stabilization. Furthermore, we predicted the m-values (ability of osmolyte to fold or unfold proteins) using Tanford's transfer-free energy model for the transfer of proteins to osmolyte solutions. We observed that, for each protein, m-value is comparable with our experimental data in cases of TMAO (trimethylamine-N-oxide) and sarcosine. However, a significant discrepancy between predicted and experimental m-values were observed in the case of glycine-betaine.

  15. Proteomic Analysis of Lettuce Seed Germination and Thermoinhibition by Sampling of Individual Seeds at Germination and Removal of Storage Proteins by Polyethylene Glycol Fractionation1

    Science.gov (United States)

    Song, Bin-Yan; Deng, Zhi-Jun; Wang, Yue; Liu, Shu-Jun; Møller, Ian Max; Song, Song-Quan

    2015-01-01

    Germination and thermoinhibition in lettuce (Lactuca sativa ‘Jianyexianfeng No. 1’) seeds were investigated by a proteomic comparison among dry seeds, germinated seeds at 15°C, at 15°C after imbibition at 25°C for 48 h, or at 25°C in KNO3 (all sampled individually at germination), and ungerminated seeds at 25°C, a thermoinhibitory temperature. Before two-dimensional gel electrophoresis analysis, storage proteins (greater than 50% of total extractable protein) were removed by polyethylene glycol precipitation, which significantly improved the detection of less abundant proteins on two-dimensional gels. A total of 108 protein spots were identified to change more than 2-fold (P ethylene production, lipid mobilization, cell elongation, and detoxification of aldehydes were revealed to be potentially related to lettuce seed germination and thermoinhibition. Accumulation of three proteins and expression of five genes participating in the mevalonate (MVA) pathway of isoprenoid biosynthesis correlated positively with seed germinability. Inhibition of this pathway by lovastatin delayed seed germination and increased the sensitivity of germination to abscisic acid. MVA pathway-derived products, cytokinins, partially reversed the lovastatin inhibition of germination and released seed thermoinhibition at 25°C. We conclude that the MVA pathway for isoprenoid biosynthesis is involved in lettuce seed germination and thermoinhibition. PMID:25736209

  16. Pressure effects on the binding of vanadate to the sarcoplasmic reticulum calcium-transport enzyme.

    Science.gov (United States)

    Ronzani, N; Stephan, L; Hasselbach, W

    1991-10-01

    The effect which hydrostatic pressure exerts on the binding of vanadate to the calcium-transport enzyme was determined. The recent unavailability of radioactive vanadate prevented direct measurements of vanadate binding. The vanadate-free enzyme fraction was instead monitored by phosphorylating it with ATP according to Medda and Hasselbach [Medda, P. & Hasselbach, W. (1983) Eur. J. Biochem. 137, 7-14]. Vanadate binding is reduced with rising pressure at first markedly and subsequently, above 30 MPa, relatively little. The biphasic pressure-binding relationship was analysed by applying a biexponential fitting procedure to the experimental data. The biphasicity of the pressure-binding relationship indicates that the description of vanadate binding requires at least a two-step reaction sequence. The volume increments which predominate at lower pressure values, range from 200-400 ml.mol-1 depending on the composition of the reaction medium containing 5 microM and 20 microM vanadate and no or 15% (by vol.) Me2SO. The binding volumes deduced for the higher pressure range amount to 20-40 ml.mol-1. Vanadate binding is reduced in the presence of 30 microM calcium, and simultaneously both binding volumes are diminished by 100 ml.mol-1 and 20 ml.mol-1 for the low and high pressure values, respectively, as one can expect for mutual interactions between the two ligands of the transport enzyme.

  17. Initialized Fractional Calculus

    Science.gov (United States)

    Lorenzo, Carl F.; Hartley, Tom T.

    2000-01-01

    This paper demonstrates the need for a nonconstant initialization for the fractional calculus and establishes a basic definition set for the initialized fractional differintegral. This definition set allows the formalization of an initialized fractional calculus. Two basis calculi are considered; the Riemann-Liouville and the Grunwald fractional calculi. Two forms of initialization, terminal and side are developed.

  18. Pasta supplemented with isolated lupin protein fractions reduces body weight gain and food intake of rats and decreases plasma glucose concentration upon glucose overload trial.

    Science.gov (United States)

    Capraro, Jessica; Magni, Chiara; Scarafoni, Alessio; Caramanico, Rosita; Rossi, Filippo; Morlacchini, Mauro; Duranti, Marcello

    2014-02-01

    The supplementation of foods with biologically active compounds can be a powerful approach for improving diet and well being. In this study we separately included in pasta matrices a concentrate of γ-conglutin, a glucose-lowering protein from Lupinus albus seeds, an isolate of the other main lupin storage proteins and ovalbumin, at a ratio corresponding to 125 mg of pure protein in 100 g of pasta. With these products we fed rats made hyperglycaemic, for 3 weeks. Among the most relevant changes measured in body and blood parameters were: (i) a significant reduction in food intake of rats fed γ-conglutin concentrate supplemented pasta and a significant limitation in the body weight increase in rats fed α, β and δ-conglutin isolate supplemented pasta, while the food conversion indices were unchanged; (ii) a reduction in glycaemia upon glucose overload trial, especially in the γ-conglutin concentrate supplemented pasta fed animals, at a dose of 45 mg per kg body weight. The correlations among the measured parameters are discussed. Overall, the results evidence the potentiality of supplementing traditional foods with exogenous nutraceutical seed proteins to control body weight gain and glycaemia.

  19. Combined N-glycome and N-glycoproteome analysis of the Lotus japonicus seed globulin fraction shows conservation of protein structure and glycosylation in legumes

    DEFF Research Database (Denmark)

    Dam, Svend Secher; Thaysen-Andersen, Morten; Stenkjær, Eva

    2013-01-01

    interactions and, due to its well characterized genotype/phenotype and easily manipulated genome, may also be suitable for studies of legume food allergy. Here we present a comprehensive study of the Lotus N-glycoproteome. The global and site-specific N-glycan structures of Lotus seed globulins were analyzed...... β-1,2-xylose and α-1,3-fucose. Two abundant Lotus seed N-glycoproteins were site-specifically profiled; a predicted lectin containing two fully occupied N-glycosylation sites carried predominantly pauci-mannosidic structures in different distributions. In contrast, Lotus convicilin storage protein 2...... the potential to serve as a model system for studying the role of seed proteins and their glycosylation in food allergy....

  20. Ryanodine receptor fragmentation and sarcoplasmic reticulum Ca2+ leak after one session of high-intensity interval exercise.

    Science.gov (United States)

    Place, Nicolas; Ivarsson, Niklas; Venckunas, Tomas; Neyroud, Daria; Brazaitis, Marius; Cheng, Arthur J; Ochala, Julien; Kamandulis, Sigitas; Girard, Sebastien; Volungevičius, Gintautas; Paužas, Henrikas; Mekideche, Abdelhafid; Kayser, Bengt; Martinez-Redondo, Vicente; Ruas, Jorge L; Bruton, Joseph; Truffert, Andre; Lanner, Johanna T; Skurvydas, Albertas; Westerblad, Håkan

    2015-12-15

    High-intensity interval training (HIIT) is a time-efficient way of improving physical performance in healthy subjects and in patients with common chronic diseases, but less so in elite endurance athletes. The mechanisms underlying the effectiveness of HIIT are uncertain. Here, recreationally active human subjects performed highly demanding HIIT consisting of 30-s bouts of all-out cycling with 4-min rest in between bouts (≤3 min total exercise time). Skeletal muscle biopsies taken 24 h after the HIIT exercise showed an extensive fragmentation of the sarcoplasmic reticulum (SR) Ca(2+) release channel, the ryanodine receptor type 1 (RyR1). The HIIT exercise also caused a prolonged force depression and triggered major changes in the expression of genes related to endurance exercise. Subsequent experiments on elite endurance athletes performing the same HIIT exercise showed no RyR1 fragmentation or prolonged changes in the expression of endurance-related genes. Finally, mechanistic experiments performed on isolated mouse muscles exposed to HIIT-mimicking stimulation showed reactive oxygen/nitrogen species (ROS)-dependent RyR1 fragmentation, calpain activation, increased SR Ca(2+) leak at rest, and depressed force production due to impaired SR Ca(2+) release upon stimulation. In conclusion, HIIT exercise induces a ROS-dependent RyR1 fragmentation in muscles of recreationally active subjects, and the resulting changes in muscle fiber Ca(2+)-handling trigger muscular adaptations. However, the same HIIT exercise does not cause RyR1 fragmentation in muscles of elite endurance athletes, which may explain why HIIT is less effective in this group.

  1. Comparative studies of cardiac and skeletal sarcoplasmic reticulum ATPases. Effect of a phospholamban antibody on enzyme activation by Ca2+.

    Science.gov (United States)

    Cantilina, T; Sagara, Y; Inesi, G; Jones, L R

    1993-08-15

    Vesicular fragments of skeletal (LSR) and cardiac (CSR) sarcoplasmic reticulum were compared with the aim of defining the effect of a monoclonal phospholamban (Pl) antibody (Ab). The Pl Ab has no effect on LSR, while enhancing the Ca2+ transport rates of CSR at Ca2+ concentrations below saturation. We found no direct effect of the Pl Ab on Ca2+ binding by the ATPase in the absence of ATP. Equilibrium measurements of Ca2+ binding yield positively cooperative isotherms which are best fit with a two-interacting sites equation. LSR and CSR display nearly identical affinities for Ca2+, and no effect of the Pl Ab is observed. Taking advantage of a stable CrATP-enzyme complex, we demonstrated that the stoichiometric ratio of occluded Ca2+ to catalytic sites is 2 in either LSR or CSR and that the addition of Pl Ab does not affect the Ca2+ concentration dependence of Ca2+ occluded after equilibration of the system. Interestingly, the cooperative interaction between the two Ca2+ sites is lost in the occluded state, with only one of the two sites acquiring lumenal exposure. The concentration dependence of Ca2+ inhibition of CSR ATPase phosphorylation with Pi is also unaffected by the Pl Ab. Contrary to the lack of Pl Ab effect on reactions measured at equilibrium, enhancement of phosphorylated intermediate formation by the Pl Ab is obtained in kinetic experiments in which nonsaturating Ca2+ and ATP are added to CSR preincubated with EGTA. Therefore, Ab binding to Pl reduces specifically the activation energy for a slow transition triggered by Ca2+ binding, with consequent enhancement of overall kinetics under conditions enhancing the rate-limiting contribution of this transition.

