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Sample records for antifreeze protein activity

  1. Antifreeze activity enhancement by site directed mutagenesis on an antifreeze protein from the beetle Rhagium mordax

    DEFF Research Database (Denmark)

    Friis, Dennis Steven; Kristiansen, Erlend; von Solms, Nicolas

    2014-01-01

    The ice binding motifs of insect antifreeze proteins (AFPs) mainly consist of repetitive TxT motifs aligned on a flat face of the protein. However, these motifs often contain non-threonines that disrupt the TxT pattern. We substituted two such disruptive amino acids located in the ice binding face...... of an AFP from Rhagium mordax with threonine. Furthermore, a mutant with an extra ice facing TxT motif was constructed. These mutants showed enhanced antifreeze activity compared to the wild type at low concentrations. However, extrapolating the data indicates that the wild type will become the most active...

  2. Structural basis of antifreeze activity of a bacterial multi-domain antifreeze protein.

    Directory of Open Access Journals (Sweden)

    Chen Wang

    Full Text Available Antifreeze proteins (AFPs enhance the survival of organisms inhabiting cold environments by affecting the formation and/or structure of ice. We report the crystal structure of the first multi-domain AFP that has been characterized. The two ice binding domains are structurally similar. Each consists of an irregular β-helix with a triangular cross-section and a long α-helix that runs parallel on one side of the β-helix. Both domains are stabilized by hydrophobic interactions. A flat plane on the same face of each domain's β-helix was identified as the ice binding site. Mutating any of the smaller residues on the ice binding site to bulkier ones decreased the antifreeze activity. The bulky side chain of Leu174 in domain A sterically hinders the binding of water molecules to the protein backbone, partially explaining why antifreeze activity by domain A is inferior to that of domain B. Our data provide a molecular basis for understanding differences in antifreeze activity between the two domains of this protein and general insight on how structural differences in the ice-binding sites affect the activity of AFPs.

  3. Antifreeze Protein Mimetic Metallohelices with Potent Ice Recrystallization Inhibition Activity.

    Science.gov (United States)

    Mitchell, Daniel E; Clarkson, Guy; Fox, David J; Vipond, Rebecca A; Scott, Peter; Gibson, Matthew I

    2017-07-26

    Antifreeze proteins are produced by extremophile species to control ice formation and growth, and they have potential applications in many fields. There are few examples of synthetic materials which can reproduce their potent ice recrystallization inhibition property. We report that self-assembled enantiomerically pure, amphipathic metallohelicies inhibited ice growth at just 20 μM. Structure-property relationships and calculations support the hypothesis that amphipathicity is the key motif for activity. This opens up a new field of metallo-organic antifreeze protein mimetics and provides insight into the origins of ice-growth inhibition.

  4. Antifreeze Proteins of Bacteria

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 12; Issue 12. Antifreeze Proteins of Bacteria. M K Chattopadhyay. General Article Volume 12 Issue 12 December 2007 pp 25-30. Fulltext. Click here to view fulltext PDF. Permanent link: https://www.ias.ac.in/article/fulltext/reso/012/12/0025-0030 ...

  5. Antifreeze Proteins of Bacteria

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 12; Issue 12. Antifreeze Proteins of Bacteria. M K Chattopadhyay. General Article Volume 12 Issue 12 December 2007 pp 25-30. Fulltext. Click here to view fulltext PDF. Permanent link: http://www.ias.ac.in/article/fulltext/reso/012/12/0025-0030. Keywords.

  6. Cloning, expression, and activity of type IV antifreeze protein from cultured subtropical olive flounder (Paralichthys olivaceus

    Directory of Open Access Journals (Sweden)

    Jong Kyu Lee

    2016-10-01

    Full Text Available Abstract Antifreeze proteins (AFPs lower the freezing point but not the melting point of aqueous solutions by inhibiting the growth of ice crystals via an adsorption-inhibition mechanism. However, the function of type IV AFP (AFP IV is questionable, as its antifreeze activity is on the verge of detectable limits, its physiological concentration in adult fish blood is too low to function as a biological antifreeze, and its homologues are present even in fish from tropic oceans as well as freshwater. Therefore, we speculated that AFP IV may have gained antifreeze activity not by selective pressure but by chance. To test this hypothesis, we cloned, expressed, and assayed AFP IV from cultured subtropical olive flounder (Paralichthys olivaceus, which do not require antifreeze protein for survival. Among the identified expressed sequence tags of the flounder liver sample, a 5′-deleted complementary DNA (cDNA sequence similar to the afp4 gene of the longhorn sculpin was identified, and its full-length cDNA and genome structure were examined. The deduced amino acid sequence of flounder AFP IV shared 55, 53, 52, and 49 % identity with those of Pleuragramma antarcticum, Myoxocephalus octodecemspinosus, Myoxocephalus scorpius, and Notothenia coriiceps, respectively. Furthermore, the genomic structure of this gene was conserved with those of other known AFP IVs. Notably, the recombinant AFP IV showed a weak but distinct thermal hysteresis of 0.07 ± 0.01 °C at the concentration of 0.5 mg/mL, and ice crystals in an AFP IV solution grew star-shaped, which are very similar to those obtained from other polar AFP IVs. Taken together, our results do not support the hypothesis of evolution of AFP IV by selective pressure, suggesting that the antifreeze activity of AFP IV may have been gained by chance.

  7. Natural and Artificial Antifreeze Proteins

    Science.gov (United States)

    Arai, Soichi; Hirao, Noriko

    In the blood of winter polar fish an antifreeze glycoprotein (AFGP) occurs which acts to protect the fish from freezing to death. The AFGP has a unique hydrophilic hydrophobic conformation, involved in non-colligative depression of the freezing temperature of water without altering the melting point of ice. This phenomenon is reportedly a reflection of the ice crystal growth inhibition by the adsorption of the AFGP onto a-axial surfaces of the ice crystal. The authors, on the other hand, have developed an enzymatically modified protein (EMG-12) by covalent attachment of leucine dodecyl ester to the C-terminal position of gelation with the aid of a reverse reaction catalyzed by a protease. EMG-12, having a hydrophilic-hydrophobic structure, is highly surface-active and acts to stabilize a supercooling state of water by antinucleation. Discussions are made on similarities and dissimilarities of structure-function relationships of these natural and artificial antifreeze proteins. The significance of using them as antifreeze agents is also discussed.

  8. A kinetic description of antifreeze glycoprotein activity.

    Science.gov (United States)

    Burcham, T S; Osuga, D T; Yeh, Y; Feeney, R E

    1986-05-15

    The antifreeze glycoproteins (AFGP) of polar fish have the ability to depress the freezing temperature of water approximately 500 times the amount expected based on the number of AFGP molecules in solution; yet AFGP solutions have a purely colligative melting point depression. The difference of solution melting and freezing temperatures is the antifreeze activity of AFGP. One characteristic of AFGP activity that requires further examination is the effect of concentration on antifreeze activity, especially whether the activity saturates at high concentrations or the measured activity increases ad infinitum. This study first surveys the activity of the various antifreeze components from both Pagothenia borchgrevinki and the Arg-containing antifreeze glycoprotein from Eleginus gracilis (EgAF). It was found that all AFGP components examined have a plateau in activity at high concentration, but the actual value of the plateau activity differs between the different length AFGP components and between AFGP and EgAF. While the low molecular weight components of both AFGP and EgAF lose activity at deep supercooling, at high concentration activity is restored. The activity data is then shown to fit a reversible kinetic model of AFGP activity, and the coefficients obtained are used to compare the activity differences between AFGP components and between AFGP and EgAF. The model is also shown to describe the activity of the antifreeze protein of the fish Pseudopleuronectes americanus and the thermal hysteresis protein of the insect, Tenebrio molitor.

  9. Operation of Kelvin Effect in the Activities of an Antifreeze Protein: A Molecular Dynamics Simulation Study.

    Science.gov (United States)

    Midya, Uday Sankar; Bandyopadhyay, Sanjoy

    2018-03-29

    Ice growth and melting inhibition activities of antifreeze proteins (AFPs) are better explained by the adsorption-inhibition mechanism. Inhibition occurs as a result of the Kelvin effect induced by adsorbed protein molecules onto the surface of seed ice crystal. However, the Kelvin effect has not been explored by the state-of-the-art experimental techniques. In this work, atomistic molecular dynamics simulations have been carried out with Tenebrio molitor antifreeze protein ( TmAFP) placed at ice-water interface to probe the Kelvin effect in the mechanism of AFPs. Simulations show that, below equilibrium melting temperature, ice growth is inhibited through the convex ice-water interface formation toward the water phase and, above equilibrium melting temperature, ice melting is inhibited through the concave ice-water interface formation inward to ice phase. Simulations further reveal that the radius of curvature of the interface formed to stop the ice growth increases with decrease in the degree of supercooling. Our results are in qualitative agreement with the theoretical prediction of the Kelvin effect and thus reveal its operation in the activities of AFPs.

  10. Antifreeze proteins enable plants to survive in freezing conditions

    Indian Academy of Sciences (India)

    Overwintering plants secrete antifreeze proteins (AFPs) to provide freezing tolerance. These proteins bind to and inhibit the growth of ice crystals that are formed in the apoplast during subzero temperatures. Antifreeze activity has been detected in more than 60 plants and AFPs have been purified from 15 of these, including ...

  11. Re-evaluation of a bacterial antifreeze protein as an adhesin with ice-binding activity.

    Directory of Open Access Journals (Sweden)

    Shuaiqi Guo

    Full Text Available A novel role for antifreeze proteins (AFPs may reside in an exceptionally large 1.5-MDa adhesin isolated from an Antarctic Gram-negative bacterium, Marinomonas primoryensis. MpAFP was purified from bacterial lysates by ice adsorption and gel electrophoresis. We have previously reported that two highly repetitive sequences, region II (RII and region IV (RIV, divide MpAFP into five distinct regions, all of which require mM Ca(2+ levels for correct folding. Also, the antifreeze activity is confined to the 322-residue RIV, which forms a Ca(2+-bound beta-helix containing thirteen Repeats-In-Toxin (RTX-like repeats. RII accounts for approximately 90% of the mass of MpAFP and is made up of ∼120 tandem 104-residue repeats. Because these repeats are identical in DNA sequence, their number was estimated here by pulsed-field gel electrophoresis. Structural homology analysis by the Protein Homology/analogY Recognition Engine (Phyre2 server indicates that the 104-residue RII repeat adopts an immunoglobulin beta-sandwich fold that is typical of many secreted adhesion proteins. Additional RTX-like repeats in RV may serve as a non-cleavable signal sequence for the type I secretion pathway. Immunodetection shows both repeated regions are uniformly distributed over the cell surface. We suggest that the development of an AFP-like domain within this adhesin attached to the bacterial outer surface serves to transiently bind the host bacteria to ice. This association would keep the bacteria within the upper reaches of the water column where oxygen and nutrients are potentially more abundant. This novel envirotactic role would give AFPs a third function, after freeze avoidance and freeze tolerance: that of transiently binding an organism to ice.

  12. Antifreeze proteins of teleost fishes.

    Science.gov (United States)

    Fletcher, G L; Hew, C L; Davies, P L

    2001-01-01

    Marine teleosts at high latitudes can encounter ice-laden seawater that is approximately 1 degrees C colder than the colligative freezing point of their body fluids. They avoid freezing by producing small antifreeze proteins (AFPs) that adsorb to ice and halt its growth, thereby producing an additional non-colligative lowering of the freezing point. AFPs are typically secreted by the liver into the blood. Recently, however, it has become clear that AFP isoforms are produced in the epidermis (skin, scales, fin, and gills) and may serve as a first line of defense against ice propagation into the fish. The basis for the adsorption of AFPs to ice is something of a mystery and is complicated by the extreme structural diversity of the five antifreeze types. Despite the recent acquisition of several AFP three-dimensional structures and the definition of their ice-binding sites by mutagenesis, no common ice-binding motif or even theme is apparent except that surface-surface complementarity is important for binding. The remarkable diversity of antifreeze types and their seemingly haphazard phylogenetic distribution suggest that these proteins might have evolved recently in response to sea level glaciation occurring just 1-2 million years ago in the northern hemisphere and 10-30 million years ago around Antarctica. Not surprisingly, the expression of AFP genes from different origins can also be quite dissimilar. The most intensively studied system is that of the winter flounder, which has a built-in annual cycle of antifreeze expression controlled by growth hormone (GH) release from the pituitary in tune with seasonal cues. The signal transduction pathway, transcription factors, and promoter elements involved in this process are just beginning to be characterized.

  13. Influence of Block Copolymerization on the Antifreeze Protein Mimetic Ice Recrystallization Inhibition Activity of Poly(vinyl alcohol).

    Science.gov (United States)

    Congdon, Thomas R; Notman, Rebecca; Gibson, Matthew I

    2016-09-12

    Antifreeze (glyco) proteins are produced by many cold-acclimatized species to enable them to survive subzero temperatures. These proteins have multiple macroscopic effects on ice crystal growth which makes them appealing for low-temperature applications-from cellular cryopreservation to food storage. Poly(vinyl alcohol) has remarkable ice recrystallization inhibition activity, but its mode of action is uncertain as is the extent at which it can be incorporated into other high-order structures. Here the synthesis and characterization of well-defined block copolymers containing poly(vinyl alcohol) and poly(vinylpyrrolidone) by RAFT/MADIX polymerization is reported, as new antifreeze protein mimetics. The effect of adding a large second hydrophilic block is studied across a range of compositions, and it is found to be a passive component in ice recrystallization inhibition assays, enabling retention of all activity. In the extreme case, a block copolymer with only 10% poly(vinyl alcohol) was found to retain all activity, where statistical copolymers of PVA lose all activity with very minor changes to composition. These findings present a new method to increase the complexity of antifreeze protein mimetic materials, while retaining activity, and also to help understand the underlying mechanisms of action.

  14. The effect of surface charge on the thermal stability and ice recrystallization inhibition activity of antifreeze protein III (AFP III).

    Science.gov (United States)

    Deller, R C; Carter, B M; Zampetakis, I; Scarpa, F; Perriman, A W

    2018-01-01

    The aim of this study was to examine the effect of chemical cationization on the structure and function of antifreeze protein III (AFP III) over an extreme temperature range (-40°C to +90°C) using far-UV synchrotron radiation circular dichroism (SRCD) and ice recrystallization inhibition (IRI) assays. Chemical cationization was able to produce a modified AFP III with a net cationic charge at physiological pH that had enhanced resistance to denaturation at elevated temperatures, with no immediate negative impact on protein structure at subzero temperatures. Furthermore, cationized AFP III retained an IRI activity similar to that of native AFP III. Consequently, chemical cationization may provide a pathway to the development of more robust antifreeze proteins as supplementary cryoprotectants in the cryopreservation of clinically relevant cells. Copyright © 2017. Published by Elsevier Inc.