  2. Calcium-sensing receptors regulate cardiomyocyte Ca2+ signaling via the sarcoplasmic reticulum-mitochondrion interface during hypoxia/reoxygenation

    Directory of Open Access Journals (Sweden)

    Lu Fang-hao

    2010-06-01

    Full Text Available Abstract Communication between the SR (sarcoplasmic reticulum, SR and mitochondria is important for cell survival and apoptosis. The SR supplies Ca2+ directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP3Rs at close contacts between the two organelles referred to as mitochondrion-associated ER membrane (MAM. Although it has been demonstrated that CaR (calcium sensing receptor activation is involved in intracellular calcium overload during hypoxia/reoxygenation (H/Re, the role of CaR activation in the cardiomyocyte apoptotic pathway remains unclear. We postulated that CaR activation plays a role in the regulation of SR-mitochondrial inter-organelle Ca2+ signaling, causing apoptosis during H/Re. To investigate the above hypothesis, cultured cardiomyocytes were subjected to H/Re. We examined the distribution of IP3Rs in cardiomyocytes via immunofluorescence and Western blotting and found that type 3 IP3Rs were located in the SR. [Ca2+]i, [Ca2+]m and [Ca2+]SR were determined using Fluo-4, x-rhod-1 and Fluo 5N, respectively, and the mitochondrial membrane potential was detected with JC-1 during reoxygenation using laser confocal microscopy. We found that activation of CaR reduced [Ca2+]SR, increased [Ca2+]i and [Ca2+]m and decreased the mitochondrial membrane potential during reoxygenation. We found that the activation of CaR caused the cleavage of BAP31, thus generating the pro-apoptotic p20 fragment, which induced the release of cytochrome c from mitochondria and the translocation of bak/bax to mitochondria. Taken together, these results reveal that CaR activation causes Ca2+ release from the SR into the mitochondria through IP3Rs and induces cardiomyocyte apoptosis during hypoxia/reoxygenation.

  3. Calcium-sensing receptors regulate cardiomyocyte Ca2+ signaling via the sarcoplasmic reticulum-mitochondrion interface during hypoxia/reoxygenation.

    Science.gov (United States)

    Lu, Fang-hao; Tian, Zhiliang; Zhang, Wei-hua; Zhao, Ya-jun; Li, Hu-lun; Ren, Huan; Zheng, Hui-shuang; Liu, Chong; Hu, Guang-xia; Tian, Ye; Yang, Bao-feng; Wang, Rui; Xu, Chang-qing

    2010-06-17

    Communication between the SR (sarcoplasmic reticulum, SR) and mitochondria is important for cell survival and apoptosis. The SR supplies Ca2+ directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP3Rs) at close contacts between the two organelles referred to as mitochondrion-associated ER membrane (MAM). Although it has been demonstrated that CaR (calcium sensing receptor) activation is involved in intracellular calcium overload during hypoxia/reoxygenation (H/Re), the role of CaR activation in the cardiomyocyte apoptotic pathway remains unclear. We postulated that CaR activation plays a role in the regulation of SR-mitochondrial inter-organelle Ca2+ signaling, causing apoptosis during H/Re. To investigate the above hypothesis, cultured cardiomyocytes were subjected to H/Re. We examined the distribution of IP3Rs in cardiomyocytes via immunofluorescence and Western blotting and found that type 3 IP3Rs were located in the SR. [Ca2+]i, [Ca2+]m and [Ca2+]SR were determined using Fluo-4, x-rhod-1 and Fluo 5N, respectively, and the mitochondrial membrane potential was detected with JC-1 during reoxygenation using laser confocal microscopy. We found that activation of CaR reduced [Ca2+]SR, increased [Ca2+]i and [Ca2+]m and decreased the mitochondrial membrane potential during reoxygenation. We found that the activation of CaR caused the cleavage of BAP31, thus generating the pro-apoptotic p20 fragment, which induced the release of cytochrome c from mitochondria and the translocation of bak/bax to mitochondria. Taken together, these results reveal that CaR activation causes Ca2+ release from the SR into the mitochondria through IP3Rs and induces cardiomyocyte apoptosis during hypoxia/reoxygenation.

  4. Decrease in sarcoplasmic reticulum calcium content, not myofilament function, contributes to muscle twitch force decline in isolated cardiac trabeculae

    Science.gov (United States)

    Milani-Nejad, Nima; Brunello, Lucia; Gyorke, Sándor; Janssen, Paul M.L.

    2014-01-01

    We set out to determine the factors responsible for twitch force decline in isolated intact rat cardiac trabeculae. The contractile force of trabeculae declined over extended periods of isometric twitch contractions. The force-frequency relationship within the frequency range of 4–8 Hz, at 37 °C, became more positive and the frequency optimum shifted to higher rates with this decline in baseline twitch tensions. The post-rest potentiation (37 °C), a phenomenon highly dependent on calcium handling mechanisms, became more pronounced with decrease in twitch tensions. We show that the main abnormality during muscle run-down was not due to a deficit in the myofilaments; maximal tension achieved using a K+ contracture protocol was either unaffected or only slightly decreased. Conversely, the sarcoplasmic reticulum (SR) calcium content, as assessed by rapid cooling contractures (from 27 °C to 0 °C), decreased, and had a close association with the declining twitch tensions (R2 ~ 0.76). SR Ca2+-ATPase, relative to Na+/Ca2+ exchanger activity, was not altered as there was no significant change in paired rapid cooling contracture ratios. Furthermore, confocal microscopy detected no abnormalities in the overall structure of the cardiomyocytes and t-tubules in the cardiac trabeculae (~23 °C). Overall, the data indicates that the primary mechanism responsible for force run-down in multi-cellular cardiac preparations is a decline in the SR calcium content and not the maximal tension generation capability of the myofilaments. PMID:25056841

  5. Reduced junctional Na+/Ca2+-exchanger activity contributes to sarcoplasmic reticulum Ca2+ leak in junctophilin-2-deficient mice

    Science.gov (United States)

    Wang, Wei; Landstrom, Andrew P.; Wang, Qiongling; Munro, Michelle L.; Beavers, David; Ackerman, Michael J.; Soeller, Christian

    2014-01-01

    Expression silencing of junctophilin-2 (JPH2) in mouse heart leads to ryanodine receptor type 2 (RyR2)-mediated sarcoplasmic reticulum (SR) Ca2+ leak and rapid development of heart failure. The mechanism and physiological significance of JPH2 in regulating RyR2-mediated SR Ca2+ leak remains elusive. We sought to elucidate the role of JPH2 in regulating RyR2-mediated SR Ca2+ release in the setting of cardiac failure. Cardiac myocytes isolated from tamoxifen-inducible conditional knockdown mice of JPH2 (MCM-shJPH2) were subjected to confocal Ca2+ imaging. MCM-shJPH2 cardiomyocytes exhibited an increased spark frequency width with altered spark morphology, which caused increased SR Ca2+ leakage. Single channel studies identified an increased RyR2 open probability in MCM-shJPH2 mice. The increase in spark frequency and width was observed only in MCM-shJPH2 and not found in mice with increased RyR2 open probability with native JPH2 expression. Na+/Ca2+-exchanger (NCX) activity was reduced by 50% in MCM-shJPH2 with no detectable change in NCX expression. Additionally, 50% inhibition of NCX through Cd2+ administration alone was sufficient to increase spark width in myocytes obtained from wild-type mice. Additionally, superresolution analysis of RyR2 and NCX colocalization showed a reduced overlap between RyR2 and NCX in MCM-shJPH2 mice. In conclusion, decreased JPH2 expression causes increased SR Ca2+ leakage by directly increasing open probability of RyR2 and by indirectly reducing junctional NCX activity through increased dyadic cleft Ca2+. This demonstrates two novel and independent cellular mechanisms by which JPH2 regulates RyR2-mediated SR Ca2+ leak and heart failure development. PMID:25193470

  6. Pressure effects on the interactions of the sarcoplasmic reticulum calcium transport enzyme with calcium and dinitrophenyl phosphate.

    Science.gov (United States)

    Hasselbach, W

    1988-01-01

    The effect of hydrostatic pressure on the calcium-dependent hydrolysis of dinitrophenyl phosphate by the sarcoplasmic calcium transport enzyme has been studied. The magnesium dinitrophenyl phosphate complex is the true substrate of the enzyme (K = 7000 M-1) by which it is hydrolyzed at 20 degrees C with a turnover rate of 4 s-1. Activation by calcium ions occurs between 0.1 and 1 microM as observed for ATP hydrolysis. The activation volume of the enzyme saturated with both ligands exhibits pronounced pressure-dependence, rising from 25 ml/mol at atmospheric pressure to 80 ml/mol at 100 MPa. The apparent binding volumes for magnesium dinitrophenyl phosphate and calcium are likewise pressure-dependent. The volume changes connected with the binding of magnesium dinitrophenyl phosphate is quite small approaching zero at 100 MPa. The apparent binding volume for calcium greatly increases with pressure from 35 ml/mol at atmospheric pressure to 150 ml/mol at 70 MPa. A nearly constant binding volume of approximately 40 ml/mol results if the effect of pressure on the respective rate constants that contribute to the apparent binding constant, is taken into account. The pressure-dependence of enzyme activity at subsaturating calcium concentrations yields an activation volume of 250 ml/mol related to the rate of calcium binding indicating the occurrence of a transient large volume expansion of the enzyme complex. The volume changes observed for the calcium-dependent interaction of the enzyme with magnesium dinitrophenyl phosphate well agree with that found for magnesium p-nitrophenyl phosphate (W. Hasselbach and L. Stephan,Z. Naturforsch. 42 c, 641-652 (1987)) indicating that the found volume changes are intrinsic properties of the transport enzyme, independent of the respective energy donor.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. Effects of Mg2+ on Ca2+ handling by the sarcoplasmic reticulum in skinned skeletal and cardiac muscle fibres.

    Science.gov (United States)

    Kabbara, A A; Stephenson, D G

    1994-10-01

    The influence of myoplasmic Mg2+ (0.05-10 mM) on Ca2+ accumulation (net Ca2+ flux) and Ca2+ uptake (pump-driven Ca2+ influx) by the intact sarcoplasmic reticulum (SR) was studied in skinned fibres from the toad iliofibularis muscle (twitch portion), rat extensor digitorum longus (EDL) muscle (fast twitch), rat soleus muscle (slow twitch) and rat cardiac trabeculae. Ca2+ accumulation was optimal between 1 and 3 mM Mg2+ in toad fibres and reached a plateau between 1 and 10 mM Mg2+ in the rat EDL fibres and between 3 and 10 mM Mg2+ in the rat cardiac fibres. In soleus fibres, optimal Ca2+ accumulation occurred at 10 mM Mg2+. The same trend was obtained with all preparations at 0.3 and 1 microM Ca2+. Experiments with 2,5-di-(tert-butyl)-1,4-benzohydroquinone, a specific inhibitor of the Ca2+ pump, revealed a marked Ca2+ efflux from the SR of toad iliofibularis fibres in the presence of 0.2 microM Ca2+ and 1 mM Mg2+. Further experiments indicated that the SR Ca2+ leak could be blocked by 10 microM ruthenium red without affecting the SR Ca2+ pump and this allowed separation between SR Ca2+ uptake and SR Ca2+ accumulation. At 0.3 microM Ca2+, Ca2+ uptake was optimal with 1 mM Mg2+ in the toad iliofibularis and rat EDL fibres and between 1 and 10 mM Mg2+ in the rat soleus and trabeculae preparations. At higher [Ca2+] (1 microM), Ca2+ uptake was optimal with 1 mM Mg2+ in the iliofibularis fibres and between 1 and 3 mM Mg2+ in the EDL fibres.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. Role of SERCA and the sarcoplasmic reticulum calcium content on calcium waves propagation in rat ventricular myocytes.

    Science.gov (United States)

    Salazar-Cantú, Ayleen; Pérez-Treviño, Perla; Montalvo-Parra, Dolores; Balderas-Villalobos, Jaime; Gómez-Víquez, Norma L; García, Noemí; Altamirano, Julio

    2016-08-15

    In Ca(2+)-overloaded ventricular myocytes, SERCA is crucial to steadily achieve the critical sarcoplasmic reticulum (SR) Ca(2+) level to trigger and sustain Ca(2+) waves, that propagate at constant rate (ʋwave). High luminal Ca(2+) sensitizes RyR2, thereby increasing Ca(2+) sparks frequency, and the larger RyR2-mediated SR Ca(2+) flux (dF/dt) sequentially activates adjacent RyR2 clusters. Recently, it was proposed that rapid SERCA Ca(2+) reuptake, ahead of the wave front, further sensitizes RyR2, increasing ʋwave. Nevertheless, this is controversial because rapid cytosolic Ca(2+) removal could instead impair RyR2 activation. We assessed whether rapid SR Ca(2+) uptake enhances ʋwave by changing SERCA activity (ҡDecay) over a large range (∼175%). We used normal (Ctrl) and hyperthyroid rat (HT; reduced phospholamban by ∼80%) myocytes treated with thapsigargin or isoproterenol (ISO). We found that ʋwave and dF/dt had a non-linear dependency with ҡDecay, while Ca(2+) waves amplitude was largely unaffected. Furthermore, SR Ca(2+) also showed a non-linear dependency with ҡDecay, however, the relationships ʋwave vs. SR Ca(2+) and ʋwave vs. dF/dt were linear, suggesting that high steady state SR Ca(2+) determines ʋwave, while rapid SERCA Ca(2+) uptake does not. Finally, ISO did not increase ʋwave in HT cells, therefore, ISO-enhanced ʋwave in Ctrl depended on high SR Ca(2+).

  9. Kinetics of calcium uptake by isolated sarcoplasmic reticulum vesicles using flash photolysis of caged adenosine 5'-triphosphate.

    Science.gov (United States)

    Pierce, D H; Scarpa, A; Topp, M R; Blasie, J K

    1983-11-08

    The kinetics of ATP-induced Ca2+ uptake by vesicular dispersions of sarcoplasmic reticulum were determined with a time resolution of about 10 ms, depending on the temperature. Ca2+ uptake was initiated by the addition of ATP through the flash photolysis of P3-1-(2-nitrophenyl)-ethyl adenosine 5'-triphosphate utilizing a frequency-doubled ruby laser and measured with two different detector systems that followed the absorbance changes of the metallochromic indicator arsenazo III sensitive to changes in the extravesicular [Ca2+]. The temperature range investigated was -2 to 26 degrees C. The Ca2+ ionophore A23187 was used to distinguish those features of the Ca2+ uptake kinetics associated with the formation of a transmembrane Ca2+ gradient. The acid-stable phosphorylated enzyme intermediate, E approximately P, was determined independently with a quenched-flow technique. Ca2+ uptake is characterized by at least two phases, a fast initial phase and a slow phase. The fast phase exhibits pseudo-first-order kinetics with a specific rate constant of 64 +/- 10 s-1 at 23-26 degrees C, an activation energy of 16 +/- 1 kcal mol-1, and a delta S* of approximately 5 cal deg-1 mol-1, is insensitive to the presence of a Ca2+ ionophore, and occurs simultaneously with the formation of the phosphorylated enzyme, E approximately P, with a stoichiometry of approximately 2 mol of Ca2+/mol of phosphorylated enzyme intermediate. The slow phase also exhibits pseudo-first-order kinetics with a specific rate constant of 0.60 +/- 0.09 s-1 at 25-26 degrees C, an activation energy of 22 +/- 1 kcal mol-1, and a delta S* of approximately 16 cal deg-1 mol-1, is inhibited by the presence of a Ca2+ ionophore, and has a stoichiometry of approximately 2 mol of Ca2+/mol of ATP hydrolyzed.

  10. Fraction Sense: Foundational Understandings.

    Science.gov (United States)

    Fennell, Francis Skip; Karp, Karen

    2016-08-09

    The intent of this commentary is to identify elements of fraction sense and note how the research studies provided in this special issue, in related but somewhat different ways, validate the importance of such understandings. Proficiency with fractions serves as a prerequisite for student success in higher level mathematics, as well as serving as a gateway to many occupations and varied contexts beyond the mathematics classroom. Fraction sense is developed through instructional opportunities involving fraction equivalence and magnitude, comparing and ordering fractions, using fraction benchmarks, and computational estimation. Such foundations are then extended to operations involving fractions and decimals and applications involving proportional reasoning. These components of fraction sense are all addressed in the studies provided in this issue, with particular consideration devoted to the significant importance of the use of the number line as a central representational tool for conceptually understanding fraction magnitude.

  11. Whey Protein

    Science.gov (United States)

    ... Fraction de Lactosérum, Fraction de Petit-Lait, Goat Milk Whey, Goat Whey, Isolat de Protéine de Lactosérum, Isolat ... Lactosérum de Lait de Chèvre, MBP, Milk Protein, Milk Protein Isolate, Mineral Whey Concentrate, Proteínas del Suero de la Leche, Protéine ...

  12. Impact of total solid content and extraction pH on enzyme-aided recovery of protein from defatted rapeseed (Brassica rapa L.) press cake and physicochemical properties of the protein fractions.

    Science.gov (United States)

    Rommi, Katariina; Ercili-Cura, Dilek; Hakala, Terhi K; Nordlund, Emilia; Poutanen, Kaisa; Lantto, Raija

    2015-03-25

    Pectinase treatment was used to facilitate protein recovery from defatted rapeseed (Brassica rapa) cold-pressing residue in water-lean conditions and without pH adjustment. Effect of extraction pH on protein yield and physiochemical properties of the protein concentrates was assessed. Enzymatic hydrolysis of carbohydrates was feasible at high (40%) solid content and improved protein recovery at pH 6. Comparable protein yields (40-41% of total protein) from enzyme-aided water extraction (pH 6) and nonenzymatic alkaline extraction (pH10) at 10% solid content suggested that after enzymatic treatment, rapeseed protein could be extracted without exposure to alkali. However, water extraction required dilute conditions, whereas alkaline extraction was feasible also at 20% solid content. The water extracts possessed better protein solubility, higher ζ-potential, and smaller particle size than isoelectric precipitates from alkaline extraction, indicating higher dispersion stability. This is suggested to be mediated by electrostatic interactions between proteins and pectic carbohydrates in the water extracts.

  13. Combined N-glycome and N-glycoproteome analysis of the Lotus japonicus seed globulin fraction shows conservation of protein structure and glycosylation in legumes.

    Science.gov (United States)

    Dam, Svend; Thaysen-Andersen, Morten; Stenkjær, Eva; Lorentzen, Andrea; Roepstorff, Peter; Packer, Nicolle H; Stougaard, Jens

    2013-07-05

    Legume food allergy, such as allergy toward peanuts and soybeans, is a health issue predicted to worsen as dietary advice recommends higher intake of legume-based foods. Lotus japonicus (Lotus) is an established legume plant model system for studies of symbiotic and pathogenic microbial interactions and, due to its well characterized genotype/phenotype and easily manipulated genome, may also be suitable for studies of legume food allergy. Here we present a comprehensive study of the Lotus N-glycoproteome. The global and site-specific N-glycan structures of Lotus seed globulins were analyzed using mass spectrometry-based glycomics and glycoproteomics techniques. In total, 19 N-glycan structures comprising high mannose (∼20%), pauci-mannosidic (∼40%), and complex forms (∼40%) were determined. The pauci-mannosidic and complex N-glycans contained high amounts of the typical plant determinants β-1,2-xylose and α-1,3-fucose. Two abundant Lotus seed N-glycoproteins were site-specifically profiled; a predicted lectin containing two fully occupied N-glycosylation sites carried predominantly pauci-mannosidic structures in different distributions. In contrast, Lotus convicilin storage protein 2 (LCP2) carried exclusively high mannose N-glycans similar to its homologue, Ara h 1, which is the major allergen in peanut. In silico investigation confirmed that peanut Ara h 1 and Lotus LCP2 are highly similar at the primary and higher protein structure levels. Hence, we suggest that Lotus has the potential to serve as a model system for studying the role of seed proteins and their glycosylation in food allergy.