  15. Antifreeze glycoprotein agents: structural requirements for activity.

    Science.gov (United States)

    Carvajal-Rondanelli, Patricio A; Marshall, Sergio H; Guzman, Fanny

    2011-11-01

    Antifreeze glycoproteins (AFGPs) are considered to be the most efficient means to reduce ice damage to cell tissues since they are able to inhibit growth and crystallization of ice. The key element of antifreeze proteins is to act in a non-colligative manner which allows them to function at concentrations 300-500 times lowers than other dissolved solutes. During the past decade, AFGPs have demonstrated tremendous potential for many pharmaceutical and food applications. Presently, the only route to obtain AFGPs involves the time consuming and expensive process of isolation and purification from deep-sea polar fishes. Unfortunately, it is not amenable to mass production and commercial applications. The lack of understanding of the mechanism through which the AFGPs inhibit ice growth has also hampered the realization of industrial and biotechnological applications. Here we report the structural motifs that are essential for antifreeze activity of AFGPs, and propose a unified mechanism based on both recent studies of short alanine peptides and structure activity relationship of synthesized AFGPs. Copyright © 2011 Society of Chemical Industry.

  16. Gold Nanoparticle Aggregation as a Probe of Antifreeze (Glyco) Protein-Inspired Ice Recrystallization Inhibition and Identification of New IRI Active Macromolecules.

    Science.gov (United States)

    Mitchell, Daniel E; Congdon, Thomas; Rodger, Alison; Gibson, Matthew I

    2015-10-26

    Antifreeze (glyco)proteins are found in polar fish species and act to slow the rate of growth of ice crystals; a property known as ice recrystallization inhibition. The ability to slow ice growth is of huge technological importance especially in the cryopreservation of donor cells and tissue, but native antifreeze proteins are often not suitable, nor easily available. Therefore, the search for new materials that mimic this function is important, but currently limited by the low-throughout assays associated with the antifreeze properties. Here 30 nm gold nanoparticles are demonstrated to be useful colorimetric probes for ice recrystallization inhibition, giving a visible optical response and is compatible with 96 well plates for high-throughout studies. This method is faster, requires less infrastructure, and has easier interpretation than the currently used 'splat' methods. Using this method, a series of serum proteins were identified to have weak, but specific ice recrystallization inhibition activity, which was removed upon denaturation. It is hoped that high-throughput tools such as this will accelerate the discovery of new antifreeze mimics.

  17. Antivirulence Properties of an Antifreeze Protein

    Directory of Open Access Journals (Sweden)

    Martin Heisig

    2014-10-01

    Full Text Available As microbial drug-resistance increases, there is a critical need for new classes of compounds to combat infectious diseases. The Ixodes scapularis tick antifreeze glycoprotein, IAFGP, functions as an antivirulence agent against diverse bacteria, including methicillin-resistant Staphylococcus aureus. Recombinant IAFGP and a peptide, P1, derived from this protein bind to microbes and alter biofilm formation. Transgenic iafgp-expressing flies and mice challenged with bacteria, as well as wild-type animals administered P1, were resistant to infection, septic shock, or biofilm development on implanted catheter tubing. These data show that an antifreeze protein facilitates host control of bacterial infections and suggest therapeutic strategies for countering pathogens.

  18. The role of endogenous antifreeze protein enhancers in the hemolymph thermal hysteresis activity of the beetle Dendroides canadensis.

    Science.gov (United States)

    Duman, John G.; Serianni, Anthony S.

    2002-01-01

    Antifreeze proteins (AFPs) lower the freezing point of water by a non-colligative mechanism, but do not lower the melting point, therefore producing a difference between the freezing and melting points termed thermal hysteresis. Thermal hysteresis activity (THA) of AFPs from overwintering larvae of the beetle Dendroides canadensis is dependent upon AFP concentration and the presence of enhancers of THA which may be either other proteins or low molecular mass enhancers. The purpose of this study was to determine the relative contributions of endogenous enhancers in winter D. canadensis hemolymph.Winter hemolymph collected over four successive winters (1997-1998 to 2000-2001) was tested. The first three of these winters were the warmest on record in this area, while December of the final year was the coldest on record. Protein and low molecular mass enhancers raised hemolymph THA 60-97% and 35-55%, respectively, based on hemolymph with peak THA for each year collected over the four successive winters. However, the hemolymph AFPs were not maximally enhanced since addition of the potent enhancer citrate (at non-physiologically high levels) resulted in large increases in THA. (13)NMR showed that glycerol was the only low molecular mass solute present in sufficiently high concentrations in the hemolymph to function as an enhancer. Maximum THA appears to be approximately 8.5 degrees C.

  19. Freeze resistance in rainbow smelt (Osmerus mordax): seasonal pattern of glycerol and antifreeze protein levels and liver enzyme activity associated with glycerol production.

    Science.gov (United States)

    Lewis, Johanne M; Ewart, K Vanya; Driedzic, William R

    2004-01-01

    Rainbow smelt (Osmerus mordax) inhabit inshore waters along the North American Atlantic coast. During the winter, these waters are frequently ice covered and can reach temperatures as low as -1.9 degrees C. To prevent freezing, smelt accumulate high levels of glycerol, which lower the freezing point via colligative means, and antifreeze proteins (AFP). The up-regulation of the antifreeze response (both glycerol and AFP) occurs in early fall, when water temperatures are 5 degrees -6 degrees C. The accumulation of glycerol appears to be the main mechanism of freeze resistance in smelt because it contributes more to the lowering of the body's freezing point than the activity of the AFP (0.5 degrees C vs. 0.25 degrees C for glycerol and AFP, respectively) at a water temperature of -1.5 degrees C. Moreover, AFP in smelt appears to be a safeguard mechanism to prevent freezing when glycerol levels are low. Significant increases in activities of the liver enzymes glycerol 3-phosphate dehydrogenase (GPDH), alanine aminotransferase (AlaAT), and phosphoenolpyruvate carboxykinase (PEPCK) during the initiation of glycerol production and significant correlations between enzyme activities and plasma glycerol levels suggest that these enzymes are closely associated with the synthesis and maintenance of elevated glycerol levels for use as an antifreeze. These findings add further support to the concept that carbon for glycerol is derived from amino acids.

  20. Antifreeze Proteins from Diverse Organisms and their Applications: An Overview.

    Science.gov (United States)

    Cheung, Randy Chi Fai; Ng, Tzi Bun; Wong, Jack Ho

    2017-01-01

    Antifreeze proteins are ice-binding or ice-structuring proteins that prevent water from freezing by adsorbing to the ice surface and stopping the growth of minute ice crystals to large crystals in a non-colligative manner. The antifreeze proteins are found in species like fish, arthropods, plants, algae, fungi, yeasts and bacteria. The diversity, distribution and classification of antifreeze proteins were highlighted in this review. Antifreeze proteins help the organisms adapt to and survive in subzero temperature environments. The distribution of antifreeze proteins in different species appears to be the outcome of a combination of independent evolutionary events, probably the convergent evolution or horizontal gene transfer. Benefits can be derived from the frost resistance of these organisms. Their potential applications have been recognized in food processing, cryopreservation, cryosurgery, fishery and agricultural industries and anti-icing materials development. This review includes information on the current understanding of antifreeze proteins. A discussion on interactions and mechanisms involving ice recognition and adsorption was also included. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  1. MINING THE PROTEIN REPERTOIRE OF A HIMALAYAN SHRUB, HIPPOPHAE RHAMNOIDES FOR ANTIFREEZE PROTEINS

    Directory of Open Access Journals (Sweden)

    Bhavana Sharma

    2016-09-01

    Full Text Available Seabuckthorn (Hippophae rhamnoides, a cold desert shrub, can survive freezing temperature conditions and is considered as frost and drought tolerant. Earlier, antifreeze activity was reported in seabuckthorn seedlings grown under laboratory conditions. No reports are available on the cold hardiness of this huge bioresource available naturally in the Himalayan region. Detection of antifreeze activity in leaves and berries (splat assay and nanoliter osmometer confirmed the presence of putative antifreeze proteins (AFPs which may help in the survival of this plant under freezing conditions. Flower shaped ice crystals were observed in both leaves and berries while hexagonal ice crystals in seedlings indicated comparatively higher antifreeze activity in the wild parts. Splat assay results confirmed highest IRI activity in leaf (a 2.75 fold decrease in mean grain size of ice crystal after annealing followed by berry (with 1.75 fold decrease and least in the seedlings (with 1.5 fold decrease. Gel filtration chromatography resolved leaf fractions exhibited antifreeze activity in 34, 36 and 41 kDa while in berry fractions a 41 kDa polypeptide showed antifreeze activity. This is the first report showing presence of AFPs in (H. rhamnoides leaf and berry. Shotgun proteomic analysis using Q-Exactive Orbitrap High Resolution Mass Spectrometer and functional annotation of leaf and berry proteins revealed their association with primary, secondary metabolism, defence/stress, redox regulation, signalling and structural remodelling majority of which are affected during cold stress. Further purification of these AFPs could open gates for commercial utilization of this plant growing abundantly in Himalayan regions of India.

  2. Structure and application of antifreeze proteins from Antarctic bacteria.

    Science.gov (United States)

    Muñoz, Patricio A; Márquez, Sebastián L; González-Nilo, Fernando D; Márquez-Miranda, Valeria; Blamey, Jenny M

    2017-08-07

    Antifreeze proteins (AFPs) production is a survival strategy of psychrophiles in ice. These proteins have potential in frozen food industry avoiding the damage in the structure of animal or vegetal foods. Moreover, there is not much information regarding the interaction of Antarctic bacterial AFPs with ice, and new determinations are needed to understand the behaviour of these proteins at the water/ice interface. Different Antarctic places were screened for antifreeze activity and microorganisms were selected for the presence of thermal hysteresis in their crude extracts. Isolates GU1.7.1, GU3.1.1, and AFP5.1 showed higher thermal hysteresis and were characterized using a polyphasic approach. Studies using cucumber and zucchini samples showed cellular protection when samples were treated with partially purified AFPs or a commercial AFP as was determined using toluidine blue O and neutral red staining. Additionally, genome analysis of these isolates revealed the presence of genes that encode for putative AFPs. Deduced amino acids sequences from GU3.1.1 (gu3A and gu3B) and AFP5.1 (afp5A) showed high similarity to reported AFPs which crystal structures are solved, allowing then generating homology models. Modelled proteins showed a triangular prism form similar to β-helix AFPs with a linear distribution of threonine residues at one side of the prism that could correspond to the putative ice binding side. The statistically best models were used to build a protein-water system. Molecular dynamics simulations were then performed to compare the antifreezing behaviour of these AFPs at the ice/water interface. Docking and molecular dynamics simulations revealed that gu3B could have the most efficient antifreezing behavior, but gu3A could have a higher affinity for ice. AFPs from Antarctic microorganisms GU1.7.1, GU3.1.1 and AFP5.1 protect cellular structures of frozen food showing a potential for frozen food industry. Modeled proteins possess a β-helix structure, and

  3. Blocking rapid ice crystal growth through nonbasal plane adsorption of antifreeze proteins.

    Science.gov (United States)

    Olijve, Luuk L C; Meister, Konrad; DeVries, Arthur L; Duman, John G; Guo, Shuaiqi; Bakker, Huib J; Voets, Ilja K

    2016-04-05

    Antifreeze proteins (AFPs) are a unique class of proteins that bind to growing ice crystal surfaces and arrest further ice growth. AFPs have gained a large interest for their use in antifreeze formulations for water-based materials, such as foods, waterborne paints, and organ transplants. Instead of commonly used colligative antifreezes such as salts and alcohols, the advantage of using AFPs as an additive is that they do not alter the physicochemical properties of the water-based material. Here, we report the first comprehensive evaluation of thermal hysteresis (TH) and ice recrystallization inhibition (IRI) activity of all major classes of AFPs using cryoscopy, sonocrystallization, and recrystallization assays. The results show that TH activities determined by cryoscopy and sonocrystallization differ markedly, and that TH and IRI activities are not correlated. The absence of a distinct correlation in antifreeze activity points to a mechanistic difference in ice growth inhibition by the different classes of AFPs: blocking fast ice growth requires rapid nonbasal plane adsorption, whereas basal plane adsorption is only relevant at long annealing times and at small undercooling. These findings clearly demonstrate that biomimetic analogs of antifreeze (glyco)proteins should be tailored to the specific requirements of the targeted application.

  4. Expression and purification of sea raven type II antifreeze protein from Drosophila melanogaster S2 cells.

    Science.gov (United States)

    Scotter, Andrew J; Kuntz, Douglas A; Saul, Michelle; Graham, Laurie A; Davies, Peter L; Rose, David R

    2006-06-01

    We present a system for the expression and purification of recombinant sea raven type II antifreeze protein, a cysteine-rich, C-type lectin-like globular protein that has proved to be a difficult target for recombinant expression and purification. The cDNAs encoding the pro- and mature forms of the sea raven protein were cloned into a modified pMT Drosophila expression vector. These constructs produced N-terminally His(6)-tagged pro- and mature forms of the type II antifreeze protein under the control of a metallothionein promoter when transfected into Drosophila melanogaster S2 cells. Upon induction of stable cell lines the two proteins were expressed at high levels and secreted into the medium. The proteins were then purified from the cell medium in a simple and rapid protocol using immobilized metal affinity chromatography and specific protease cleavage by tobacco etch virus protease. The proteins demonstrated antifreeze activity indistinguishable from that of wild-type sea raven antifreeze protein purified from serum as illustrated by ice affinity purification, ice crystal morphology, and their ability to inhibit ice crystal growth. This expression and purification system gave yields of 95 mg/L of fully active mature sea raven type II AFP and 9.6 mg/L of the proprotein. This surpasses all previous attempts to express this protein in Escherichia coli, baculovirus-infected fall armyworm cells and Pichia pastoris and will provide sufficient protein for structural analysis.

  5. Antifreeze proteins enable plants to survive in freezing conditions

    Indian Academy of Sciences (India)

    2014-10-20

    Oct 20, 2014 ... 0.52°C. TH. Zhang et al. 2011. 4. Daucus carrota. Tap. Root extract. 36. Apoplastic. Polygalacturonase inhibitor protein. N-glycosylated,. TH-0.36 at. 150 μ ...... na R 2011 A framework for classification of antifreeze proteins in over wintering plants based on their sequence and structural features. J. Bioinform.

  6. Studies on new antifreeze protein from the psychrophilic diatom ...

    African Journals Online (AJOL)

    Antifreeze proteins (AFPs) are found in a wide range of species including fishes, plants, etc. They have very characteristic feature that inhibit the growth and recrystallization of ice that forms in intercellular spaces. Two expressed sequence tags (ESTs) were previously identified from salt stress cDNA library in Fragilariopsis ...

  7. In silico characterization of antifreeze proteins using computational ...

    Indian Academy of Sciences (India)

    In this paper, seventeen different fish Antifreeze Proteins (AFPs) retrieved from Swiss-Prot database are analysed and characterized using In silico tools. ... Department of Chemistry, Sri Chandrasekharendra Saraswathi Viswa Maha Vidyalaya (Deemed University), Enathur, Kanchipuram 631 561; EXCEL and Polymer ...

  8. Structural characteristics of a novel antifreeze protein from the longhorn beetle Rhagium inquisitor

    DEFF Research Database (Denmark)

    Kristiansen, E; Ramløv, Hans; Højrup, Peter

    2011-01-01

    beetle Rhagium inquisitor is known to express AFPs in its body fluids. In this work we report on the primary structure and structural characteristics of a 12.8 kDa AFP from this beetle (RiAFP). It has a high capacity to evoke antifreeze activity as compared to other known insect AFPs......Antifreeze proteins (AFPs) are characterized by their capacity to inhibit the growth of ice and are produced by a variety of polar fish, terrestrial arthropods and other organisms inhabiting cold environments. This capacity reflects their role as stabilizers of supercooled body fluids. The longhorn...