  14. Inhibition of sarcoplasmic Ca2+-ATPase increases caffeine- and halothane-induced contractures in muscle bundles of malignant hyperthermia susceptible and healthy individuals

    Science.gov (United States)

    Schuster, Frank; Müller, Rainer; Hartung, Edmund; Roewer, Norbert; Anetseder, Martin

    2005-01-01

    Background Malignant hyperthermia (MH) is triggered by halogenated anaesthetics and depolarising muscle relaxants, leading to an uncontrolled hypermetabolic state of skeletal muscle. An uncontrolled sarcoplasmic Ca2+ release is mediated via the ryanodine receptor. A compensatory mechanism of increased sarcoplasmic Ca2+-ATPase activity was described in pigs and in transfected cell lines. We hypothesized that inhibition of Ca2+ reuptake via the sarcoplasmic Ca2+-ATPase (SERCA) enhances halothane- and caffeine-induced muscle contractures in MH susceptible more than in non-susceptible skeletal muscle. Methods With informed consent, surplus muscle bundles of 7 MHS (susceptible), 7 MHE (equivocal) and 16 MHN (non-susceptible) classified patients were mounted to an isometric force transducer, electrically stimulated, preloaded and equilibrated. Following 15 min incubation with cyclopiazonic acid (CPA) 25 μM, the European MH standard in-vitro-contracture test protocol with caffeine (0.5; 1; 1.5; 2; 3; 4 mM) and halothane (0.11; 0.22; 0.44; 0.66 mM) was performed. Data as median and quartiles; Friedman- and Wilcoxon-test for differences with and without CPA; p muscle bundles did not differ between groups. CPA increased halothane- and caffeine-induced contractures significantly. This increase was more pronounced in MHS and MHE than in MHN muscle bundles. Conclusion Inhibition of the SERCA activity by CPA enhances halothane- and caffeine-induced contractures especially in MHS and MHE skeletal muscle and may help for the diagnostic assignment of MH susceptibility. The status of SERCA activity may play a significant but so far unknown role in the genesis of malignant hyperthermia. PMID:15946384

  15. Inhibition of sarcoplasmic Ca2+-ATPase increases caffeine- and halothane-induced contractures in muscle bundles of malignant hyperthermia susceptible and healthy individuals

    Directory of Open Access Journals (Sweden)

    Roewer Norbert

    2005-06-01

    Full Text Available Abstract Background Malignant hyperthermia (MH is triggered by halogenated anaesthetics and depolarising muscle relaxants, leading to an uncontrolled hypermetabolic state of skeletal muscle. An uncontrolled sarcoplasmic Ca2+ release is mediated via the ryanodine receptor. A compensatory mechanism of increased sarcoplasmic Ca2+-ATPase activity was described in pigs and in transfected cell lines. We hypothesized that inhibition of Ca2+ reuptake via the sarcoplasmic Ca2+-ATPase (SERCA enhances halothane- and caffeine-induced muscle contractures in MH susceptible more than in non-susceptible skeletal muscle. Methods With informed consent, surplus muscle bundles of 7 MHS (susceptible, 7 MHE (equivocal and 16 MHN (non-susceptible classified patients were mounted to an isometric force transducer, electrically stimulated, preloaded and equilibrated. Following 15 min incubation with cyclopiazonic acid (CPA 25 μM, the European MH standard in-vitro-contracture test protocol with caffeine (0.5; 1; 1.5; 2; 3; 4 mM and halothane (0.11; 0.22; 0.44; 0.66 mM was performed. Data as median and quartiles; Friedman- and Wilcoxon-test for differences with and without CPA; p Results Initial length, weight, maximum twitch height, predrug resting tension and predrug twitch height of muscle bundles did not differ between groups. CPA increased halothane- and caffeine-induced contractures significantly. This increase was more pronounced in MHS and MHE than in MHN muscle bundles. Conclusion Inhibition of the SERCA activity by CPA enhances halothane- and caffeine-induced contractures especially in MHS and MHE skeletal muscle and may help for the diagnostic assignment of MH susceptibility. The status of SERCA activity may play a significant but so far unknown role in the genesis of malignant hyperthermia.

  16. Utilization of Different Corn Fractions by Broilers

    Directory of Open Access Journals (Sweden)

    SIFR Costa

    2015-09-01

    Full Text Available ABSTRACTThis study was conducted to evaluate the nutritional values of fractions of damaged corn. One hundred and eighty 22-d-old Cobb 500 male broilers were distributed in batteries according to a completely randomized design with six treatments of six replicates each. The treatments consisted of diets containing five corn fractions, classified as sound, fermented, insect-damaged, mold-damaged, or reference corn. The test diets consisted of 60% of reference diet + 40% of each corn fraction. Only the reference corn fraction included all the fractions at different proportions (0.8% fermented, 0.05% insect-damaged, 3.3% mold-damaged, and 95.85% sound grains. The method of total excreta collection was used to determine AMEn values and metabolizability coefficients of dry matter (MDM, crude protein (MCP, ether extract (MEE, and gross energy (MGE of the reference corn and its fractions. The density values of the corn fractions were used to calculate the correlations among the evaluated parameters. The evaluated corn fractions presented different compositions values. The insect-damaged and mold-damaged grains presented higher CP level, lower density, and MDM and MCP coefficients compared with the other fractions. However, calculated AMEn values were not significantly different (p>0.05 among corn fractions. A low correlation between density and AMEn content (r0.8 were calculated. Although the evaluated corn fractions presented different nutritional values, there were no marked differences in their utilization by broilers.

  17. Meaning of Fractions

    Science.gov (United States)

    Dewi, D. A. K.; Suryadi, D.; Suratno, T.; Mulyana, E.; Kurniawan, H.

    2017-02-01

    Introducing fractions is identical to divide an object. Suppose we divide the apple into two parts. One divided into two parts, the question arises whether one part can be called a half or not. Based on this activity, how can students give meaning to fractions. This study aims at designing a different fractions lesson by applying Didactical Design Research. In doing so, we undertook several research phases: 1) thinking what is fractions and why students should learn this concept; 2) designing didactical situation based on identified learning obstacles; and 3) reflecting retrospectively on the lesson design and its implementation as to redesign the fractions lesson. Our analysis revealed that most students held epistemological obstacles in giving meaning of fractions because they only know fractions as numbers that have numerator and denominator. By positioning ourselves as students, we discuss the ideal design to help students in constructing the meaning of fractions.

  18. Fractional Dynamical Systems

    CERN Document Server

    Edelman, Mark

    2014-01-01

    In this paper the author presents the results of the preliminary investigation of fractional dynamical systems based on the results of numerical simulations of fractional maps. Fractional maps are equivalent to fractional differential equations describing systems experiencing periodic kicks. Their properties depend on the value of two parameters: the non-linearity parameter, which arises from the corresponding regular dynamical systems; and the memory parameter which is the order of the fractional derivative in the corresponding non-linear fractional differential equations. The examples of the fractional Standard and Logistic maps demonstrate that phase space of non-linear fractional dynamical systems may contain periodic sinks, attracting slow diverging trajectories, attracting accelerator mode trajectories, chaotic attractors, and cascade of bifurcations type trajectories whose properties are different from properties of attractors in regular dynamical systems. The author argues that discovered properties s...

  19. Instrument and method to determine the electrophoretic mobility of nanoparticles and proteins by combining electrical and flow field-flow fractionation.

    Science.gov (United States)

    Johann, Christoph; Elsenberg, Stephan; Schuch, Horst; Rösch, Ulrich

    2015-04-21

    A new FFF method is presented which combines asymmetrical flow-FFF (AF4) and electrical FFF (ElFFF) in one channel to electrical asymmetrical flow-FFF (EAF4) to overcome the restrictions of pure ElFFF. It allows for measuring electrophoretic mobility (μ) as a function of size. The method provides an absolute value and does not require calibration. Results of μ for two particle standards are in good agreement with values determined by phase analysis light scattering (PALS). There is no requirement for low ionic strength carriers with EAF4. This overcomes one of the main limitations of ElFFF, making it feasible to measure proteins under physiological conditions. EAF4 has the capability to determine μ for individual populations which are resolved into separate peaks. This is demonstrated for a mixture of three polystyrene latex particles with different sizes as well as for the monomer and dimer of BSA and an antibody. The experimental setup consists of an AF4 channel with added electrodes; one is placed beneath the frit at the bottom wall and the other covers the inside of the upper channel plate. This design minimizes contamination from the electrolysis reactions by keeping the particles distant from the electrodes. In addition the applied voltage range is low (1.5-5 V), which reduces the quantity of gaseous electrolysis products below a threshold that interferes with the laminar flow profile or detector signals. Besides measuring μ, the method can be useful to improve the separation between sample components compared to pure flow-FFF. For two proteins (BSA and a monoclonal antibody), enhanced resolution of the monomer and dimer is achieved by applying an electric field.

  20. On continued fraction algorithms

    NARCIS (Netherlands)

    Smeets, Ionica

    2010-01-01

    Is there a good continued fraction approximation between every two bad ones? What is the entropy of the natural extension for alpha-Rosen fractions? How do you find multi-dimensional continued fractions with a guaranteed quality in polynomial time? These, and many more, questions are answered in thi

  1. DIY Fraction Pack.

    Science.gov (United States)

    Graham, Alan; Graham, Louise

    2003-01-01

    Describes a very successful attempt to teach fractions to year 5 pupils based on pupils making their own fraction pack. Children decided for themselves how to make the fractional slices used in the activity using colored cardboard sheets and templates of a paper circle consisting of 24 equal slices. (Author/NB)

  2. On continued fraction algorithms

    NARCIS (Netherlands)

    Smeets, Ionica

    2010-01-01

    Is there a good continued fraction approximation between every two bad ones? What is the entropy of the natural extension for alpha-Rosen fractions? How do you find multi-dimensional continued fractions with a guaranteed quality in polynomial time? These, and many more, questions are answered in thi

  3. Protein carbonylation and heat shock proteins in human skeletal muscle: relationships to age and sarcopenia.

    Science.gov (United States)

    Beltran Valls, Maria R; Wilkinson, Daniel J; Narici, Marco V; Smith, Kenneth; Phillips, Bethan E; Caporossi, Daniela; Atherton, Philip J

    2015-02-01

    Aging is associated with a gradual loss of muscle mass termed sarcopenia, which has significant impact on quality-of-life. Because oxidative stress is proposed to negatively impact upon musculoskeletal aging, we investigated links between human aging and markers of oxidative stress, and relationships to muscle mass and strength in young and old nonsarcopenic and sarcopenic adults. Sixteen young and 16 old males (further subdivided into "old" and "old sarcopenic") were studied. The abundance of protein carbonyl adducts within skeletal muscle sarcoplasmic, myofibrillar, and mitochondrial protein subfractions from musculus vastus lateralis biopsies were determined using Oxyblot immunoblotting techniques. In addition, concentrations of recognized cytoprotective proteins (eg, heat shock proteins [HSP], αβ-crystallin) were also assayed. Aging was associated with increased mitochondrial (but not myofibrillar or sarcoplasmic) protein carbonyl adducts, independently of (stage-I) sarcopenia. Correlation analyses of all subjects revealed that mitochondrial protein carbonyl abundance negatively correlated with muscle strength ([1-repetition maximum], p = .02, r (2) = -.16), but not muscle mass (p = .13, r (2) = -.08). Abundance of cytoprotective proteins, including various HSPs (HSP 27 and 70), were unaffected by aging/sarcopenia. To conclude, these data reveal that mitochondrial protein carbonylation increases moderately with age, and that this increase may impact upon skeletal muscle function, but is not a hallmark of (stage-I) sarcopenia, per se. © The Author 2014. Published by Oxford University Press on behalf of The Gerontological Society of America.

  4. Improvement in cardiac function after sarcoplasmic reticulum Ca2+-ATPase gene transfer in a beagle heart failure model

    Institute of Scientific and Technical Information of China (English)

    MI Ya-fei; LI Xiao-ying; TANG Li-jiang; LU Xiao-chun; FU Zhi-qing; YE Wei-hua

    2009-01-01

    Background Heart failure (HF) is a major cause of morbidity and mortality worldwide, but current treatment modalities cannot reverse the underlying pathological state of the heart. Gene-based therapies are emerging as promising therapeutic modalities in HF patients. Our previous studies have shown that recombinant adeno-associated viral (rAAV) gene transfer of Sarco-endoplasmic reticulum calcium ATPase (SERCA2a) can be effective in treating rats with chronic heart failure (CHF). The aim of this study was to examine the effects of SERCA2a gene transfer in a large HF animal model.Methods HF was induced in beagles by rapid right ventricular pacing (230 beats/min) for 30 days. A reduced rate ventricular pacing (180 beats/min) was continued for another 30 days. The beagles were assigned to four groups: (a) control group (n=4); (b) HF group (n=4); (c) enhanced green fluorescent protein group (n=4); and (d) SERCA2.a group (n=4). rAAVl-EGFP (lx1012 μg) and rAAVl-SERCA2a (lx1012 μg) were delivered intramyocardially. SERCA2.a expression was assessed by Western blotting and immunohistochemistry.Results Following 30 days of SERCA2a gene transfer in HF beagles its protein expression was significantly higher than in the HF group than in the control group (P <0.05). Heart function improved along with the increase in SERCA2a expression. Left ventricular systolic function significantly improved, including the ejection fraction, left ventricular systolic pressure, maximal rate of rise of left ventricular pressure (+dp/dtmax), and the maximal rate of decline of left ventricular pressure (-dp/dtmax) (P <0.05). Left ventricular end-diastole pressure significantly decreased (P <0.05). The expression of SERCA2a in the myocardial tissue was higher in the SERCA2a group than in the HF group (P<0.05). Conclusions Intramyocardial injection of rAAVl-SERCA2a can improve the cardiac function in beagles induced with HE We expect further studies on SERCA2a's long-term safety, efficacy, dosage

  5. Fractional Differential Equations

    Directory of Open Access Journals (Sweden)

    Jianping Zhao

    2012-01-01

    Full Text Available An extended fractional subequation method is proposed for solving fractional differential equations by introducing a new general ansätz and Bäcklund transformation of the fractional Riccati equation with known solutions. Being concise and straightforward, this method is applied to the space-time fractional coupled Burgers’ equations and coupled MKdV equations. As a result, many exact solutions are obtained. It is shown that the considered method provides a very effective, convenient, and powerful mathematical tool for solving fractional differential equations.

  6. Fractional smith chart theory

    KAUST Repository

    Shamim, Atif

    2011-03-01

    For the first time, a generalized Smith chart is introduced here to represent fractional order circuit elements. It is shown that the standard Smith chart is a special case of the generalized fractional order Smith chart. With illustrations drawn for both the conventional integer based lumped elements and the fractional elements, a graphical technique supported by the analytical method is presented to plot impedances on the fractional Smith chart. The concept is then applied towards impedance matching networks, where the fractional approach proves to be much more versatile and results in a single element matching network for a complex load as compared to the two elements in the conventional approach. © 2010 IEEE.

  7. Conducting and voltage-dependent behaviors of potassium ion channels reconstituted from diaphragm sarcoplasmic reticulum: comparison with the cardiac isoform.

    Science.gov (United States)

    Picher, M; Decrouy, A; Rousseau, E

    1996-02-21

    Sarcoplasmic reticulum (SR) K+ channels from canine diaphragm were studied upon fusion of longitudinal and junctional membrane vesicles into planar lipid bilayers (PLB). The large-conductance cation selective channel (gamma(max) = 250 pS; Km = 33 mM) displays long-lasting open events which are much more frequent at positive than at negative voltages. A major subconducting state about 45% of the fully-open state current amplitude was occasionally observed at all voltages. The voltage-dependence of the open probability displays a sigmoid relationship that was fitted by the Boltzmann equation and expressed in terms of thermodynamic parameters, namely the free energy (delta Gi) and the effective gating charge (Zs): delta Gi = 0.27 kcal/mol and Zs = -1.19 in 250 mM potassium gluconate (K-gluconate). Kinetic analyses also confirmed the voltage-dependent gating behavior of this channel, and indicate the implication of at least two open and three closed states. The diaphragm SR K+ channel shares several biophysical properties with the cardiac isoform: g = 180 pS, delta Gi = 0.75 kcal/mol, Zs = -1.45 in 150 mM K-gluconate, and a similar sigmoid P(o)/voltage relationship. Little is known about the regulation of the diaphragm and cardiac SR K+ channels. The conductance and gating of these channels were not influenced by physiological concentrations of Ca2+ (0.1 microM-1 mM) or Mg2+ (0.25-1 mM), as well as by cGMP (25-100 microM), lemakalim (1-100 microM), glyburide (up to 10 microM) or charybdotoxin (45-200 nM), added either to the cis or to the trans chamber. The apparent lack of biochemical or pharmacological modulation of these channels implies that they are not related to any of the well characterized surface membrane K+ channels. On the other hand, their voltage sensitivity strongly suggests that their activity could be modulated by putative changes in SR membrane potential that might occur during calcium fluxes.

  8. Interaction of phosphatidic acid and phosphatidylserine with the Ca2+-ATPase of sarcoplasmic reticulum and the mechanism of inhibition.

    Science.gov (United States)

    Dalton, K A; East, J M; Mall, S; Oliver, S; Starling, A P; Lee, A G

    1998-02-01

    The sarcoplasmic reticulum of skeletal muscle contains anionic phospholipids as well as the zwitterionic phosphatidylcholine and phosphatidylethanolamine. Here we study the effects of anionic phospholipids on the activity of the Ca2+-ATPase purified from the membrane. Reconstitution of the Ca2+-ATPase into dioleoylphosphatidylserine [di(C18:1)PS] or dioleoylphosphatidic acid [di(C18:1)PA] leads to a decrease in ATPase activity. Measurements of the quenching of the tryptophan fluorescence of the ATPase by brominated phospholipids give a relative binding constant for the anionic lipids compared with dioleoylphosphatidylcholine close to 1 and suggest that phosphatidic acid only binds to the ATPase at the bulk lipid sites around the ATPase. Addition of di(C18:1)PS or di(C18:1)PA to the ATPase in the short-chain dimyristoleoylphosphatidylcholine [di(C14:1)PC] reverse the effects of the short-chain lipid on ATPase activity and on Ca2+ binding, as revealed by the response of tryptophan fluorescence intensity to Ca2+ binding. It is concluded that the lipid headgroup and lipid fatty acyl chains have separate effects on the function of the ATPase. The anionic phospholipids have no significant effect on Ca2+ binding to the ATPase; the level of Ca2+ binding to the ATPase, the affinity of binding and the rate of dissociation of Ca2+ are unchanged by reconstitution into di(C18:1)PA. The major effect of the anionic lipids is a reduction in the maximal level of binding of MgATP. This is attributed to the formation of oligomers of the Ca2+-ATPase, in which only one molecule of the ATPase can bind MgATP dimers in di(C18:1)PS and trimers or tetramers in di(C18:1)PA. The rates of phosphorylation and dephosphorylation for the proportion of the ATPase still able to bind ATP are unaffected by reconstitution. Larger changes were observed in the level of phosphorylation of the ATPase by Pi, which became very low in the anionic phospholipids. The fluorescence response to Mg2+ for the ATPase

  9. Effect of saponin treatment on the sarcoplasmic reticulum of rat, cane toad and crustacean (yabby) skeletal muscle.

    Science.gov (United States)

    Launikonis, B S; Stephenson, D G

    1997-10-15

    1. Mechanically skinned fibres from skeletal muscles of the rat, toad and yabby were used to investigate the effect of saponin treatment on sarcoplasmic reticulum (SR) Ca2+ loading properties. The SR was loaded submaximally under control conditions before and after treatment with saponin and SR Ca2+ was released with caffeine. 2. Treatment with 10 micrograms ml-1 saponin greatly reduced the SR Ca2+ loading ability of skinned fibres from the extensor digitorum longus muscle of the rat with a rate constant of 0.24 min-1. Saponin concentrations up to 150 micrograms ml-1 and increased exposure time up to 30 min did not further reduce the SR Ca2+ loading ability of the SR, which indicates that the inhibitory action of 10-150 micrograms ml-1 saponin is not dose dependent. The effect of saponin was also not dependent on the state of polarization of the transverse-tubular system. 3. Treatment with saponin at concentrations up to 100 micrograms ml-1 for 30 min did not affect the Ca2+ loading ability of SR in skinned skeletal muscle fibres from the twitch portion of the toad iliofibularis muscle but SR Ca2+ loading ability decreased markedly with a time constant of 0.22 min-1 in the presence of 150 micrograms ml-1 saponin. 4. The saponin dependent increase in permeability could be reversed in both rat and toad fibres by short treatment with 6 microM Ruthenium Red, a potent SR Ca2+ channel blocker, suggesting that saponin does affect the SR Ca2+ channel properties in mammalian and anuran skeletal muscle. 5. Treatment of skinned fibres of long sarcomere length (> 6 microns) from the claw muscle of the yabby (a freshwater decapod crustacean) with 10 micrograms ml-1 saponin for 30 min abolished the ability of the SR to load Ca2+, indicating that saponin affects differently the SR from skeletal muscles of mammals, anurans and crustaceans. 6. It is concluded that at relatively low concentrations, saponin causes inhibition of the skeletal SR Ca2+ loading ability in a species