  9. Two domains of RD3 antifreeze protein diffuse independently.

    Science.gov (United States)

    Holland, Nolan B; Nishimiya, Yoshiyuki; Tsuda, Sakae; Sönnichsen, Frank D

    2008-06-03

    Antifreeze proteins (AFPs) make up a class of structurally diverse proteins that help to protect many organisms from freezing temperatures by inhibiting ice crystal growth at temperatures below the colligative freezing point. AFPs are typically small proteins with a relatively flat, slightly hydrophobic binding region that matches the lattice structure of a specific ice crystal plane. The only known two-domain AFP is RD3 from the Antarctic eel pout. It consists of two nearly identical type III domains connected by a nine-residue linker. This protein exhibits higher activity than the single-domain protein at low concentrations. The initial solution structure of RD3 revealed that the domains were aligned so that the binding regions were nearly coplanar, effectively doubling the surface area for binding. A more recent report suggests that the domains may not be aligned in solution but rather diffuse independently. To resolve the issue, we have measured the NMR residual dipolar couplings using alignment media of stretched gels and filamentous phage to determine the relative orientation of the domains. We find that the two domains of RD3 are free to move relative to each other, within the constraint of the flexible nine-residue linker. Our data show that there is no strongly preferred alignment in solution. Furthermore, the flexibility and length of the linker are sufficient to allow the two domains to have their binding faces in the same orientation and coplanar for simultaneous binding to an ice crystal surface.

  10. Antifreeze proteins: Adsorption to ice, silica and gas hydrates

    Energy Technology Data Exchange (ETDEWEB)

    Zeng, Huang; Brown, Alan; Wathen, Brent; Ripmeester, John A.; Walker, VIrginia K.

    2005-07-01

    Certain organisms survive under freezing conditions that could otherwise prove fatal by the synthesis of antifreeze proteins (AFPs). AFPs adsorb to the surface of microscopic ice crystals and prevent further ice growth, resulting in a noncolligative freezing point depression. Type I AFP from the winter flounder (wfAFP) is an alfa-helical, alanine-rich serum protein that helps protect against innoculative freezing from ice-laden seas. The AFP of a moth from the boreal forest, Choristoneura fumiferana (Cf), is a beta-helical threonine-rich protein that helps prevent freezing at the overwintering, caterpillar stage. In contrast, the beta-roll AFP from the grass, Lolium perenne (Lp), confers little freezing point depression and the plants readily freeze. Remarkably, AFPs also adsorb to tetrahyrofuran (THF) hydrate, changing the hydrate's octahedral morphology and, as well, inhibiting the growth of THF and gas hydrates. The hyperactive CfAFP, with 30-100 times the activity of wfAFP toward ice, showed far greater nucleation inhibition for THF hydrate than did a commercial hydrate inhibitor, poly(N-vinylpyrrolidone) (PVP). Active AFPs were also judged to be superior to PVP in that they inhibited the memory effect, a phenomenon whereby hydrate reforms at a faster rate soon after melting. An inactive mutant wfAFP, with an amino acid substitution at the ice-binding site, also reduced the growth of THF hydrate but was ineffective at suppressing hydrate reformation. These results suggest that the molecular properties important for ice adsorption and inhibition of hydrate reformation may be similar, and are distinct from those required for hydrate growth inhibition. The different AFPs also show markedly different aggregations on a third hydrophilic substrate, silica. Together these studies suggest that AFP adsorption to ice, hydrates and silica depends on the overall structure, specific residues and protein-protein interactions. (Author)

  11. Marine Antifreeze Proteins: Structure, Function, and Application to Cryopreservation as a Potential Cryoprotectant

    Directory of Open Access Journals (Sweden)

    Hak Jun Kim

    2017-01-01

    Full Text Available Antifreeze proteins (AFPs are biological antifreezes with unique properties, including thermal hysteresis(TH,ice recrystallization inhibition(IRI,and interaction with membranes and/or membrane proteins. These properties have been utilized in the preservation of biological samples at low temperatures. Here, we review the structure and function of marine-derived AFPs, including moderately active fish AFPs and hyperactive polar AFPs. We also survey previous and current reports of cryopreservation using AFPs. Cryopreserved biological samples are relatively diverse ranging from diatoms and reproductive cells to embryos and organs. Cryopreserved biological samples mainly originate from mammals. Most cryopreservation trials using marine-derived AFPs have demonstrated that addition of AFPs can improve post-thaw viability regardless of freezing method (slow-freezing or vitrification, storage temperature, and types of biological sample type.

  12. Animal ice-binding (antifreeze) proteins and glycolipids: an overview with emphasis on physiological function.

    Science.gov (United States)

    Duman, John G

    2015-06-01

    Ice-binding proteins (IBPs) assist in subzero tolerance of multiple cold-tolerant organisms: animals, plants, fungi, bacteria etc. IBPs include: (1) antifreeze proteins (AFPs) with high thermal hysteresis antifreeze activity; (2) low thermal hysteresis IBPs; and (3) ice-nucleating proteins (INPs). Several structurally different IBPs have evolved, even within related taxa. Proteins that produce thermal hysteresis inhibit freezing by a non-colligative mechanism, whereby they adsorb onto ice crystals or ice-nucleating surfaces and prevent further growth. This lowers the so-called hysteretic freezing point below the normal equilibrium freezing/melting point, producing a difference between the two, termed thermal hysteresis. True AFPs with high thermal hysteresis are found in freeze-avoiding animals (those that must prevent freezing, as they die if frozen) especially marine fish, insects and other terrestrial arthropods where they function to prevent freezing at temperatures below those commonly experienced by the organism. Low thermal hysteresis IBPs are found in freeze-tolerant organisms (those able to survive extracellular freezing), and function to inhibit recrystallization - a potentially damaging process whereby larger ice crystals grow at the expense of smaller ones - and in some cases, prevent lethal propagation of extracellular ice into the cytoplasm. Ice-nucleator proteins inhibit supercooling and induce freezing in the extracellular fluid at high subzero temperatures in many freeze-tolerant species, thereby allowing them to control the location and temperature of ice nucleation, and the rate of ice growth. Numerous nuances to these functions have evolved. Antifreeze glycolipids with significant thermal hysteresis activity were recently identified in insects, frogs and plants. © 2015. Published by The Company of Biologists Ltd.

  13. An insight into the molecular basis for convergent evolution in fish antifreeze Proteins.

    Science.gov (United States)

    Nath, Abhigyan; Chaube, Radha; Subbiah, Karthikeyan

    2013-08-01

    Antifreeze proteins (AFPs) prevent the growth of ice-crystals in order to enable certain organisms to survive under sub-zero temperature surroundings. These AFPs have evolved from different types of proteins without having any significant structural and sequence similarities among them. However, all the AFPs perform the same function of anti-freeze activity and are a classical example of convergent evolution. We have analyzed fish AFPs at the sequence level, the residue level and the physicochemical property group composition to discover molecular basis for this convergent evolution. Our study on amino acid distribution does not reveal any distinctive feature among AFPs, but comparative study of the AFPs with their close non-AFP homologs based on the physicochemical property group residues revealed some useful information. In particular (a) there is a similar pattern of avoidance and preference of amino acids in Fish AFP subtypes II, III and IV-Aromatic residues are avoided whereas small residues are preferred, (b) like other psychrophilic proteins, AFPs have a similar pattern of preference/avoidance for most of the residues except for Ile, Leu and Arg, and (c) most of the computed amino acids in preferred list are the key functional residues as obtained in previous predicted model of Doxey et al. For the first time this study revealed common patterns of avoidance/preference in fish AFP subtypes II, III and IV. These avoidance/preference lists can further facilitate the identification of key functional residues and can shed more light into the mechanism of antifreeze function. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. Solvation structure of ice-binding antifreeze proteins

    Science.gov (United States)

    Hansen-Goos, Hendrik; Wettlaufer, John

    2009-03-01

    Antifreeze proteins (AFPs) can be found in organisms which survive at subzero temperatures. They were first discovered in polar fishes since the 1950's [1] and have been isolated meanwhile also from insects, plants, and bacteria. While AFPs shift the freezing point of water below the bulk melting point and hence can prevent recrystallization; the effect is non-colligative and there is a pronounced hysteresis between freezing and melting. For many AFPs it is generally accepted that they function through an irreversible binding to the ice-water interface which leads to a piecewise convex growth front with a lower nonequilibrium freezing point due to the Kelvin effect. Recent molecular dynamics simulations of the AFP from Choristoneura fumiferana reveal that the solvation structures of water at ice-binding and non-ice-binding faces of the protein are crucial for understanding how the AFP binds to the ice surface and how it is protected from being overgrown [2]. We use density functional theory of classical fluids in order to assess the microscopic solvent structure in the vicinity of protein faces with different surface properties. With our method, binding energies of different protein faces to the water-ice-interface can be computed efficiently in a simplified model. [1] Y. Yeh and R.E. Feeney, Chem. Rev. 96, 601 (1996). [2] D.R. Nutt and J.C. Smith, J. Am. Chem. Soc. 130, 13066 (2008).

  15. Purification, crystal structure determination and functional characterization of type III antifreeze proteins from the European eelpout Zoarces viviparus

    DEFF Research Database (Denmark)

    Wilkens, Casper; Poulsen, Jens-Christian Navarro; Ramløv, Hans

    2014-01-01

    Antifreeze proteins (AFPs) are essential components of many organisms adaptation to cold temperatures. Fish type III AFPs are divided into two groups, SP isoforms being much less active than QAE1 isoforms. Two type III AFPs from Zoarces viviparus, a QAE1 (ZvAFP13) and an SP (ZvAFP6) isoform...... this is the first report of dimerization of AFP type III proteins....

  16. Low thermodynamic but high kinetic stability of an antifreeze protein from Rhagium mordax

    DEFF Research Database (Denmark)

    Friis, Dennis Steven; Johnsen, Johannes Lørup; Kristiansen, Erlend

    2014-01-01

    , the RmAFP1 has only one disulfide bridge. The melting temperature, Tm, of the protein is determined to be 28.5°C (pH 7.4), which is much lower than most of those reported for AFPs or globular proteins in general. Despite its low melting temperature, both biophysical and activity measurements show......The equilibrium heat stability and the kinetic heat tolerance of a recombinant antifreeze protein (AFP) from the beetle Rhagium mordax (RmAFP1) are studied through differential scanning calorimetry and circular dichroism spectroscopy. In contrast to other insect AFPs studied with this respect...... that the protein almost completely refolds into the native state after repeated exposure of 70°C. RmAFP1 thus appears to be kinetically stable even far above its melting temperature. Thermodynamically, the insect AFPs seem to be dividable in three groups, relating to their content of disulfide bridges and widths...

  17. Using Antifreeze Proteins to understand ice microstructure evolution

    Science.gov (United States)

    Bayer-Giraldi, Maddalena; Azuma, Nobuhiko; Takata, Morimasa; Weikusat, Christian; Kondo, Hidemasa; Kipfstuhl, Sepp

    2017-04-01

    Polar ice sheets are considered a unique climate archive. The chemical analysis of its impurities and the development of its microstructure with depth give insight in past climate conditions as well as in the development of the ice sheet with time and deformation. Microstructural patterns like small grain size observed in specific depths are thought to be linked to the retarding effect of impurities on ice grain growth. Clear evidence of size or chemical composition of the impurities causing this effect is missing, but in this context a major role of nanoparticles has been suggested. In order to shed light on different mechanisms by which nanoparticles can control microstructure development we used antifreeze proteins (AFPs) as proxies for particles in ice. These proteins are small nanoparticles, approx. 5 nm in size, with the special characteristics of firmly binding to ice through several hydrogen bonds. We used AFPs from the sea-ice microalgae Fragilariopsis cylindrus (fcAFPs) in bubble-free, small-grained polycrystalline ice obtained by the phase-transition size refinement method. We explain how fcAFP bind to ice by presenting the 3-D-protein structure model inferred by X-ray structure analysis, and show the importance of the chemical interaction between particles and ice in controlling normal grain growth, comparing fcAFPs to other protein nanoparticles. We used modifications of fcAFPs for particle localization through fluorescence spectroscopy. Furthermore, the effect of fcAFPs on the driving factors for ice deformation during creep, i.e. on internal dislocations due to incorporation within the lattice and on the mobility of grain boundaries due to pinning, makes these proteins particularly interesting in studying the process of ice deformation.

  18. Anchored Clathrate Waters Bind Antifreeze Proteins to Ice

    Energy Technology Data Exchange (ETDEWEB)

    C Garnham; R Campbell; P Davies

    2011-12-31

    The mechanism by which antifreeze proteins (AFPs) irreversibly bind to ice has not yet been resolved. The ice-binding site of an AFP is relatively hydrophobic, but also contains many potential hydrogen bond donors/acceptors. The extent to which hydrogen bonding and the hydrophobic effect contribute to ice binding has been debated for over 30 years. Here we have elucidated the ice-binding mechanism through solving the first crystal structure of an Antarctic bacterial AFP. This 34-kDa domain, the largest AFP structure determined to date, folds as a Ca{sup 2+}-bound parallel beta-helix with an extensive array of ice-like surface waters that are anchored via hydrogen bonds directly to the polypeptide backbone and adjacent side chains. These bound waters make an excellent three-dimensional match to both the primary prism and basal planes of ice and in effect provide an extensive X-ray crystallographic picture of the AFP{vert_ellipsis}ice interaction. This unobstructed view, free from crystal-packing artefacts, shows the contributions of both the hydrophobic effect and hydrogen bonding during AFP adsorption to ice. We term this mode of binding the 'anchored clathrate' mechanism of AFP action.

  19. Revealing Surface Waters on an Antifreeze Protein by Fusion Protein Crystallography Combined with Molecular Dynamic Simulations.

    Science.gov (United States)

    Sun, Tianjun; Gauthier, Sherry Y; Campbell, Robert L; Davies, Peter L

    2015-10-08

    Antifreeze proteins (AFPs) adsorb to ice through an extensive, flat, relatively hydrophobic surface. It has been suggested that this ice-binding site (IBS) organizes surface waters into an ice-like clathrate arrangement that matches and fuses to the quasi-liquid layer on the ice surface. On cooling, these waters join the ice lattice and freeze the AFP to its ligand. Evidence for the generality of this binding mechanism is limited because AFPs tend to crystallize with their IBS as a preferred protein-protein contact surface, which displaces some bound waters. Type III AFP is a 7 kDa globular protein with an IBS made up two adjacent surfaces. In the crystal structure of the most active isoform (QAE1), the part of the IBS that docks to the primary prism plane of ice is partially exposed to solvent and has clathrate waters present that match this plane of ice. The adjacent IBS, which matches the pyramidal plane of ice, is involved in protein-protein crystal contacts with few surface waters. Here we have changed the protein-protein contacts in the ice-binding region by crystallizing a fusion of QAE1 to maltose-binding protein. In this 1.9 Å structure, the IBS that fits the pyramidal plane of ice is exposed to solvent. By combining crystallography data with MD simulations, the surface waters on both sides of the IBS were revealed and match well with the target ice planes. The waters on the pyramidal plane IBS were loosely constrained, which might explain why other isoforms of type III AFP that lack the prism plane IBS are less active than QAE1. The AFP fusion crystallization method can potentially be used to force the exposure to solvent of the IBS on other AFPs to reveal the locations of key surface waters.