  10. Fractional Dynamics and Control

    CERN Document Server

    Machado, José; Luo, Albert

    2012-01-01

    Fractional Dynamics and Control provides a comprehensive overview of recent advances in the areas of nonlinear dynamics, vibration and control with analytical, numerical, and experimental results. This book provides an overview of recent discoveries in fractional control, delves into fractional variational principles and differential equations, and applies advanced techniques in fractional calculus to solving complicated mathematical and physical problems.Finally, this book also discusses the role that fractional order modeling can play in complex systems for engineering and science. Discusses how fractional dynamics and control can be used to solve nonlinear science and complexity issues Shows how fractional differential equations and models can be used to solve turbulence and wave equations in mechanics and gravity theories and Schrodinger’s equation  Presents factional relaxation modeling of dielectric materials and wave equations for dielectrics  Develops new methods for control and synchronization of...

  11. Fractional factorial plans

    CERN Document Server

    Dey, Aloke

    2009-01-01

    A one-stop reference to fractional factorials and related orthogonal arrays.Presenting one of the most dynamic areas of statistical research, this book offers a systematic, rigorous, and up-to-date treatment of fractional factorial designs and related combinatorial mathematics. Leading statisticians Aloke Dey and Rahul Mukerjee consolidate vast amounts of material from the professional literature--expertly weaving fractional replication, orthogonal arrays, and optimality aspects. They develop the basic theory of fractional factorials using the calculus of factorial arrangements, thereby providing a unified approach to the study of fractional factorial plans. An indispensable guide for statisticians in research and industry as well as for graduate students, Fractional Factorial Plans features: * Construction procedures of symmetric and asymmetric orthogonal arrays. * Many up-to-date research results on nonexistence. * A chapter on optimal fractional factorials not based on orthogonal arrays. * Trend-free plans...

  12. Extracellular matrix proteins, integrin receptors (VLA-beta 1, VLA-alpha 2 and VLA-alpha 5) and growth fraction in atypical macroregenerative nodules of the liver: an immunocytochemical case study.

    Science.gov (United States)

    Patriarca, C; Roncalli, M; Viale, G; Alfano, R M; Braidotti, P; Guddo, F; Coggi, G

    1994-08-01

    A case of cirrhotic liver harbouring three atypical macroregenerative nodules and an hepatocellular carcinoma was immunocytochemically investigated for the expression of VLA-beta 1, VLA-alpha 2 and VLA-alpha 5 integrins and for different extracellular matrix (ECM) components (collagen I, collagen IV, laminin, fibronectin and tenascin). In addition, the proliferative activity within the nodules was evaluated, using the MIB 1 monoclonal antibody (MAb). The cirrhotic liver disclosed a continuous staining pattern of the ECM proteins investigated, as well as a "sinusoidal" immunostaining of VLA-beta 1, VLA-alpha 2 and VLA-alpha 5. The macroregenerative nodules showed a discontinued immunoreactivity for ECM proteins while maintaining a VLA-beta 1 sinusoidal immunostaining, coupled with intercellular immunostaining. VLA-alpha 2 and VLA-alpha 5 expression was lacking. The growth fraction was low in both the above pathological conditions. The hepatocellular carcinoma was devoid of any ECM immunostaining. VLA-beta 1 immunoreactivity exhibited a honeycomb pattern of staining, whereas VLA alpha subunits were absent. MIB1 expression was high, being present in 30% of neoplastic nuclei. A possible relationship between atypical macroregenerative nodules and hepatocellular carcinoma is discussed.

  13. Dividing Fractions: A Pedagogical Technique

    Science.gov (United States)

    Lewis, Robert

    2016-01-01

    When dividing one fraction by a second fraction, invert, that is, flip the second fraction, then multiply it by the first fraction. To multiply fractions, simply multiply across the denominators, and multiply across the numerators to get the resultant fraction. So by inverting the division of fractions it is turned into an easy multiplication of…

  14. Subcellular fractionation of human liver reveals limits in global proteomic quantification from isolated fractions.

    Science.gov (United States)

    Wiśniewski, Jacek R; Wegler, Christine; Artursson, Per

    2016-09-15

    The liver plays an important role in metabolism and elimination of xenobiotics, including drugs. Determination of concentrations of proteins involved in uptake, distribution, metabolism, and excretion of xenobiotics is required to understand and predict elimination mechanisms in this tissue. In this work, we have fractionated homogenates of snap-frozen human liver by differential centrifugation and performed quantitative mass spectrometry-based proteomic analysis of each fraction. Concentrations of proteins were calculated by the "total protein approach". A total of 4586 proteins were identified by at least five peptides and were quantified in all fractions. We found that the xenobiotics transporters of the canalicular and basolateral membranes were differentially enriched in the subcellular fractions and that phase I and II metabolizing enzymes, the cytochrome P450s and the UDP-glucuronyl transferases, have complex subcellular distributions. These findings show that there is no simple way to scale the data from measurements in arbitrarily selected membrane fractions using a single scaling factor for all the proteins of interest. This study also provides the first absolute quantitative subcellular catalog of human liver proteins obtained from frozen tissue specimens. Our data provide quantitative insights into the subcellular distribution of proteins and can be used as a guide for development of fractionation procedures. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  15. Fractional Pure Birth Processes

    CERN Document Server

    Orsingher, Enzo; 10.3150/09-BEJ235

    2010-01-01

    We consider a fractional version of the classical non-linear birth process of which the Yule-Furry model is a particular case. Fractionality is obtained by replacing the first-order time derivative in the difference-differential equations which govern the probability law of the process, with the Dzherbashyan-Caputo fractional derivative. We derive the probability distribution of the number $ \\mathcal{N}_\

  16. Fractional vortex Hilbert's Hotel

    CERN Document Server

    Gbur, Greg

    2015-01-01

    We demonstrate how the unusual mathematics of transfinite numbers, in particular a nearly perfect realization of Hilbert's famous hotel paradox, manifests in the propagation of light through fractional vortex plates. It is shown how a fractional vortex plate can be used, in principle, to create any number of "open rooms," i.e. topological charges, simultaneously. Fractional vortex plates are therefore demonstrated to create a singularity of topological charge, in which the vortex state is completely undefined and in fact arbitrary.

  17. Fractional Electromagnetic Waves

    CERN Document Server

    Gómez, J F; Bernal, J J; Tkach, V I; Guía, M

    2011-01-01

    In the present work we consider the electromagnetic wave equation in terms of the fractional derivative of the Caputo type. The order of the derivative being considered is 0 <\\gamma<1. A new parameter \\sigma, is introduced which characterizes the existence of the fractional components in the system. We analyze the fractional derivative with respect to time and space, for \\gamma = 1 and \\gamma = 1/2 cases.

  18. Fractional and noncommutative spacetimes

    NARCIS (Netherlands)

    Arzano, M.; Calcagni, M.; Oriti, D.; Scalisi, M.

    2011-01-01

    We establish a mapping between fractional and noncommutative spacetimes in configuration space. Depending on the scale at which the relation is considered, there arise two possibilities. For a fractional spacetime with log-oscillatory measure, the effective measure near the fundamental scale determi

  19. Can Kindergartners Do Fractions?

    Science.gov (United States)

    Cwikla, Julie

    2014-01-01

    Mathematics professor Julie Cwikla decided that she needed to investigate young children's understandings and see what precurricular partitioning notions young minds bring to the fraction table. Cwikla realized that only a handful of studies have examined how preschool-age and early elementary school-age students solve fraction problems (Empson…

  20. Can Kindergartners Do Fractions?

    Science.gov (United States)

    Cwikla, Julie

    2014-01-01

    Mathematics professor Julie Cwikla decided that she needed to investigate young children's understandings and see what precurricular partitioning notions young minds bring to the fraction table. Cwikla realized that only a handful of studies have examined how preschool-age and early elementary school-age students solve fraction problems (Empson…

  1. An Appetite for Fractions

    Science.gov (United States)

    Wilkerson, Trena L.; Bryan, Tommy; Curry, Jane

    2012-01-01

    This article describes how using candy bars as models gives sixth-grade students a taste for learning to represent fractions whose denominators are factors of twelve. Using paper models of the candy bars, students explored and compared fractions. They noticed fewer different representations for one-third than for one-half. The authors conclude…

  2. Categories of Fractions Revisited

    OpenAIRE

    Fritz, Tobias

    2008-01-01

    The theory of categories of fractions as originally developed by Gabriel and Zisman is reviewed in a pedagogical manner giving detailed proofs of all statements. A weakening of the category of fractions axioms used by Higson is discussed and shown to be equivalent to the original axioms.

  3. On fractional programming

    Energy Technology Data Exchange (ETDEWEB)

    Bajona-Xandri, C.; Martinez-Legaz, J.E.

    1994-12-31

    This paper studies the minimax fractional programming problem, assuming quasiconvexity of the objective function, under the lower subdifferentiability viewpoint. Necessary and sufficient optimality conditions and dual properties are found. We present applications of this theory to find the Pareto efficient solutions of a multiobjective fractional problem and to solve several economic models.

  4. A computational model of spatio-temporal cardiac intracellular calcium handling with realistic structure and spatial flux distribution from sarcoplasmic reticulum and t-tubule reconstructions.