  20. The mysteries of memory effect and its elimination with antifreeze proteins

    Energy Technology Data Exchange (ETDEWEB)

    Walker, V.; Gordienko, R.; Kuiper, M.; Huva, E.; Wu, Z. [Queen' s Univ., Kingston, ON (Canada). Dept. of Biology; Zeng, H.; Ripmeester, J. [Queen' s Univ., Kingston, ON (Canada). Dept. of Biology]|[National Research Council of Canada, Ottawa, ON (Canada). Steacie Inst. for Molecular Sciences

    2008-07-01

    With the decline in easily accessible and conventional hydrocarbon supplies, exploration will focus on hydrocarbons in deep offshore waters, in permafrost or in crystalline water as gas hydrates. Crystallization of water or water-encaged gas molecules takes place when nuclei reach a critical size, but the crystal growth may be inhibited by certain antifreeze proteins (AFPs). In this study, the authors hypothesized that the crystal lattice of gas hydrates may act as an alternative for substrate antifreeze proteins (AFPs). AFP-mediated inhibition of ice and clathrate hydrate crystallization was examined. Since the AFPs had a notable ability to eliminate the memory effect (ME) or the faster reformation of clathrate hydrates after melting, the authors were prompted to examine heterogeneous nucleation. Silica, served as a model nucleator hydrophilic surface. Quartz crystal microbalance-dissipation (QCM-D) experiments showed that an active AFP was tightly adsorbed to the silica surface. However, polyvinylpyrrolidone (PVP) and polyvinylcaprolactam (PVCap), 2 commercial hydrate kinetic inhibitors that do not eliminate ME, were not as tightly adsorbed. A mutant AFP inhibited tetrahydrofuran clathrate hydrate growth, but not ME. QCM-D analysis showed that adsorption of the mutant AFP was more similar to PVCap than the active AFP. It was concluded that although there is no evidence for memory in ice reformation, the crystallization of ice and hydrates, and the elimination of the more rapid recrystallization of hydrates, can be mediated by the same proteins. The properties of adsorbed layers can be effectively monitored by QCM-D. These study results provided useful information about the inhibition mechanism of heterogeneous nucleation of clathrate hydrate. The technique facilitates the screening of potential low dose hydrate inhibitors and residues in AFPs that are involved in silica adsorption. 24 refs., 1 tab., 4 figs.

  1. Cloning and expression of a novel antifreeze protein AFP72 from the beetle Tenebrio molitor.

    Science.gov (United States)

    Yan, Qing-Hua; Yang, Li; Wang, Qing; Zhang, Hui-Rong; Shao, Qiang

    2012-01-01

    A novel antifreeze protein AFP72 cDNA (GenBbank accession No. AY929389) was obtained by RT-PCR from Tenebrio molitor. The 216 bp fragment encodes a protein of 72 amino acid residues. Sequence analysis revealed that the cDNA displays a high degree of homology with T. molitor antifreeze proteins, ranging up to 90.78%. Recombinant plasmids pMAL-p2X-afp72 and pMAL-c2X-afp72 were transferred into E. coil TBI to induce a MBP fusion protein by IPTG. The target fusion protein was released from the periplasm and cytoplasm by the cold osmotic shock procedure and sonication respectively. The content of the fusion protein came up to 38.9 and 41.5% of the total dissolved protein, respectively. The fusion protein was purified through an amylose affinity column, and incised by factor Xa. Molecular sieve chromatography was used to achieve a high state of purity of the target protein. The purified target protein displayed a single band in SDS-PAGE. The fusion protein was shown to increase resistance to low temperatures in bacteria. This finding could help in further investigations of the properties and function of antifreeze proteins.

  2. Applications of type I antifreeze proteins: studies with model membranes & cryoprotectant properties.

    Science.gov (United States)

    Inglis, Steven R; Turner, Jennifer J; Harding, Margaret M

    2006-12-01

    Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs), found in the body fluids of many species of polar fish allow them to survive in waters colder than the equilibrium freezing point of their blood and other internal fluids. Despite their structural diversity, all AF(G)Ps kinetically depress the temperature at which ice grows in a non-colligative manner and hence exhibit thermal hysteresis. AF(G)Ps also share the ability to interact with and protect mammalian cells and tissues from hypothermic damage (e.g., improved storage of human blood platelets at low temperatures), and are able to stabilize or disrupt membrane composition during low temperature and freezing stress (e.g., cryoprotectant properties in stabilization of sperm and oocytes). This review will summarize studies of AFPs with phospholipids and plant lipids, proposed mechanisms for inhibition of leakage from membranes, and cryoprotectant studies with biological samples. The major focus will be on the alpha-helical type I antifreeze proteins, and synthetic mutants, that have been most widely studied. For completeness, data on glycoproteins will also be presented. While a number of models to explain stabilization and destabilization of different lipid systems have been proposed, it is currently not possible to predict whether a particular AFP will stabilize or destabilize a given lipid system. Furthermore the relationship between the antifreeze property of thermal hysteresis and membrane stabilization is unknown. This lack of detailed knowledge about how AFPs function in the presence of different types of materials has hampered progress toward the development of antifreezes for cold storage of cells, tissues, and organs.

  3. Interaction of Tenebrio Molitor Antifreeze Protein with Ice Crystal: Insights from Molecular Dynamics Simulations.

    Science.gov (United States)

    Ramya, L; Ramakrishnan, Vigneshwar

    2016-07-01

    Antifreeze proteins (AFP) observed in cold-adapting organisms bind to ice crystals and prevent further ice growth. However, the molecular mechanism of AFP-ice binding and AFP-inhibited ice growth remains unclear. Here we report the interaction of the insect antifreeze protein (Tenebrio molitor, TmAFP) with ice crystal by molecular dynamics simulation studies. Two sets of simulations were carried out at 263 K by placing the protein near the primary prism plane (PP) and basal plane (BL) of the ice crystal. To delineate the effect of temperatures, both the PP and BL simulations were carried out at 253 K as well. The analyses revealed that the protein interacts strongly with the ice crystal in BL simulation than in PP simulation both at 263 K and 253 K. Further, it was observed that the interactions are primarily mediated through the interface waters. We also observed that as the temperature decreases, the interaction between the protein and the ice increases which can be attributed to the decreased flexibility and the increased structuring of the protein at low temperature. In essence, our study has shed light on the interaction mechanism between the TmAFP antifreeze protein and the ice crystal. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Balance between hydration enthalpy and entropy is important for ice binding surfaces in Antifreeze Proteins.

    Science.gov (United States)

    Schauperl, Michael; Podewitz, Maren; Ortner, Teresa S; Waibl, Franz; Thoeny, Alexander; Loerting, Thomas; Liedl, Klaus R

    2017-09-19

    Antifreeze Proteins (AFPs) inhibit the growth of an ice crystal by binding to it. The detailed binding mechanism is, however, still not fully understood. We investigated three AFPs using Molecular Dynamics simulations in combination with Grid Inhomogeneous Solvation Theory, exploring their hydration thermodynamics. The observed enthalpic and entropic differences between the ice-binding sites and the inactive surface reveal key properties essential for proteins in order to bind ice: While entropic contributions are similar for all sites, the enthalpic gain for all ice-binding sites is lower than for the rest of the protein surface. In contrast to most of the recently published studies, our analyses show that enthalpic interactions are as important as an ice-like pre-ordering. Based on these observations, we propose a new, thermodynamically more refined mechanism of the ice recognition process showing that the appropriate balance between entropy and enthalpy facilitates ice-binding of proteins. Especially, high enthalpic interactions between the protein surface and water can hinder the ice-binding activity.

  5. Growth-melt asymmetry in ice crystals under the influence of spruce budworm antifreeze protein

    Energy Technology Data Exchange (ETDEWEB)

    Pertaya, Natalya [Department of Physics and Astronomy, Ohio University, Athens, OH 45701 (United States); Celik, Yeliz [Department of Physics and Astronomy, Ohio University, Athens, OH 45701 (United States); DiPrinzio, Carlos L [Department of Physics and Astronomy, Ohio University, Athens, OH 45701 (United States); Wettlaufer, J S [Department of Geology and Geophysics, Yale University, New Haven, CT 06520-8109 (United States); Davies, Peter L [Department of Biochemistry, Queen' s University, Kingston, ON K7L 3N6 (Canada); Braslavsky, Ido [Department of Physics and Astronomy, Ohio University, Athens, OH 45701 (United States)

    2007-10-17

    Here we describe studies of the crystallization behavior of ice in an aqueous solution of spruce budworm antifreeze protein (sbwAFP) at atmospheric pressure. SbwAFP is an ice binding protein with high thermal hysteresis activity, which helps protect Choristoneura fumiferana (spruce budworm) larvae from freezing as they overwinter in the spruce and fir forests of the north eastern United States and Canada. Different types of ice binding proteins have been found in many other species. They have a wide range of applications in cryomedicine and cryopreservation, as well as the potential to protect plants and vegetables from frost damage through genetic engineering. However, there is much to learn regarding the mechanism of action of ice binding proteins. In our experiments, a solution containing sbwAFP was rapidly frozen and then melted back, thereby allowing us to produce small single crystals. These maintained their hexagonal shapes during cooling within the thermal hysteresis gap. Melt-growth-melt sequences in low concentrations of sbwAFP reveal the same shape transitions as are found in pure ice crystals at low temperature (-22 deg. C) and high pressure (2000 bar) (Cahoon et al 2006 Phys. Rev. Lett. 96 255502); while both growth and melt shapes display faceted hexagonal morphology, they are rotated 30 deg. relative to one another. Moreover, the initial melt shape and orientation is recovered in the sequence. To visualize the binding of sbwAFP to ice, we labeled the antifreeze protein with enhanced green fluorescent protein (eGFP) and observed the sbwAFP-GFP molecules directly on ice crystals using confocal microscopy. When cooling the ice crystals, facets form on the six primary prism planes (slowest growing planes) that are evenly decorated with sbwAFP-GFP. During melting, apparent facets form on secondary prism planes (fastest melting planes), leaving residual sbwAFP at the six corners of the hexagon. Thus, the same general growth-melt behavior of an apparently

  6. HEAT INDUCIBLE EXPRESSION OF ANTIFREEZE PROTEIN GENES FROM THE BEETLES Tenebrio molitor AND Microdera punctipennis.

    Science.gov (United States)

    Li, Jieqiong; Ma, Wenjing; Ma, Ji

    2016-01-01

    Antifreeze proteins (AFPs) play important roles in protecting poikilothermic organisms from cold damage. The expression of AFP genes (afps) is induced by low temperature. However, it is reported that heat can influence the expression of afps in the desert beetle Microdera punctipennis. To further detect whether heat also induce the expression of afps in other insects, and to determine the expression profiling of insect afps at different temperatures. The expression of antifreeze protein genes in the two beetles, Microdera punctipennis and Tenebrio molitor that have quite different living environment, under different temperatures were studied by using real-time quantitative PCR. Mild low temperatures (5~15 degree C), high temperature (38~47 degree C for M. punctipennis, or 37~42 degree C for T. molitor) and temperature difference (10~30 degree C) all stimulated strongly to the expression of AFP genes (Mpafps) in M. punctipennis which lives in the wild filed in desert. The mRNA level of Mpafps after M. punctipennis were exposed to these temperatures for 1h~5h was at least 30-fold of the control at 25 degree C. For T. molitor which is breeding in door with wheat bran all these temperatures stimulated significantly to the expression of Tmafps, while the extent and degree of the temperature stimulation on Tmafps expression were much lower than on Mpafps. After T. molitor were exposed to 5 degree C and 15 degree C for 1h~5h, the mRNA level of Tmafps was over 6-fold and 45-fold of the control at 25 degree C. High temperature (37~42 degree C) for 1h~3h treatments increased Tmafps mRNA level 4.8-fold of the control. Temperature difference of 10 degree C was effective in stimulating Tmafps expression. The expression of insect antifreeze protein genes both in M. punctipennis and T. molitor was induced by heat, suggesting that this phenomenon may be common in insects; the extent and degree of the influence differ in species that have different living conditions. The heat

  7. Hydration behavior at the ice-binding surface of the Tenebrio molitor antifreeze protein.

    Science.gov (United States)

    Midya, Uday Sankar; Bandyopadhyay, Sanjoy

    2014-05-08

    Molecular dynamics (MD) simulations have been carried out at two different temperatures (300 and 220 K) to study the conformational rigidity of the hyperactive Tenebrio molitor antifreeze protein (TmAFP) in aqueous medium and the structural arrangements of water molecules hydrating its surface. It is found that irrespective of the temperature the ice-binding surface (IBS) of the protein is relatively more rigid than its nonice-binding surface (NIBS). The presence of a set of regularly arranged internally bound water molecules is found to play an important role in maintaining the flat rigid nature of the IBS. Importantly, the calculations reveal that the strategically located hydroxyl oxygens of the threonine (Thr) residues in the IBS influence the arrangements of five sets of ordered waters around it on two parallel planes that closely resemble the basal plane of ice. As a result, these waters can register well with the ice basal plane, thereby allowing the IBS to preferentially bind at the ice interface and inhibit its growth. This provides a possible molecular reason behind the ice-binding activity of TmAFP at the basal plane of ice.

  8. Electro-Optical Properties Characterization of Fish Type III Antifreeze Protein

    OpenAIRE

    Salvay, Andrés G.; Santos, Javier; Howard, Eduardo I.

    2007-01-01

    Antifreeze proteins (AFPs) are ice-binding proteins that depress the freezing point of water in a non-colligative manner without a significant modification of the melting point. Found in the blood and tissues of some organisms (such as fish, insects, plants, and soil bacteria), AFPs play an important role in subzero temperature survival. Fish Type III AFP is present in members of the subclass Zoarcoidei. AFPIII are small 7-kDa—or 14-kDa tandem—globular proteins. In the present work, we study ...

  9. De novo DESIGN AND SYNTHESIS OF AN ICE-BINDING, DENDRIMERIC, POLYPEPTIDE BASED ON INSECT ANTIFREEZE PROTEINS

    Directory of Open Access Journals (Sweden)

    Ricardo Vera Bravo

    2011-12-01

    Full Text Available A new strategy is presented for the designand synthesis of peptides that exhibitice-binding and antifreeze activity. Apennant-type dendrimer polypeptidescaffold combining an α-helical backbonewith four short β-strand branches wassynthesized in solid phase using Fmocchemistry in a divergent approach. The51-residue dendrimer was characterizedby reverse phase high performance liquidchromatography, mass spectrometry andcircular dichroism. Each β-strand branchcontained three overlapping TXT aminoacid repeats, an ice-binding motif foundin the ice-binding face of the sprucebudworm (Choristoneura fumiferanaand beetle (Tenebrio molitor antifreezeproteins. Ice crystals in the presence ofthe polypeptide monomer displayed flat,hexagonal plate morphology, similar tothat produced by weakly active antifreezeproteins. An oxidized dimeric form of thedendrimer polypeptide also produced flathexagonal ice crystals and was capableof inhibiting ice crystal growth upontemperature reduction, a phenomenontermed thermal hysteresis, a definingproperty of antifreeze proteins. Linkageof the pennant-type dendrimer to a trifunctionalcascade-type polypeptideproduced a trimeric macromolecule thatgave flat hexagonal ice crystals withhigher thermal hysteresis activity thanthe dimer or monomer and an ice crystal burst pattern similar to that producedby samples containing insect antifreezeproteins. This macromolecule was alsocapable of inhibiting ice recrystallization.

  10. Perdeuteration, purification, crystallization and preliminary neutron diffraction of an ocean pout type III antifreeze protein

    International Nuclear Information System (INIS)

    Petit-Haertlein, Isabelle; Blakeley, Matthew P.; Howard, Eduardo; Hazemann, Isabelle; Mitschler, Andre; Haertlein, Michael; Podjarny, Alberto

    2009-01-01

    Perdeuterated type III antifreeze protein has been expressed, purified and crystallized. Preliminary neutron data collection showed diffraction to 1.85 Å resolution from a 0.13 mm 3 crystal. The highly homologous type III antifreeze protein (AFP) subfamily share the capability to inhibit ice growth at subzero temperatures. Extensive studies by X-ray crystallography have been conducted, mostly on AFPs from polar fishes. Although interactions between a defined flat ice-binding surface and a particular lattice plane of an ice crystal have now been identified, the fine structural features underlying the antifreeze mechanism still remain unclear owing to the intrinsic difficulty in identifying H atoms using X-ray diffraction data alone. Here, successful perdeuteration (i.e. complete deuteration) for neutron crystallographic studies of the North Atlantic ocean pout (Macrozoarces americanus) AFP in Escherichia coli high-density cell cultures is reported. The perdeuterated protein (AFP D) was expressed in inclusion bodies, refolded in deuterated buffer and purified by cation-exchange chromatography. Well shaped perdeuterated AFP D crystals have been grown in D 2 O by the sitting-drop method. Preliminary neutron Laue diffraction at 293 K using LADI-III at ILL showed that with a few exposures of 24 h a very low background and clear small spots up to a resolution of 1.85 Å were obtained using a ‘radically small’ perdeuterated AFP D crystal of dimensions 0.70 × 0.55 × 0.35 mm, corresponding to a volume of 0.13 mm 3

  11. In silico characterization of antifreeze proteins using computational ...

    Indian Academy of Sciences (India)

    WINTEC

    GRAVY, Grand Average Hydropathy. structure of protein (3D coordinates data). The 3D structure of AFPs Q01758 and P05140 were gener- ated by homology modelling using Esypred34 server. The similar 3D structures (for the AFPs Q01758 and. P05140 sequences) in the Protein Data bank. (www.rscb.org) were identified ...

  12. Ice-surface adsorption enhanced colligative effect of antifreeze proteins in ice growth inhibition

    Science.gov (United States)

    Mao, Yougang; Ba, Yong

    2006-09-01

    This Communication describes a mechanism to explain antifreeze protein's function to inhibit the growth of ice crystals. We propose that the adsorption of antifreeze protein (AFP) molecules on an ice surface induces a dense AFP-water layer, which can significantly decrease the mole fraction of the interfacial water and, thus, lower the temperature for a seed ice crystal to grow in a super-cooled AFP solution. This mechanism can also explain the nearly unchanged melting point for the ice crystal due to the AFP's ice-surface adsorption. A mathematical model combining the Langmuir theory of adsorption and the colligative effect of thermodynamics has been proposed to find the equilibrium constants of the ice-surface adsorptions, and the interfacial concentrations of AFPs through fitting the theoretical curves to the experimental thermal hysteresis data. This model has been demonstrated by using the experimental data of serial size-mutated beetle Tenebrio molitor (Tm) AFPs. It was found that the AFP's ice-surface adsorptions could increase the interfacial AFP's concentrations by 3 to 4 orders compared with those in the bulk AFP solutions.

  13. Agrobacterium-mediated transformation of modified antifreeze protein gene in strawberry

    Directory of Open Access Journals (Sweden)

    Srisulak Dheeranupattana

    2005-07-01

    Full Text Available The optimum condition for shoot regeneration from leaf explants of strawberry cultivar Tiogar was investigated. It was found that the best regeneration condition was MS medium containing N6-Benzyladenine (BA and 2,4-Dichlorophenoxy acetic acid (2,4-D at concentrations of 1 mg.l-1 and 0.2 mg.l-1, respectively. Antibiotics sensitivity test found that shoot regeneration from leaf explant was inhibited more than 90% at the concentration of kanamycin (Km as low as 5 mg.l-1. The modified gene encoding antifreeze protein isoform HPLC 6 was successfully constructed using codons which were optimally expressed in the strawberry plant. The antifreeze protein genes, naturally in plasmid pSW1 and modified in plasmid BB, were transformed to strawberry leaf explants by Agrobacterium tumefaciens LBA 4404. The strawberry plants, transformed with both AFP genes, were able to root in MS media containing 50 mg.l-1 Km, while no roots grew from nontransformed plant in this condition. Polymerase chain reaction indicated that the transgenes were integrated in the genome of transformants.

  14. An Effective Antifreeze Protein Predictor with Ensemble Classifiers and Comprehensive Sequence Descriptors

    Directory of Open Access Journals (Sweden)

    Runtao Yang

    2015-09-01

    Full Text Available Antifreeze proteins (AFPs play a pivotal role in the antifreeze effect of overwintering organisms. They have a wide range of applications in numerous fields, such as improving the production of crops and the quality of frozen foods. Accurate identification of AFPs may provide important clues to decipher the underlying mechanisms of AFPs in ice-binding and to facilitate the selection of the most appropriate AFPs for several applications. Based on an ensemble learning technique, this study proposes an AFP identification system called AFP-Ensemble. In this system, random forest classifiers are trained by different training subsets and then aggregated into a consensus classifier by majority voting. The resulting predictor yields a sensitivity of 0.892, a specificity of 0.940, an accuracy of 0.938 and a balanced accuracy of 0.916 on an independent dataset, which are far better than the results obtained by previous methods. These results reveal that AFP-Ensemble is an effective and promising predictor for large-scale determination of AFPs. The detailed feature analysis in this study may give useful insights into the molecular mechanisms of AFP-ice interactions and provide guidance for the related experimental validation. A web server has been designed to implement the proposed method.

  15. An open source cryostage and software analysis method for detection of antifreeze activity

    DEFF Research Database (Denmark)

    Lørup Buch, Johannes; Ramløv, H

    2016-01-01

    The aim of this study is to provide the reader with a simple setup that can detect antifreeze proteins (AFP) by inhibition of ice recrystallisation in very small sample sizes. This includes an open source cryostage, a method for preparing and loading samples as well as a software analysis method....... The entire setup was tested using hyperactive AFP from the cerambycid beetle, Rhagium mordax. Samples containing AFP were compared to buffer samples, and the results are visualised as crystal radius evolution over time and in absolute change over 30 min. Statistical analysis showed that samples containing...

  16. Antifreeze glycopeptides: from structure and activity studies to current approaches in chemical synthesis.

    Science.gov (United States)

    Urbańczyk, Małgorzata; Góra, Jerzy; Latajka, Rafał; Sewald, Norbert

    2017-02-01

    Antifreeze glycopeptides (AFGPs) are a class of biological antifreeze agents found predominantly in Arctic and Antarctic species of fish. They possess the ability to regulate ice nucleation and ice crystal growth, thus creating viable life conditions at temperatures below the freezing point of body fluids. AFGPs usually consist of 4-55 repetitions of the tripeptide unit Ala-Ala-Thr that is O-glycosylated at the threonine side chains with β-D-galactosyl-(1 → 3)-α-N-acetyl-D-galactosamine. Due to their interesting properties and high antifreeze activity, they have many potential applications, e.g., in food industry and medicine. Current research is focused towards understanding the relationship between the structural preferences and the activity of the AFGPs, as well as developing time and cost efficient ways of synthesis of this class of molecules. Recent computational studies in conjunction with experimental results from NMR and THz spectroscopies were a possible breakthrough in understanding the mechanism of action of AFGPs. At the moment, as a result of these findings, the focus of research is shifted towards the analysis of behaviour of the hydration shell around AFGPs and the impact of water-dynamics retardation caused by AFGPs on ice crystal growth. In the field of organic synthesis of AFGP analogues, most of the novel protocols are centered around solid-phase peptide synthesis and multiple efforts are made to optimize this approach. In this review, we present the current state of knowledge regarding the structure and activity of AFGPs, as well as approaches to organic synthesis of these molecules with focus on the most recent developments.

  17. Electro-optical properties characterization of fish type III antifreeze protein.

    Science.gov (United States)

    Salvay, Andrés G; Santos, Javier; Howard, Eduardo I

    2007-12-01

    Antifreeze proteins (AFPs) are ice-binding proteins that depress the freezing point of water in a non-colligative manner without a significant modification of the melting point. Found in the blood and tissues of some organisms (such as fish, insects, plants, and soil bacteria), AFPs play an important role in subzero temperature survival. Fish Type III AFP is present in members of the subclass Zoarcoidei. AFPIII are small 7-kDa-or 14-kDa tandem-globular proteins. In the present work, we study the behavior of several physical properties, such as the low-frequency dielectric permittivity spectrum, circular dichroism, and electrical conductivity of Fish Type III AFP solutions measured at different concentrations. The combination of the information obtained from these measurements could be explained through the formation of AFP molecular aggregates or, alternatively, by the existence of some other type of interparticle interactions. Thermal stability and electro-optical behavior, when proteins are dissolved in deuterated water, were also investigated.

  18. Antifreeze poisoning

    Science.gov (United States)

    The poisonous ingredients in antifreeze are: Ethylene glycol Methanol Propylene glycol ... For ethylene glycol: Death may occur within the first 24 hours. If ... little as 2 tablespoons (1 ounce or 30 milliliters) can kill a ...

  19. Fluorescence and confocal microscopy studies of the ice surface - antifreeze protein interactions.

    Science.gov (United States)

    Pertaya, N.; Thomson, E.; Davies, P. L.; Braslavsky, I.

    2005-03-01

    Biomineralization is a phenomenon in which biological material influences mineral growth on the molecular level. A compelling example involves antifreeze proteins (AFPs) known to prevent fish and insects from freezing. AFPs have many potential applications in agriculture, biomedical science, and can be used as a model platform to understand biomineralization processes for future nanotechnology applications. Here we describe a new approach to study the interaction between AFPs and ice using fluorescence and confocal microscopy combined with a unique ice growth cell. After conjugating green fluorescent protein (GFP) to Type III AFP, we imaged the fluorescence signal around and inside of the ice crystals that emerged from the cooled AFP-GFP solution, and have observed an enhanced fluorescence signal at the edge of the ice crystal. In a second cell we observed a dramatic change in the ice growth morphology when AFPs were introduced into an initially pure system. Further developments of these methods will permit the direct imaging of the location and concentration of the AFPs on ice surfaces and enable a better understanding of their operation. Supported by CIHR, the Bosack and Kruger Foundation, Ohio and Yale Universities.

  20. ANTIFREEZE PROTEINS IN PLANTS: AN OVERVIEW WITH AN INSIGHT INTO THE DETECTION TECHNIQUES INCLUDING NANOBIOTECHNOLOGY

    Directory of Open Access Journals (Sweden)

    Bhavana Sharma

    2014-08-01

    Full Text Available Antifreeze proteins (AFPs are a class of polypeptides which enables various organisms to survive subzero temperatures and have been found in vertebrates, invertebrates, plants, fungi and lichens. AFPs possess the characteristic thermal hysteresis (TH and ice recrystallization inhibition (IRI properties which allow them to adsorb the surface of ice crystals and inhibit their growth and recrystallization. AFPs are also known as ice restructuring proteins due to their ability to modify ice crystal morphology which leads to formation of hexagonal shape ice crystals in the presence of AFPs and disc shape AFPs in its absence. AFPs have various applications in medical, agricultural, industrial and biotechnological field. This review provides an overview of the AFPs, their TH and IRI properties and potential biotechnological applications of AFPs. Various conventional detection methods like Capillary assay and Differential Scanning Calorimetry (DSC with their advantages and disadvantages are discussed in detail along with the commonly used Splat assay and Nanoliter osmometer. Moreover, a novel, high-throughput and efficient nanobiotechnological method for AFP detection is also discussed. The method is based on colorimetric detection of freeze-labile gold nanoparticles and can provide an alternative to overcome the limitations of conventional methods by providing quick and easy way to screen AFPs in multiple systems simultaneously

  1. Calorimetric determination of inhibition of ice crystal growth by antifreeze protein in hydroxyethyl starch solutions.

    Science.gov (United States)

    Hansen, T N; Carpenter, J F

    1993-01-01

    Differential scanning calorimetry and cryomicroscopy were used to investigate the effects of type I antifreeze protein (AFP) from winter flounder on 58% solutions of hydroxyethyl starch. The glass, devitrification, and melt transitions noted during rewarming were unaffected by 100 micrograms/ml AFP. Isothermal annealing experiments were undertaken to detect the effects of AFP-induced inhibition of ice crystal growth using calorimetry. A premelt endothermic peak was detected during warming after the annealing procedure. Increasing the duration or the temperature of the annealing for the temperature range from -28 and -18 degrees C resulted in a gradual increase in the enthalpy of the premelt endotherm. This transition was unaffected by 100 micrograms/ml AFP. Annealing between -18 and -10 degrees C resulted in a gradual decrease in the premelt peak enthalpy. This process was inhibited by 100 micrograms/ml AFP. Cryomicroscopic examination of the samples revealed that AFP inhibited ice recrystallization during isothermal annealing at -10 degrees C. Annealing at lower temperatures resulted in minimal ice recrystallization and no visible effect of AFP. Thus, the 100 micrograms/ml AFP to have a detectable influence on thermal events in the calorimeter, conditions must be used that result in significant ice growth without AFP and visible inhibition of this process by AFP. Images FIGURE 8 PMID:7690257

  2. Lateral transfer of a lectin-like antifreeze protein gene in fishes.

    Directory of Open Access Journals (Sweden)

    Laurie A Graham

    Full Text Available Fishes living in icy seawater are usually protected from freezing by endogenous antifreeze proteins (AFPs that bind to ice crystals and stop them from growing. The scattered distribution of five highly diverse AFP types across phylogenetically disparate fish species is puzzling. The appearance of radically different AFPs in closely related species has been attributed to the rapid, independent evolution of these proteins in response to natural selection caused by sea level glaciations within the last 20 million years. In at least one instance the same type of simple repetitive AFP has independently originated in two distant species by convergent evolution. But, the isolated occurrence of three very similar type II AFPs in three distantly related species (herring, smelt and sea raven cannot be explained by this mechanism. These globular, lectin-like AFPs have a unique disulfide-bonding pattern, and share up to 85% identity in their amino acid sequences, with regions of even higher identity in their genes. A thorough search of current databases failed to find a homolog in any other species with greater than 40% amino acid sequence identity. Consistent with this result, genomic Southern blots showed the lectin-like AFP gene was absent from all other fish species tested. The remarkable conservation of both intron and exon sequences, the lack of correlation between evolutionary distance and mutation rate, and the pattern of silent vs non-silent codon changes make it unlikely that the gene for this AFP pre-existed but was lost from most branches of the teleost radiation. We propose instead that lateral gene transfer has resulted in the occurrence of the type II AFPs in herring, smelt and sea raven and allowed these species to survive in an otherwise lethal niche.