    Directory of Open Access Journals (Sweden)

    Michael A Colman

    2017-08-01

    Full Text Available Intracellular calcium cycling is a vital component of cardiac excitation-contraction coupling. The key structures responsible for controlling calcium dynamics are the cell membrane (comprising the surface sarcolemma and transverse-tubules, the intracellular calcium store (the sarcoplasmic reticulum, and the co-localisation of these two structures to form dyads within which calcium-induced-calcium-release occurs. The organisation of these structures tightly controls intracellular calcium dynamics. In this study, we present a computational model of intracellular calcium cycling in three-dimensions (3-D, which incorporates high resolution reconstructions of these key regulatory structures, attained through imaging of tissue taken from the sheep left ventricle using serial block face scanning electron microscopy. An approach was developed to model the sarcoplasmic reticulum structure at the whole-cell scale, by reducing its full 3-D structure to a 3-D network of one-dimensional strands. The model reproduces intracellular calcium dynamics during control pacing and reveals the high-resolution 3-D spatial structure of calcium gradients and intracellular fluxes in both the cytoplasm and sarcoplasmic reticulum. We also demonstrated the capability of the model to reproduce potentially pro-arrhythmic dynamics under perturbed conditions, pertaining to calcium-transient alternans and spontaneous release events. Comparison with idealised cell models emphasised the importance of structure in determining calcium gradients and controlling the spatial dynamics associated with calcium-transient alternans, wherein the probabilistic nature of dyad activation and recruitment was constrained. The model was further used to highlight the criticality in calcium spark propagation in relation to inter-dyad distances. The model presented provides a powerful tool for future investigation of structure-function relationships underlying physiological and pathophysiological

  5. Extraction and fractionation of wheat flour proteins

    NARCIS (Netherlands)

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

    1982-01-01

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

  6. Effect of NaCl concentration on fractionation of lipophilic protein and 7S protein%氯化钠浓度对亲脂性蛋白和7S蛋白分级分离效果的影响

    Institute of Scientific and Technical Information of China (English)

    郑二丽; 曲家妮; 杨晓泉; 吴娜娜; 顾炜

    2011-01-01

    研究了氯化钠浓度对大豆蛋白中亲脂性蛋白(LP)与7S蛋白分级分离效果的影响,并采用差示扫描量热法(DSC)探讨了氯化钠浓度对蛋白质热变性的影响.研究表明:当氯化钠浓度低于100 mmol/L时,7S级分蛋白质得率会显著增加且蛋白质含量基本不变,所得7S级分纯度也有所提高(特别是50 mmol/L时);氯化钠浓度在0~100 mmol/L范围内LP蛋白质得率几乎不受影响,但蛋白质含量有所降低.各级分的热变性分析表明:7S组分的热变性温度不受氯化钠浓度的影响,但吸热峰焓值变化较大.当氯化钠浓度为50 mmol/L时对LP和7S的分级分离效果最好.%Effects of different NaCl concentration on the isolate of lipophilic protein (LP) and 7S fractions from soybean protein were studied, and the influences of NaCl concentration on the thermal properties of LP and 7S were investigated. The results showed that the recovery of 7S were improved obviously and the content of protein almost unchanged when the concentration of NaCl was lower than 100 mmoL/L, whereas the purity of 7S fraction increased ( especially at 50 mmol/L). When the NaC1 concentration was at the range of 0 -100 mmol/L, the recovery of LP was almost unaffected,but the protein content reduced slightly. The denaturation temperature of the 7S protein was not changed, but the endothermic peak enthalpies were significant different among the different concentration. The optimal concentration of NaCl for the fractionation of LP and 7S was 50 mmol/L.

  7. Functional analysis of mildly refined fractions from yellow pea

    NARCIS (Netherlands)

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

    2015-01-01

    Dry fractionation offers an attractive route to sustainably produce protein-enriched plant-based ingredients. For example, fine milling of peas followed by air classification separates starch granules from the protein matrix. Unlike conventional wet isolates, dry-enriched pea fractions consist of a

  8. Asymmetric Flow-Field Flow Fractionation Hyphenated ICP-MS as an Alternative to Cloud Point Extraction for Quantification of Silver Nanoparticles and Silver Speciation: Application for Nanoparticles with a Protein Corona.

    Science.gov (United States)

    Mudalige, Thilak K; Qu, Haiou; Linder, Sean W

    2015-07-21

    Production and application of nanoparticles in consumer products is at an all-time high due to the emerging field of nanotechnology. Direct detection and quantification of trace levels of nanoparticles within consumer products is very challenging and problematic. Although multiple methodologies are available for this purpose, each method has its own set of limitations. Herein, we developed an analytical platform consisting of asymmetric flow-field flow fractionation (AF4) coupled with inductively coupled plasma mass spectroscopy (ICP-MS) for the speciation and quantification of silver ions and silver nanoparticles at the ng/kg level (ppt). AF4 is utilized to concentrate the nanoparticles, and ICP-MS acts as the detector. The protein corona that forms upon exposure of nanoparticles to bovine serum albumin was utilized as a nanoparticle stabilization and AF4 recovery enhancement mechanism. Speciation of silver ions and nanoparticles was achieved with the assistance of penicillamine as a complexation ligand. The effect of nanoparticle size, surface coating, and ionization state toward the detection and quantification of the developed methodology was evaluated. The detection limit was found to be 4 ng/kg with the application of a 5 mL sample loop. Further application of this developed methodology on environmentally relevant samples was demonstrated by the analysis of Arkansas River water spiked with silver nanoparticles and nanoparticle spiked into humic acid solution (50 mg/L) at an environmentally relevant level.

  9. Fractional calculus in bioengineering.

    Science.gov (United States)

    Magin, Richard L

    2004-01-01

    Fractional calculus (integral and differential operations of noninteger order) is not often used to model biological systems. Although the basic mathematical ideas were developed long ago by the mathematicians Leibniz (1695), Liouville (1834), Riemann (1892), and others and brought to the attention of the engineering world by Oliver Heaviside in the 1890s, it was not until 1974 that the first book on the topic was published by Oldham and Spanier. Recent monographs and symposia proceedings have highlighted the application of fractional calculus in physics, continuum mechanics, signal processing, and electromagnetics, but with few examples of applications in bioengineering. This is surprising because the methods of fractional calculus, when defined as a Laplace or Fourier convolution product, are suitable for solving many problems in biomedical research. For example, early studies by Cole (1933) and Hodgkin (1946) of the electrical properties of nerve cell membranes and the propagation of electrical signals are well characterized by differential equations of fractional order. The solution involves a generalization of the exponential function to the Mittag-Leffler function, which provides a better fit to the observed cell membrane data. A parallel application of fractional derivatives to viscoelastic materials establishes, in a natural way, hereditary integrals and the power law (Nutting/Scott Blair) stress-strain relationship for modeling biomaterials. In this review, I will introduce the idea of fractional operations by following the original approach of Heaviside, demonstrate the basic operations of fractional calculus on well-behaved functions (step, ramp, pulse, sinusoid) of engineering interest, and give specific examples from electrochemistry, physics, bioengineering, and biophysics. The fractional derivative accurately describes natural phenomena that occur in such common engineering problems as heat transfer, electrode/electrolyte behavior, and sub

  10. Fracionamento dos carboidratos pelas equações do Cornell Net Carbohydrate and Protein System de três cultivares de girassol na presença ou não de irrigação Carbohydrate fractionation of three sunflower cultivars in the presence or absence of irrigation using the Cornell Net Carbohydrate and Protein System equations

    Directory of Open Access Journals (Sweden)

    Mário Adriano Ávila Queiroz

    2008-12-01

    Full Text Available Objetivou-se quantificar as frações de carboidratos pelas equações do Cornell Net Carbohydrate and Protein System (CNCPS de três cultivares de girassol (Helianthus annuus L. cultivados na presença ou não de irrigação. A utilização de uma preparação fibrosa, denominada parede celular (PC, nas equações da CNCPS, em substituição à fibra em detergente neutro (FDN não promoveu diferenças nas frações de carboidratos B1 e C, mas influenciou as frações A e B2. Como os valores da fração B1, obtidos pelo modelo CNCPS foram menores que os teores de amido e pectina determinados em laboratório, supõe-se que a pectina e outros oligossacarídeos da parede celular, solubilizados pela solução de detergente neutro (fibra solúvel, nunca fizeram parte da fração B1, e sim da fração A. Apesar de os carboidratos da fibra solúvel apresentarem elevadas taxas de degradação, não parece adequada a caracterização da fibra solúvel na fração A. Parece mais adequado que a fibra solúvel (que inclui a pectina seja alocada a uma fração exclusivamente sua, que pode ser a fração B2, e que seja criada uma nova fração, a B3, para os carboidratos digeríveis da parede celular. Assim, a fração B1 seria composta apenas de amido. A equação da fração C, que estima os carboidratos indigeríveis da parede celular, pode ser simplificada, relacionando a fração indigerível ao teor de lignina na matéria seca, e não à FDN isenta de cinzas e proteína, como atualmente utilizado. Esta proposta tem implicações práticas, uma vez que a fração indigerível da parede celular tem sido expressa em relação à FDN, e não na MS, com base no fato de que os efeitos inibitórios da lignina ocorrem sobre os componentes fibrosos da parede celular vegetal, e não sobre o conteúdo celular.This work aimed to estimate the carbohydrate fractions in three sunflower (Helianthus annuus L. cultivars in the presence or absence of irrigation, using the

  11. Social Trust and Fractionalization:

    DEFF Research Database (Denmark)

    Bjørnskov, Christian

    2008-01-01

    This paper takes a closer look at the importance of fractionalization for the creation of social trust. It first argues that the determinants of trust can be divided into two categories: those affecting individuals' trust radii and those affecting social polarization. A series of estimates using...... a much larger country sample than in previous literature confirms that fractionalization in the form of income inequality and political diversity adversely affects social trust while ethnic diversity does not. However, these effects differ systematically across countries, questioning standard...... interpretations of the influence of fractionalization on trust....

  12. The effect of monovalent and divalent cations on the ATP-dependent Ca2+-binding and phosphorylation during the reaction cycle of the sarcoplasmic reticulum Ca2+-transport ATPase.