  3. Synthetic antifreeze peptide

    OpenAIRE

    1991-01-01

    A synthetic antifreeze peptide and a synthetic gene coding for the antifreeze peptide have been produced. The antifreeze peptide has a greater number of repeating amino acid sequences than is present in the native antifreeze peptides from winter flounder upon which the synthetic antifreeze peptide was modeled. Each repeating amino acid sequence has two polar amino acid residues which are spaced a controlled distance apart so that the antifreeze peptide may inhibit ice formation. The synthetic...

  4. Characterization of threonine side chain dynamics in an antifreeze protein using natural abundance {sup 13}C NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Daley, Margaret E.; Sykes, Brian D. [University of Alberta, Department of Biochemistry, CIHR Group in Protein Structure and Function and Protein Engineering Network of Centres of Excellence (Canada)

    2004-06-15

    The dynamics of threonine side chains of the Tenebrio molitor antifreeze protein (TmAFP) were investigated using natural abundance {sup 13}C NMR. In TmAFP, the array of threonine residues on one face of the protein is responsible for conferring its ability to bind crystalline ice and inhibit its growth. Heteronuclear longitudinal and transverse relaxation rates and the {sup 1}H-{sup 13}C NOE were determined in this study. The C{alpha}H relaxation measurements were compared to the previously measured {sup 15}N backbone parameters and these are found to be in agreement. For the analysis of the threonine side chain motions, the model of restricted rotational diffusion about the {chi}{sub 1} dihedral angle was employed [London and Avitabile (1978) J. Am. Chem. Soc., 100, 7159-7165]. We demonstrate that the motion experienced by the ice binding threonine side chains is highly restricted, with an approximate upper limit of less than {+-}25 deg.

  5. Effects of three different types of antifreeze proteins on mouse ovarian tissue cryopreservation and transplantation.

    Directory of Open Access Journals (Sweden)

    Jaewang Lee

    Full Text Available Ovarian tissue (OT cryopreservation is effective in preserving fertility in cancer patients who have concerns about fertility loss due to cancer treatment. However, the damage incurred at different steps during the cryopreservation procedure may cause follicular depletion; hence, preventing chilling injury would help maintain ovarian function.This study was designed to investigate the beneficial effects of different antifreeze proteins (AFPs on mouse ovarian tissue cryopreservation and transplantation.Ovaries were obtained from 5-week-old B6D2F1 mice, and each ovary was cryopreserved using two-step vitrification and four-step warming procedures. In Experiment I, ovaries were randomly allocated into fresh, vitrification control, and nine experimental groups according to the AFP type (FfIBP, LeIBP, type III and concentration (0.1, 1, 10 mg/mL used. After vitrification and warming, 5,790 ovarian follicles were evaluated using histology and TUNEL assays, and immunofluorescence for τH2AX and Rad51 was used to detect DNA double-strand breaks (DSBs and repair (DDR, respectively. In Experiment II, 20 mice were randomly divided into two groups: one where the vitrification and warming media were supplemented with 10 mg/mL LeIBP, and the other where media alone were used (control. Ovaries were then autotransplanted under both kidney capsules 7 days after vitrification together with the addition of 10 mg/mL LeIBP in the vitrification-warming media. After transplantation, the ovarian follicles, the percentage of apoptotic follicles, the extent of the CD31-positive area, and the serum FSH levels of the transplanted groups were compared.In Experiment I, the percentage of total grade 1 follicles was significantly higher in the 10 mg/mL LeIBP group than in the vitrification control, while all AFP-treated groups had significantly improved grade 1 primordial follicle numbers compared with those of the vitrification control. The number of apoptotic (TUNEL

  6. Functional importance of short-range binding and long-range solvent interactions in helical antifreeze peptides.

    Science.gov (United States)

    Ebbinghaus, Simon; Meister, Konrad; Prigozhin, Maxim B; Devries, Arthur L; Havenith, Martina; Dzubiella, Joachim; Gruebele, Martin

    2012-07-18

    Short-range ice binding and long-range solvent perturbation both have been implicated in the activity of antifreeze proteins and antifreeze glycoproteins. We study these two mechanisms for activity of winter flounder antifreeze peptide. Four mutants are characterized by freezing point hysteresis (activity), circular dichroism (secondary structure), Förster resonance energy transfer (end-to-end rigidity), molecular dynamics simulation (structure), and terahertz spectroscopy (long-range solvent perturbation). Our results show that the short-range model is sufficient to explain the activity of our mutants, but the long-range model provides a necessary condition for activity: the most active peptides in our data set all have an extended dynamical hydration shell. It appears that antifreeze proteins and antifreeze glycoproteins have reached different evolutionary solutions to the antifreeze problem, utilizing either a few precisely positioned OH groups or a large quantity of OH groups for ice binding, assisted by long-range solvent perturbation. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  7. Reversible binding of the HPLC6 isoform of type I antifreeze proteins to ice surfaces and the antifreeze mechanism studied by multiple quantum filtering-spin exchange NMR experiment.

    Science.gov (United States)

    Ba, Yong; Wongskhaluang, Jeff; Li, Jiabo

    2003-01-15

    Antifreeze proteins (AFPs) protect organisms from freezing damage by inhibiting the growth of seed-ice crystals. It has long been hypothesized that irreversible binding of AFPs to ice surfaces is responsible for inhibiting the growth of seed-ice crystals as such a mechanism supports the popularly accepted Kelvin effect for the explanation of local freezing-point depression. However, whether the binding is reversible or irreversible is still under debate due to the lack of direct experimental evidence. Here, we report the first direct experimental result, by using the newly developed multiple quantum (MQ) filtering-spin exchange NMR experiment, that shows that the binding of HPLC6 peptides to ice surfaces is reversible. It was found that the reversible process can be explained by the model of monolayer adsorption. These results suggest that the Kelvin effect is not suitable for explaining the antifreeze mechanism, and direct interactions between the peptides and the ice-surface binding sites are the driving forces for the binding of AFPs to ice surfaces. We propose that there exists a concentration gradient of AFP from an ice-binding surface to the solution due to the affinity of ice surfaces to AFPs. This concentration gradient creates a dense layer of AFP in contact with the ice-binding surface, which depresses the local freezing point because of the colligative property, but not the Kelvin effect.

  8. Saccharide antifreeze compositions

    Energy Technology Data Exchange (ETDEWEB)

    Walters, Kent; Duman, John G; Serianni, Anthony S

    2013-12-10

    The invention provides an antifreeze glycolipid compounds and composition comprising a polysaccharide moiety of Formula I; ##STR00001## wherein D-Manp represents a D-mannopyranose moiety, D-Xylp represents a D-xylopyranose moiety, and n is about 5 to about 70; and one or more lipid moieties covalently linked to the polysaccharide moiety of Formula I or electrostatically associated with the polysaccaride moiety for Formula I. The antifreeze glycolipid compounds and compositions can be used for a variety of industrial, agricultural, medical, and cosmetic applications where recrystallization-inhibition, cyroprotection, or cryopreservation is desired. The antifreeze glycolipid compounds or compositions can be used as, for example, as cryoprotectants for tissue preservation and transplantation, improving the texture of processed frozen food and frozen meats, frostbit protection, crop protection, and green alternatives for land vehicle antifreeze and aircraft de-icing.

  9. Crystallization and preliminary X-ray crystallographic analysis of Ca{sup 2+}-independent and Ca{sup 2+}-dependent species of the type II antifreeze protein

    Energy Technology Data Exchange (ETDEWEB)

    Nishimiya, Yoshiyuki; Kondo, Hidemasa [Functional Protein Research Group, Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira, Sapporo 062-8517 (Japan); Yasui, Masanori [Functional Protein Research Group, Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira, Sapporo 062-8517 (Japan); Division of Biological Sciences, Graduate School of Science, Hokkaido University, N8W5, Kita, Sapporo 060-0808 (Japan); Sugimoto, Hiroshi [Biometal Science Laboratory, Riken SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148 (Japan); Noro, Natsuko; Sato, Ryoko [Functional Protein Research Group, Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira, Sapporo 062-8517 (Japan); Suzuki, Mamoru [Insititute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Miura, Ai [Functional Protein Research Group, Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira, Sapporo 062-8517 (Japan); Tsuda, Sakae, E-mail: s.tsuda@aist.go.jp [Functional Protein Research Group, Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira, Sapporo 062-8517 (Japan); Division of Biological Sciences, Graduate School of Science, Hokkaido University, N8W5, Kita, Sapporo 060-0808 (Japan)

    2006-06-01

    Crystallization and X-ray data analyses were successful for both Ca{sup 2+}-independent and Ca{sup 2+}-dependent species of the type II antifreeze protein. The resolution of the crystal was 1.35 Å for the Ca{sup 2+}-independent species, and was 1.25 and 1.06 Å for the Ca{sup 2+}-dependent species in the Ca{sup 2+}-free and -bound states, respectively. Ca{sup 2+}-independent and Ca{sup 2+}-dependent species of the type II antifreeze protein (AFP) were both crystallized using the hanging-drop vapour-diffusion method. It appeared that the crystal of the Ca{sup 2+}-independent species from Brachyosis rostratus belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 43.3, b = 48.4, c = 59.7 Å, and diffraction data were collected to 1.34 Å resolution. For the Ca{sup 2+}-dependent type II AFP species from Hypomesus nipponensis, crystallization was carried out for its Ca{sup 2+}-free and Ca{sup 2+}-bound states. 1.25 Å resolution data were collected from the crystal in the Ca{sup 2+}-free state, which exhibited P3{sub 1}21 (or P3{sub 2}21) symmetry, with unit-cell parameters a = b = 66.0, c = 50.3 Å. Data collection could be extended to 1.06 Å resolution for the crystal in the Ca{sup 2+} -bound state, which appeared to be isomorphous to the crystal in the Ca{sup 2+}-free state (unit-cell parameters a = b = 66.0, c = 49.8 Å). These data will allow us to determine the high-resolution structures of the two species of type II AFP.

  10. Inhibition of ice recrystallization and cryoprotective activity of wheat proteins in liver and pancreatic cells.

    Science.gov (United States)

    Chow-Shi-Yée, Mélanie; Briard, Jennie G; Grondin, Mélanie; Averill-Bates, Diana A; Ben, Robert N; Ouellet, François

    2016-05-01

    Efficient cryopreservation of cells at ultralow temperatures requires the use of substances that help maintain viability and metabolic functions post-thaw. We are developing new technology where plant proteins are used to substitute the commonly-used, but relatively toxic chemical dimethyl sulfoxide. Recombinant forms of four structurally diverse wheat proteins, TaIRI-2 (ice recrystallization inhibition), TaBAS1 (2-Cys peroxiredoxin), WCS120 (dehydrin), and TaENO (enolase) can efficiently cryopreserve hepatocytes and insulin-secreting INS832/13 cells. This study shows that TaIRI-2 and TaENO are internalized during the freeze-thaw process, while TaBAS1 and WCS120 remain at the extracellular level. Possible antifreeze activity of the four proteins was assessed. The "splat cooling" method for quantifying ice recrystallization inhibition activity (a property that characterizes antifreeze proteins) revealed that TaIRI-2 and TaENO are more potent than TaBAS1 and WCS120. Because of their ability to inhibit ice recrystallization, the wheat recombinant proteins TaIRI-2 and TaENO are promising candidates and could prove useful to improve cryopreservation protocols for hepatocytes and insulin-secreting cells, and possibly other cell types. TaENO does not have typical ice-binding domains, and the TargetFreeze tool did not predict an antifreeze capacity, suggesting the existence of nontypical antifreeze domains. The fact that TaBAS1 is an efficient cryoprotectant but does not show antifreeze activity indicates a different mechanism of action. The cryoprotective properties conferred by WCS120 depend on biochemical properties that remain to be determined. Overall, our results show that the proteins' efficiencies vary between cell types, and confirm that a combination of different protection mechanisms is needed to successfully cryopreserve mammalian cells. © 2016 The Protein Society.

  11. Stable transmission and transcription of newfoundland ocean pout type III fish antifreeze protein (AFP) gene in transgenic mice and hypothermic storage of transgenic ovary and testis.

    Science.gov (United States)

    Bagis, Haydar; Aktoprakligil, Digdem; Mercan, Hande Odaman; Yurdusev, Nevzat; Turgut, Gazi; Sekmen, Sakir; Arat, Sezen; Cetin, Seyfettin

    2006-11-01

    Here we describe the generation of transgenic mice carrying type III fish antifreeze protein (AFP) gene and evaluate whether AFP type III protects transgenic mouse ovaries and testes from hypothermic storage. AFPs exist in many different organisms. In fish, AFPs protect the host from freezing at temperatures below the colligative freezing point by adsorbing to the surface of nucleating ice crystals and inhibiting their growth. The transgenic expression of AFP holds great promise for conferring freeze-resistant plant and animal species. AFP also exhibits a potential for the cryopreservation of tissues and cells. In this study, we have generated 42 founder mice harboring the Newfoundland ocean pout (OP5A) type III AFP transgene and established one transgenic line (the line #6). This study demonstrated that AFP gene construct has been stably transmitted to the mouse progeny in the F3 generations in the line #6. Furthermore, the presence of AFP transcripts was confirmed by RT-PCR analysis on cDNAs from liver, kidney, ovarian, and testis tissues of the mouse from F3 generation in this line. These results indicate that ocean pout type III AFP gene could be integrated and transmitted to the next generation and stably transcribed in transgenic mice. In histological analysis of testis and ovarian tissues of nontransgenic control and AFP transgenic mice it has been shown that both tissues of AFP transgenic mice were protected from hypothermic storage (+4 degrees C). The AFP III transgenic mice obtained for the first time in this study would be useful for investigating the biological functions of AFP in mammalian systems and also its potential role in cryopreservation.

  12. Testing antifreeze protein from the longhorn beetle Rhagium mordax as a kinetic gas hydrate inhibitor using a high-pressure micro differential scanning calorimeter

    DEFF Research Database (Denmark)

    Daraboina, Nagu; Perfeldt, Christine Malmos; von Solms, Nicolas

    2015-01-01

    pressure micro differential scanning calorimeter HP-mu DSC VII (Setaram Inc.) containing two 50 cc high pressure cells (maximum operating pressure 40 MPa; temperature range -40 to 120 degrees C) was employed to observe methane hydrate formation and decomposition in the presence of hyperactive antifreeze...

  13. Improved viability and reduced apoptosis in sub-zero 21-hour preservation of transplanted rat hearts using anti-freeze proteins.

    Science.gov (United States)

    Amir, Gabriel; Rubinsky, Boris; Basheer, Sheick Yousif; Horowitz, Liana; Jonathan, Leor; Feinberg, Micha S; Smolinsky, Aram K; Lavee, Jacob

    2005-11-01

    Freeze-tolerant fish survive sub-zero temperatures by non-colligatively lowering the freezing temperature of their body fluids using anti-freeze proteins (AFPs). We sought to evaluate and compare the effects of prolonged sub-zero cryopreservation of transplanted rat hearts using AFP I or AFP III. Two heterotopic rat heart transplantation protocols were used. In Protocol 1 (n = 104), hearts (n = 8/group) were preserved for 12, 18 and 24 hours in University of Wisconsin solution (UW) at 4 degrees C, UW at -1.3 degrees C, UW/AFP I at -1.3 degrees C and UW/AFP III at -1.3 degrees C, with and without nucleation. Post-operative evaluation consisted of visual viability scoring of the hearts after 60 minutes. Protocol 2 (n = 58) involved evaluation of 24-hour post-transplant viability, echocardiography (fractional shortening [FS], left ventricular end-systolic and -diastolic diameter [ESD, EDD] and anterior and posterior wall systolic and diastolic thickness [AWT-S, AWT-D, PWT-S, PWT-D]), TUNEL staining and electron microscopy (EM) findings for hearts preserved for 18, 21 and 24 hours in UW at 4 degrees C or UW/AFP III at -1.3 degrees C. Hearts preserved in UW at -1.3 degrees C with nucleation froze and died. Three of 8 hearts preserved in UW at 4 degrees C for 24 hours died, whereas all hearts preserved at -1.3 degrees C survived. Hearts preserved in UW/AFP for 18 and 24 hours at -1.3 degrees C had superior viability scores compared with those in UW at 4 degrees C. Hearts in AFP III at -1.3 degrees C displayed greater AWT-S and AWT-D (3.5 +/- 0.2 vs 2.4 +/- 0.2, p hour preservation. In the 21-hour preservation group, AFP-treated hearts displayed improved echocardiographic systolic contraction indices, including: improved FS (27 +/- 3.7 vs 15 +/- 4, p = 0.04); diminished ESD (0.28 +/- 0.57 vs 0.47 +/- 0.6, p zero cryopreservation, AFPs protect the heart from freezing, improve survival and hemodynamics, and reduce apoptotic cell death.

  14. Hydration layer dynamics and association mechanisms of food and antifreeze proteins : A Molecular Dynamics and Transition Path Sampling study

    NARCIS (Netherlands)

    Brotzakis, Z.F.

    2017-01-01

    By the time the reader reads this line, billions of protein association events just occurred in our body, such as the ones regulating cell communication, signaling pathways, or in initiating a self-assembly processes, such as tissue fabrication, etc. The timescale of such transitions is slow,

  15. Molecular Characterization, Structural Modeling, and Evaluation of Antimicrobial Activity of Basrai Thaumatin-Like Protein against Fungal Infection.

    Science.gov (United States)

    Yasmin, Nusrat; Saleem, Mahjabeen; Naz, Mamoona; Gul, Roquyya; Rehman, Hafiz Muzzammel

    2017-01-01

    A thaumatin-like protein gene from Basrai banana was cloned and expressed in Escherichia coli . Amplified gene product was cloned into pTZ57R/T vector and subcloned into expression vector pET22b(+) and resulting pET22b-basrai TLP construct was introduced into E. coli BL21. Maximum protein expression was obtained at 0.7 mM IPTG concentration after 6 hours at 37°C. Western blot analysis showed the presence of approximately 20 kDa protein in induced cells. Basrai antifungal TLP was tried as pharmacological agent against fungal disease. Independently Basrai antifungal protein and amphotericin B exhibited their antifungal activity against A. fumigatus ; however combined effect of both agents maximized activity against the pathogen. Docking studies were performed to evaluate the antimicrobial potential of TLP against A. fumigatus by probing binding pattern of antifungal protein with plasma membrane ergosterol of targeted fungal strain. Ice crystallization primarily damages frozen food items; however addition of antifreeze proteins limits the growth of ice crystal in frozen foods. The potential of Basrai TLP protein, as an antifreezing agent, in controlling the ice crystal formation in frozen yogurt was also studied. The scope of this study ranges from cost effective production of pharmaceutics to antifreezing and food preserving agent as well as other real life applications.

  16. Molecular Characterization, Structural Modeling, and Evaluation of Antimicrobial Activity of Basrai Thaumatin-Like Protein against Fungal Infection

    Directory of Open Access Journals (Sweden)

    Nusrat Yasmin

    2017-01-01

    Full Text Available A thaumatin-like protein gene from Basrai banana was cloned and expressed in Escherichia coli. Amplified gene product was cloned into pTZ57R/T vector and subcloned into expression vector pET22b(+ and resulting pET22b-basrai TLP construct was introduced into E. coli BL21. Maximum protein expression was obtained at 0.7 mM IPTG concentration after 6 hours at 37°C. Western blot analysis showed the presence of approximately 20 kDa protein in induced cells. Basrai antifungal TLP was tried as pharmacological agent against fungal disease. Independently Basrai antifungal protein and amphotericin B exhibited their antifungal activity against A. fumigatus; however combined effect of both agents maximized activity against the pathogen. Docking studies were performed to evaluate the antimicrobial potential of TLP against A. fumigatus by probing binding pattern of antifungal protein with plasma membrane ergosterol of targeted fungal strain. Ice crystallization primarily damages frozen food items; however addition of antifreeze proteins limits the growth of ice crystal in frozen foods. The potential of Basrai TLP protein, as an antifreezing agent, in controlling the ice crystal formation in frozen yogurt was also studied. The scope of this study ranges from cost effective production of pharmaceutics to antifreezing and food preserving agent as well as other real life applications.

  17. Synthetic antifreeze peptide and synthetic gene coding for its production

    OpenAIRE

    1991-01-01

    A synthetic antifreeze peptide and a synthetic gene coding for the antifreeze peptide have been produced. The antifreeze peptide has a greater number of repeating amino acid sequences than is present in the native antifreeze peptides from winter flounder upon which the synthetic antifreeze peptide was modeled. Each repeating amino acid sequence has two polar amino acid residues which are spaced a controlled distance apart so that the antifreeze peptide may inhibit ice formation. The synthetic...

  18. Antifreeze life cycle assessment (LCA

    Directory of Open Access Journals (Sweden)

    Kesić Jelena

    2005-01-01

    Full Text Available Antifreeze based on ethylene glycol is a commonly used commercial product The classification of ethylene glycol as a toxic material increased the disposal costs for used antifreeze and life cycle assessment became a necessity. Life Cycle Assessment (LCA considers the identification and quantification of raw materials and energy inputs and waste outputs during the whole life cycle of the analyzed product. The objectives of LCA are the evaluation of impacts on the environment and improvements of processes in order to reduce and/or eliminate waste. LCA is conducted through a mathematical model derived from mass and energy balances of all the processes included in the life cycle. In all energy processes the part of energy that can be transformed into some other kind of energy is called exergy. The concept of exergy considers the quality of different types of energy and the quality of different materials. It is also a connection between energy and mass transformations. The whole life cycle can be described by the value of the total loss of exergy. The physical meaning of this value is the loss of material and energy that can be used. The results of LCA are very useful for the analyzed products and processes and for the determined conditions under which the analysis was conducted. The results of this study indicate that recycling is the most satisfactory solution for the treatment of used antifreeze regarding material and energy consumption but the re-use of antifreeze should not be neglected as a solution.

  19. Effects of preheating on ice growth in antifreeze polypeptides solutions in a narrow space

    Science.gov (United States)

    Miyamoto, T.; Nishi, N.; Waku, T.; Tanaka, N.; Hagiwara, Y.

    2016-09-01

    We conducted measurements on the unidirectional freezing of aqueous solutions of polypeptide or of winter flounder antifreeze protein. The polypeptide was based on a part of the antifreeze protein. We measured temperatures in the solutions and ice with a small thermocouple, and defined the interface temperature as the temperature at the tip of the serrated or pectinate interface. It was found that the interface temperature of these solutions was lower than that of pure water. To vary the activity of these solutes, we preheated the solutions and cooled them before conducting the measurements. We found that preheating for several hours caused further decreases in the interface temperature and a decrease in the interface velocity. In addition, the inclined interfaces became wider as a result of the preheating. Thus, the supercooled states in the solutions were enhanced by the preheating. To investigate the reasons for these changes, we measured the aggregates of the solutes in the solutions. These aggregates became larger as a result of preheating. It can therefore be concluded that these large aggregates attenuated the ice growth by their interaction with the ice surfaces.

  20. A nonprotein thermal hysteresis-producing xylomannan antifreeze in the freeze-tolerant Alaskan beetle Upis ceramboides

    OpenAIRE

    Walters, Kent R.; Serianni, Anthony S.; Sformo, Todd; Barnes, Brian M.; Duman, John G.

    2009-01-01

    Thermal hysteresis (TH), a difference between the melting and freezing points of a solution that is indicative of the presence of large-molecular-mass antifreezes (e.g., antifreeze proteins), has been described in animals, plants, bacteria, and fungi. Although all previously described TH-producing biomolecules are proteins, most thermal hysteresis factors (THFs) have not yet been structurally characterized, and none have been characterized from a freeze-tolerant animal. We isolated a highly a...

  1. [Antifreeze glycoproteins in fishes: structure, mode of action and possible applications].

    Science.gov (United States)

    Wöhrmann, A

    1996-02-01

    Two types of antifreezes have been isolated from polar and northern temperate fishes so far. They are either glycopeptides or peptides. Whereas these proteins have only a very small effect on the melting temperature of ice, the temperature of these fish can fall to nearly 1 degree below the melting point before ice crystals grow. This phenomenon is called thermal hysteresis, in contrast to the normal colligative effect of solutes. All Antarctic notothenioids (perches) investigated so far have the typical antifreeze glycoproteins (AFGP) with the tripeptide Ala-Ala-Thr and the disaccharide Gal-GalNAc. In the Antarctic silverfish Pleuragramma antarcticum there could be found a novel GlcNAc containing antifreeze glycoprotein, the PAGP. The antifreezes not only lower the freezing temperature, but they also retard recrystallization on frozen storage. Antifreeze proteins thus could be useful for biotechnology and cryomedicine in the future. Since some are now synthesized chemically or by genetic engineering, they no longer have to be isolated from fish blood.

  2. Antifreeze Glycoproteins Alter the Molecular Scale Surface Morphology of Ice

    Science.gov (United States)

    Zepeda, Salvador; Orme, Christine A.; Qiu, Roger; Yeh, Yin

    2003-03-01

    Trematomas borchgrevinki live in the harsh super-cooled waters of the Antarctic. Critical to their survival are antifreeze glycoproteins (AFGPs) that further suppress the freezing temperature of their blood serum in addition to the colligative action of salts found in the ocean. These proteins also modify ice crystal growth habits as well as inhibit recrystallization in polycrystalline ice. To date many other types of antifreeze proteins have been identified in cold weather insects, plants, and other fish, but the exact mechanism is not entirely understood. The mechanism is non-colligative since only a few mg/ml are required for ice crystal growth inhibition and a non-equilibrium melting/freezing point hysteresis is observed. Atomic force microscopy (AFM) can yield a wealth of surface information that can reveal molecular scale information of biomineralization processes. We use AFM to directly probe the surface of ice crystals grown from the vapor in the pure phase and in the presence of growth inhibitors/modifiers, AFGPs. Results show that the AFGPs heavily pin the surface of ice.

  3. Ice growth in supercooled solutions of antifreeze glycoprotein.

    Science.gov (United States)

    Harrison, K; Hallett, J; Burcham, T S; Feeney, R E; Kerr, W L; Yeh, Y

    Inhibition of ice growth in supercooled solution by certain proteins is vital to the survival of many living organisms. Some fish, native to both subzero northern and southern waters, have special proteins or glycoproteins in their blood serum that inhibit ice formation. Whereas these proteins have only a very small effect on the melting temperature of ice, the temperature of these fish can fall to nearly 1 K below the melting point before ice crystals grow. This phenomenon is called freezing hysteresis, in contrast to the normal colligative effect of solutes that depresses the equilibrium temperature, around which small changes lead to crystal growth or melting depending on sign. Some insects also exhibit a serum freezing hysteresis. We report the effects of different degrees of supercooling on the habit and rates of growth of ice crystals from solutions of these antifreeze glycoproteins (AFGPs). We find that the crystallization rate is up to five times greater than that in pure water.

  4. Efficiency of Composite Binders with Antifreezing Agents

    Science.gov (United States)

    Ogurtsova, Y. N.; Zhernovsky, I. V.; Botsman, L. N.

    2017-11-01

    One of the non-heating methods of cold-weather concreting is using concretes hardening at negative temperatures. This method consists in using chemical additives which reduce the freezing temperature of the liquid phase and provide for concrete hardening at negative temperatures. The non-heating cold-weather concreting, due to antifreezing agents, allows saving heat and electric energy at the more flexible work performance technology. At selecting the antifreezing components, the possibility of concreting at temperatures up to minus 20 °C and combination with a plasticizer contained in the composite binder were taken into account. The optimal proportions of antifreezing and complex agents produced by MC-Bauchemie Russia for fine-grained concretes were determined. So, the introduction of antifreezing and complex agents allows obtaining a structure of composite characteristic for cement stone in the conditions of below zero temperatures at using different binders; the hydration of such composite proceeded naturally. Low-water-demand binders (LWDB) based composites are characterized by a higher density and homogeneity due to a high dispersity of a binder and its complicated surface providing for a lot of crystallization centers. LWDB contains small pores keeping water in a liquid form and promoting a more complete hydration process.

  5. Isolation of an antifreeze peptide from the Antarctic sponge Homaxinella balfourensis

    OpenAIRE

    Wilkins, S. P.; Blum, A. J.; Burkepile, D. E.; Rutland, T. J.; Wierzbicki, A.; Kelly, M.; Hamann, M.T.

    2002-01-01

    Polar plants and animals survive in subzero waters (−2°C) and many of these marine organisms produce antifreeze proteins (AFPs) to better adapt themselves to these conditions. AFPs prevent the growth of ice crystals which disrupt cellular membranes and destroy cells by inhibiting crystallization of water within the organism. The hydrophilic extract of an Antarctic sponge Homaxinella balfourensis exhibited a non-colligative freezing point depression effect on the crystal morphology of water. T...

  6. Synthesis of Structures Related to Antifreeze Glycoproteins

    OpenAIRE

    Fyrner, Timmy

    2005-01-01

    In this thesis, synthesis of structures related to antifreeze glycoproteins (AFGPs) are presented. Synthetic routes to a protected carbohydrate derivative, 2,3,4,6-tetra-O-benzyl-β-galactopyranosyl-(1→3)-2-deoxy-2-azido-4,6-di-O-benzyl-β-D-thio-1-galactopyranoside, and a tBu-Ala-Thr-Ala-Fmoc tripeptide, are described. These compounds are meant to be used in the assembly of AFGPs and analogues thereof. A Gal-GlcN disaccharide was synthesized via glycosylation between the donor, bromo-2-O-benzo...

  7. De novo gene evolution of antifreeze glycoproteins in codfishes revealed by whole genome sequence data.

    Science.gov (United States)

    Baalsrud, Helle Tessand; Tørresen, Ole Kristian; Hongrø Solbakken, Monica; Salzburger, Walter; Hanel, Reinhold; Jakobsen, Kjetill S; Jentoft, Sissel

    2017-12-05

    New genes can arise through duplication of a pre-existing gene or de novo from non-coding DNA, providing raw material for evolution of new functions in response to a changing environment. A prime example is the independent evolution of antifreeze glycoprotein genes (afgps) in the Arctic codfishes and Antarctic notothenioids to prevent freezing. However, the highly repetitive nature of these genes complicates studies of their organization. In notothenioids, afgps evolved from an extant gene, yet the evolutionary origin of afgps in codfishes is unknown. Here, we demonstrate that afgps in codfishes have evolved de novo from non-coding DNA 13-18 Ma, coinciding with the cooling of the Northern Hemisphere. Using whole-genome sequence data from several codfishes and notothenioids, we find higher copy number of afgp in species exposed to more severe freezing suggesting a gene dosage effect. Notably, antifreeze function is lost in one lineage of codfishes analogous to the afgp losses in non-Antarctic notothenioids. This indicates that selection can eliminate the antifreeze function when freezing is no longer imminent. Additionally, we show that evolution of afgp-assisting antifreeze potentiating protein genes (afpps) in notothenioids coincides with origin and lineage-specific losses of afgp. The origin of afgps in codfishes is one of the first examples of an essential gene born from non-coding DNA in a non-model species. Our study underlines the power of comparative genomics to uncover past molecular signatures of genome evolution, and further highlights the impact of de novo gene origin in response to a changing selection regime. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  8. Effects of short-time preheating on ice growth in antifreeze polypeptides solutions in a narrow space

    Science.gov (United States)

    Miyamoto, T.; Nishi, N.; Waku, T.; Tanaka, N.; Hagiwara, Y.