    Science.gov (United States)

    Medda, P; Fassold, E; Hasselbach, W

    1987-06-01

    The coupling of Ca2+ movements and phosphate fluxes as well as the time-dependent occurrence of sequential reaction intermediates in the forward mode of the Ca,Mg-dependent ATPase reaction have been investigated using leaky vesicles (A23187) in the presence of varying Ca2+, Mg2+, and K+ concentrations. The employed ATP concentration of 2 microM does not allow more than one reaction cycle to occur. The respective fractions of ADP-sensitive and ADP-insensitive phosphoenzyme have been determined. The chosen experimental conditions (0-1 degree C, pH 6.0, absence of solubilizers) allow a prolonged time of observation and exclude interfering alterations of coupling and binding parameters, respectively. It is shown that under the experimental conditions K+ interacts with at least four different reaction steps (phosphoenzyme formation, E1P----E2P transition, E2P hydrolysis, and E2----E1 transformation). Mg2+ represents the sole ionic co-factor for the formation of the substrate MgATP if it is present in high concentrations (5 mM). Additional Ca2+ is bound to the substrate as well as to unspecific sites otherwise occupied by Mg2+ if Mg2+ is reduced to 0.1 mM. In this case the E1P----E2P transition rate (including Ca2+ translocation and Ca2+ release from low-affinity sites) is little diminished. If, in the absence of K+, both Mg2+ and Ca2+ are deficient E2P hydrolysis is vastly retarded. We find Ca2+ release to occur time-coincidently with E1P formation and not concomitantly with the comparably slow appearance of E2P; the molar amount of Ca2+ released, however, rather agreed with that of E2P formed. This suggests that under the prevailing conditions of a high proton concentration, phosphoenzyme states containing occluded Ca2+ or Ca2+ bound to low-affinity sites are transitional and not detectable. Preliminary findings on this subject have been published by us and colleagues from this laboratory [Hasselbach, W., Agostini, B., Medda, P., Migala, A. & Waas, W. (1985) in The

  13. Discrete fractional calculus

    CERN Document Server

    Goodrich, Christopher

    2015-01-01

    This text provides the first comprehensive treatment of the discrete fractional calculus. Experienced researchers will find the text useful as a reference for discrete fractional calculus and topics of current interest. Students who are interested in learning about discrete fractional calculus will find this text to provide a useful starting point. Several exercises are offered at the end of each chapter and select answers have been provided at the end of the book. The presentation of the content is designed to give ample flexibility for potential use in a myriad of courses and for independent study. The novel approach taken by the authors includes a simultaneous treatment of the fractional- and integer-order difference calculus (on a variety of time scales, including both the usual forward and backwards difference operators). The reader will acquire a solid foundation in the classical topics of the discrete calculus while being introduced to exciting recent developments, bringing them to the frontiers of the...

  14. Fractional Derivative Cosmology

    CERN Document Server

    Roberts, Mark D

    2009-01-01

    The degree by which a function can be differentiated need not be restricted to integer values. Usually most of the field equations of physics are taken to be second order, curiosity asks what happens if this is only approximately the case and the field equations are nearly second order. For Robertson-Walker cosmology there is a simple fractional modification of the Friedman and conservation equations. In general fractional gravitational equations similar to Einstein's are hard to define as this requires fractional derivative geometry. What fractional derivative geometry might entail is briefly looked at and it turns out that even asking very simple questions in two dimensions leads to ambiguous or intractable results. A two dimensional line element which depends on the Gamma-function is looked at.

  15. Intracellular Cadmium Isotope Fractionation

    Science.gov (United States)

    Horner, T. J.; Lee, R. B.; Henderson, G. M.; Rickaby, R. E.

    2011-12-01

    Recent stable isotope studies into the biological utilization of transition metals (e.g. Cu, Fe, Zn, Cd) suggest several stepwise cellular processes can fractionate isotopes in both culture and nature. However, the determination of fractionation factors is often unsatisfactory, as significant variability can exist - even between different organisms with the same cellular functions. Thus, it has not been possible to adequately understand the source and mechanisms of metal isotopic fractionation. In order to address this problem, we investigated the biological fractionation of Cd isotopes within genetically-modified bacteria (E. coli). There is currently only one known biological use or requirement of Cd, a Cd/Zn carbonic anhydrase (CdCA, from the marine diatom T. weissfloggii), which we introduce into the E. coli genome. We have also developed a cleaning procedure that allows for the treating of bacteria so as to study the isotopic composition of different cellular components. We find that whole cells always exhibit a preference for uptake of the lighter isotopes of Cd. Notably, whole cells appear to have a similar Cd isotopic composition regardless of the expression of CdCA within the E. coli. However, isotopic fractionation can occur within the genetically modified E. coli during Cd use, such that Cd bound in CdCA can display a distinct isotopic composition compared to the cell as a whole. Thus, the externally observed fractionation is independent of the internal uses of Cd, with the largest Cd isotope fractionation occurring during cross-membrane transport. A general implication of these experiments is that trace metal isotopic fractionation most likely reflects metal transport into biological cells (either actively or passively), rather than relating to expression of specific physiological function and genetic expression of different metalloenzymes.

  16. Isolation and Suborganellar Fractionation of Arabidopsis Chloroplasts.

    Science.gov (United States)

    Flores-Pérez, Úrsula; Jarvis, Paul

    2017-01-01

    Chloroplasts are structurally complex organelles containing ~2000-3000 proteins. They are delimited by a double membrane system or envelope, have an inner aqueous compartment called the stroma, and possess a second internal membrane system called the thylakoids. Thus, determining the suborganellar location of a chloroplast protein is vital to understanding or verifying its function. One way in which protein localization can be addressed is through fractionation. Here we present two rapid and simple methods that may be applied sequentially on the same day: (a) The isolation of intact chloroplasts from Arabidopsis thaliana plants that may be used directly (e.g., for functional studies such as protein import analysis), or for further processing as follows; (b) separation of isolated chloroplasts into three suborganellar fractions (envelope membranes, a soluble fraction containing stromal proteins, and the thylakoids). These methods are routinely used in our laboratory, and they provide a good yield of isolated chloroplasts and suborganellar fractions that can be used for various downstream applications.

  17. New Fractional Complex Transform for Conformable Fractional Partial Differential Equations

    Directory of Open Access Journals (Sweden)

    Çenesiz Y.

    2016-12-01

    Full Text Available Conformable fractional complex transform is introduced in this paper for converting fractional partial differential equations to ordinary differential equations. Hence analytical methods in advanced calculus can be used to solve these equations. Conformable fractional complex transform is implemented to fractional partial differential equations such as space fractional advection diffusion equation and space fractional telegraph equation to obtain the exact solutions of these equations.

  18. Changes in proteins during the ripening of Spanish dried beef 'Cecina'.

    Science.gov (United States)

    García, I; Díez, V; Zumalacárregui, J M

    1997-08-01

    Changes in the solubility of sarcoplasmic and myofibrillar proteins were tracked in Semitendinosus and Rectus femoris muscles during the ripening process of Spanish 'Cecina'. The extractability of both types of proteins decreased during the ripening. This phenomenon was more marked in the initial stages of processing. Electrophoretic studies of the myofibrillar proteins showed the virtual disappearance of the myosin heavy chain, troponin C and myosin light chain 2 from the smoking phase onward and the appearance of three components of molecular weight of about 65, 70 and 75 kda during ripening. The remaining proteins did not suffer appreciable changes.

  19. Differential Association of the Na+/H+ Exchanger Regulatory Factor (NHERF Family of Adaptor Proteins with the Raft- and the Non-Raft Brush Border Membrane Fractions of NHE3

    Directory of Open Access Journals (Sweden)

    Ayesha Sultan

    2013-11-01

    Full Text Available Background/Aims: Trafficking, brush border membrane (BBM retention, and signal-specific regulation of the Na+/H+ exchanger NHE3 is regulated by the Na+/H+ Exchanger Regulatory Factor (NHERF family of PDZ-adaptor proteins, which enable the formation of multiprotein complexes. It is unclear, however, what determines signal specificity of these NHERFs. Thus, we studied the association of NHE3, NHERF1 (EBP50, NHERF2 (E3KARP, and NHERF3 (PDZK1 with lipid rafts in murine small intestinal BBM. Methods: Detergent resistant membranes (“lipid rafts” were isolated by floatation of Triton X-incubated small intestinal BBM from a variety of knockout mouse strains in an Optiprep step gradient. Acid-activated NHE3 activity was measured fluorometrically in BCECF-loaded microdissected villi, or by assessment of CO2/HCO3- mediated increase in fluid absorption in perfused jejunal loops of anethetized mice. Results: NHE3 was found to partially associate with lipid rafts in the native BBM, and NHE3 raft association had an impact on NHE3 transport activity and regulation in vivo. NHERF1, 2 and 3 were differentially distributed to rafts and non-rafts, with NHERF2 being most raft-associated and NHERF3 entirely non-raft associated. NHERF2 expression enhanced the localization of NHE3 to membrane rafts. The use of acid sphingomyelinase-deficient mice, which have altered membrane lipid as well as lipid raft composition, allowed us to test the validity of the lipid raft concept in vivo. Conclusions: The differential association of the NHERFs with the raft-associated and the non-raft fraction of NHE3 in the brush border membrane is one component of the differential and signal-specific NHE3 regulation by the different NHERFs.

  20. 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

  1. Effect of Interactions between Water Management and Nitrogen Fertilizer on Wheat Processing Quality and Protein Fractions%水氮互作对小麦籽粒蛋白质组分和品质的影响

    Institute of Scientific and Technical Information of China (English)

    王小燕; 于振文

    2009-01-01

    This experiment was carried out at the Experiment Station of Shandong Agricultural University, under a rain-proof through shelter. Winter wheat cultivar Jimai 20 with high grain yield and strong gluten was used to determine the effects of irrigation rate and nitrogen fertilizer rate on wheat processing quality and protein fractions. The results were as follows: when the nitrogen rate increased from 120 kg*ha-1 to 240 kg*ha-1, grain protein content, albumin content and globulin content increased, while the ratio of gliadin & glutenin content to albumin & globulin content decreased, which lead to the decrease of the dough stability time. Under the same nitrogen rate, when the irrigation rate changed from W0(no irrigation) to W1(twice irrigation at before planting and jointing stages), grain protein content increased, but the ratio of gliadin & glutenin content to albumin & globulin content decreased from W1 to W2 (three times irrigation at before planting, jointing stage and anthesis stage) and W3(five times irrigation at before planting, before winter, jointing stage, and anthesis and grain filling stages), grain protein content decreased, and gliadin & glutenin content decreased as well, but albumin & globulin content increased, and at last, the ratio of gliadin & glutenin content to alb