    2018-03-01

    We conducted experiments on the unidirectional freezing of solutions of winter flounder antifreeze protein or of a polypeptide which was based on twelve amino-acid residues of this protein. The temperature in the solutions and ice was measured with a small thermocouple. The interface temperature was defined as the temperature at the tip of the serrated or pectinate interface. The interface temperature of these solutions was lower than that of pure water. To vary this supercooling activity of these solutes, we preheated the solutions and cooled them before conducting identical experiments. It was found that short-time preheating caused further decreases in the interface temperature and interface velocities. Furthermore, the inclined interfaces and the narrow liquid regions inside the ice area became wider. To investigate the reasons for these changes, we measured aggregates of the solutes in the solutions. These aggregates were found to become larger as a result of preheating. Thus, it can be concluded that these large aggregates attenuated the ice growth by their interaction with ice. Finally, we carried out similar measurements by using pH-adjusted solutions of the protein to produce aggregates without preheating, and obtained similar supercooling enhancement by the aggregates. Thus, the effects of thermal denaturation on the supercooling were not significant in the preheating.

  9. Renal conservation of antifreeze peptide in Antarctic eelpout, Rhigophila dearborni.

    Science.gov (United States)

    Eastman, J T; De Vries, A L; Coalson, R E; Nordquist, R E; Boyd, R B

    1979-11-08

    In Antarctic notothenioid fishes large amounts (3% w/v) of small molecular weights of 2,600-23,500 and would be expected to be filtered into the urine, they remain in the blood because the kidneys of these fishes contain only aglomerular nephrons. Unlike the situation in most fishes, urine formation is the result of secretion rather than filtration and reabsorption. On the other hand, the peptide antifreezes in Northern Hemisphere fishes such as the winter flounder. Pseudopleuronectes americanus, are retained by the glomerular kidney even though inulin, of comparable weight, is rapidly filtered from the blood into the urine. The Antarctic eelpout (zoarcid), Rhigophila dearborni, which is unrelated to either the Antarctic notothenioids or P. americanus, also uses a peptide antifreeze (molecular weight 6,000) which is maintained at a concentration of 3% (w/v) in the blood plasma. We report here that the lack of antifreeze in the urine of R. dearborni probably reflects the fact that the glomeruli are not functional and cannot filter. We support this conclusion with morphological and physiological evidence and relate our findings to the conservation of biological antifreeze necessary for life in ice-laden polar waters.

  10. RAPID TEST METHOD FOR EVALUATION OF ANTIFREEZE ADDITIVE EFFICIENCY

    Directory of Open Access Journals (Sweden)

    S. V. Gushchin

    2015-01-01

    Full Text Available Usage of chemical additives while executing concrete works at negative temperatures is considered as a convenient and economical method. Range of the used antifreeze additives is rather wide. A great number of new additives are advertised but their characteristics have not been practically studied. Evaluation of the antifreeze additive efficiency is unfortunately rather long process and it does not provide comprehensive data on concrete structure formation processes. Due to this development of rapid and comprehensive methodology for construction companies is urgently required.Freezing processes of antifreeze additive aqueous solutions and hardening of cement paste with them have been investigated in the paper. The paper proposes a methodology for determination of freezing point for aqueous solutions of chemical additives of various applications. Identity of  freezing point for a chemical additive aqueous solution and cement paste with an equal concentration of the additive in the paste pore fluid has been determined while taking  calcium nitrate and sodium formate additives as an example. The paper demonstrates the possibility to evaluate efficiency of antifreeze additive action on the basis of kinetics in temperature changes of the cement paste with additives by its consecutive freezing and defrosting.  A methodology for operational evaluation in the field of chemical additive application for concreting items at negative temperatures has been offered in the paper.  The methodology does not require  deficient and expensive test-equipment. It can be applied at ordinary construction companies and it is comprehensible for personnel of low-qualification.  The paper shows the possibility to develop an original methodology for designing concrete structure which is based on operating efficiency determinations  for single and integrated antifreeze additives.

  11. Isolation of an antifreeze peptide from the Antarctic sponge Homaxinella balfourensis.

    Science.gov (United States)

    Wilkins, S P; Blum, A J; Burkepile, D E; Rutland, T J; Wierzbicki, A; Kelly, M; Hamann, M T

    2002-12-01

    Polar plants and animals survive in subzero waters (-2 degrees C) and many of these marine organisms produce antifreeze proteins (AFPs) to better adapt themselves to these conditions. AFPs prevent the growth of ice crystals which disrupt cellular membranes and destroy cells by inhibiting crystallization of water within the organism. The hydrophilic extract of an Antarctic sponge Homaxinella balfourensis exhibited a non-colligative freezing point depression effect on the crystal morphology of water. The extract was purified by repeated reverse phase high-pressure liquid chromatography, then assayed and shown to contain several AFPs. The major peptide was isolated, analyzed using matrix-assisted laser desorption ionization mass spectrometry and the partial structure of the peptide identified through amino acid sequencing. AFPs have potential applications in agriculture, medicine and the food industry.

  12. Potential Antifreeze Compounds in Present-Day Martian Seepage Groundwater

    Directory of Open Access Journals (Sweden)

    Jiin-Shuh Jean

    2008-01-01

    Full Text Available Is the recently found seepage groundwater on Mars pure H2O, or mixed with salts and other antifreeze compounds? Given the surface conditions of Mars, it is unlikely that pure water could either exist in its liquid state or have shaped Mars¡¦ fluid erosional landforms (gullies, channels, and valley networks. More likely is that Mars¡¦ seepage groundwater contains antifreeze and salt compounds that resist freezing and suppress evaporation. This model better accounts for Mars¡¦ enigmatic surface erosion. This paper suggests 17 antifreeze compounds potentially present in Martian seepage groundwater. Given their liquid state and physical properties, triethylene glycol, diethylene glycol, ethylene glycol, and 1,3-propylene glycol are advanced as the most likely candidate compounds. This paper also explores how a mixing of glycol or glycerol with salts in the Martian seepage groundwater may have lowered water¡¦s freezing point and raised its boiling point, with consequences that created fluid gully and channel erosion. Ethylene glycol and related hydrocarbon compounds have been identified in Martian and other interstellar meteorites. We suggest that these compounds and their proportions to water be included for detection in future explorations.

  13. Chitin and stress induced protein kinase activation

    DEFF Research Database (Denmark)

    Kenchappa, Chandra Shekar; Azevedo da Silva, Raquel; Bressendorff, Simon

    2017-01-01

    The assays described here are pertinent to protein kinase studies in any plant. They include an immunoblot phosphorylation/activation assay and an in-gel activity assay for MAP kinases (MPKs) using the general protein kinase substrate myelin basic protein. They also include a novel in-gel peptide...... substrate assay for Snf1-related kinase family 2 members (SnRK2s). This kinase family-specific assay overcomes some limitations of in-gel assays and permits the identification of different types of kinase activities in total protein extracts....

  14. Anti-freeze proteins: characteristics, mechanism of action and applications

    NARCIS (Netherlands)

    Stolle-Smits, T.; Kreuwels, M.J.J.; Wichers, H.J.

    1998-01-01

    Antivrieseiwitten hebben de eigenschap om het vriespunt te verlagen en rekristallisatie van ijs tegen te gaan. Deze eiwitten komen voor in poolvissen, insekten, planten en micro-organismen. Bovendien geven ze de ijskristallen een andere morfologie

  15. Antifreeze proteins enable plants to survive in freezing conditions

    Indian Academy of Sciences (India)

    1. Acaenamagellanica. Prickly burr. Doucet et al. 2000. 2. Acer saccharoides. Maple. Doucet et al. 2000. 3. Agrostistenuis. Creeping bentgrass. Doucet et al. 2000. 4. Alliarapetiolata. Garlic mustard. Urrutia et al. 1992. 5. Ammopiptanthusmongolicus. Evergreen legume. Wang et al. 2003. 6. Aster cordifolius. Wood aster.

  16. In silico characterization of antifreeze proteins using computational ...

    Indian Academy of Sciences (India)

    WINTEC

    organs.12 Chao et al13 have reported the relative effi- cacy of AFP types I, II and III in protecting the red blood cells. Numerous structure and function studies ..... Table 7. PDB templates (first 2 hits with maximum % identity) obtained using BLASTP search against the Pro- tein Data Bank. Accession number. PDB code.

  17. Studies on new antifreeze protein from the psychrophilic diatom ...

    African Journals Online (AJOL)

    Administrator

    2011-09-12

    Sep 12, 2011 ... forward primer and XhoI to reverse primer for cloning into pET vector. Bacterial and plant expressions. PCR products were analyzed and resolved on 1% agarose gel. (w/v). Bands with expected lengths were excised from the gel with razor blade and purified using the QIAquick gel extraction kit. (Qiagen, CA ...

  18. Antifreeze proteins enable plants to survive in freezing conditions

    Indian Academy of Sciences (India)

    Common name. References. 28. Hordeumvulgare. Barley. Antikainen and Griffith 1997. 29. Hydrophyllumvirginianum. Virginia waterleaf. Urrutia et al. 1992. 30. Juncussquarrosus. Heath rush. Doucet et al. 2000. 31. Loliumperenne. Perennial ryegrass. Sidebottom et al. 2000; Pudney et al. 2003. 32. Northofagusantarctica.

  19. Structure/Function Studies of Insect Antifreeze Proteins

    National Research Council Canada - National Science Library

    Duman, John

    1997-01-01

    ...-x5-x6-Cys-X8-X9-Ala-X11-Th-X13 where X3 and X1 tend toward charged residues, X5 toward threonine or serine, X9 toward asparagine or aspartate, X6 toward asparagine or lysine, and X13 toward alanine...

  20. A plasma coagulation assay for an activated protein C-independent anticoagulant activity of protein S

    NARCIS (Netherlands)

    van Wijnen, M.; van 't Veer, C.; Meijers, J. C.; Bertina, R. M.; Bouma, B. N.

    1998-01-01

    To study the physiological importance of the activated protein C (APC)-independent anticoagulant activity of protein S, we developed an assay specific for this activity. The ability of protein S to prolong the clotting time in an APC-independent way was expressed as the ratio of the clotting time in

  1. Dietary protein considerations to support active aging.

    Science.gov (United States)

    Wall, Benjamin T; Cermak, Naomi M; van Loon, Luc J C

    2014-11-01

    Given our rapidly aging world-wide population, the loss of skeletal muscle mass with healthy aging (sarcopenia) represents an important societal and public health concern. Maintaining or adopting an active lifestyle alleviates age-related muscle loss to a certain extent. Over time, even small losses of muscle tissue can hinder the ability to maintain an active lifestyle and, as such, contribute to the development of frailty and metabolic disease. Considerable research focus has addressed the application of dietary protein supplementation to support exercise-induced gains in muscle mass in younger individuals. In contrast, the role of dietary protein in supporting the maintenance (or gain) of skeletal muscle mass in active older persons has received less attention. Older individuals display a blunted muscle protein synthetic response to dietary protein ingestion. However, this reduced anabolic response can largely be overcome when physical activity is performed in close temporal proximity to protein consumption. Moreover, recent evidence has helped elucidate the optimal type and amount of dietary protein that should be ingested by the older adult throughout the day in order to maximize the skeletal muscle adaptive response to physical activity. Evidence demonstrates that when these principles are adhered to, muscle maintenance or hypertrophy over prolonged periods can be further augmented in active older persons. The present review outlines the current understanding of the role that dietary protein occupies in the lifestyle of active older adults as a means to increase skeletal muscle mass, strength and function, and thus support healthier aging.

  2. Recombinant human activated protein C (Xigris)

    NARCIS (Netherlands)

    Levi, M. [=Marcel M.; de Jonge, E.; van der Poll, T.

    2002-01-01

    An impaired function of the protein C pathway plays a central role in the pathogenesis of sepsis. Administration of human recombinant activated protein C (Xigris) may restore the dysfunctional anticoagulant mechanism and prevent amplification and propagation of thrombin generation and formation of

  3. Modulation of mitogen-activated protein kinase-activated protein kinase 3 by hepatitis C virus core protein

    DEFF Research Database (Denmark)

    Ngo, HT; Pham, Long; Kim, JW

    2013-01-01

    and protein levels of MAPKAPK3 were elevated in both HCV subgenomic replicon cells and cell culture-derived HCV (HCVcc)-infected cells. Silencing of MAPKAPK3 expression resulted in decreases in both protein and HCV infectivity levels but not in the intracellular HCV RNA level. We showed that MAPKAPK3......Hepatitis C virus (HCV) is highly dependent on cellular proteins for its own propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assays using the HCV core protein as a probe. Of ~9,000 host proteins immobilized in a microarray......, approximately 100 cellular proteins were identified as HCV core-interacting partners. Of these candidates, mitogen-activated protein kinase-activated protein kinase 3 (MAPKAPK3) was selected for further characterization. MAPKAPK3 is a serine/threonine protein kinase that is activated by stress and growth...

  4. Inhibition of Condensation Frosting by Arrays of Hygroscopic Antifreeze Drops.

    Science.gov (United States)

    Sun, Xiaoda; Damle, Viraj G; Uppal, Aastha; Linder, Rubin; Chandrashekar, Sriram; Mohan, Ajay R; Rykaczewski, Konrad

    2015-12-29

    The formation of frost and ice can have negative impacts on travel and a variety of industrial processes and is typically addressed by dispensing antifreeze substances such as salts and glycols. Despite the popularity of this anti-icing approach, some of the intricate underlying physical mechanisms are just being unraveled. For example, recent studies have shown that in addition to suppressing ice formation within its own volume, an individual salt saturated water microdroplet forms a region of inhibited condensation and condensation frosting (RIC) in its surrounding area. This occurs because salt saturated water, like most antifreeze substances, is hygroscopic and has water vapor pressure at its surface lower than water saturation pressure at the substrate. Here, we demonstrate that for macroscopic drops of propylene glycol and salt saturated water, the absolute RIC size can remain essentially unchanged for several hours. Utilizing this observation, we demonstrate that frost formation can be completely inhibited in-between microscopic and macroscopic arrays of propylene glycol and salt saturated water drops with spacing (S) smaller than twice the radius of the RIC (δ). Furthermore, by characterizing condensation frosting dynamics around various hygroscopic drop arrays, we demonstrate that they can delay complete frosting over of the samples 1.6 to 10 times longer than films of the liquids with equivalent volume. The significant delay in onset of ice nucleation achieved by dispensing propylene glycol in drops rather than in films is likely due to uniform dilution of the drops driven by thermocapillary flow. This transport mode is absent in the films, leading to faster dilution, and with that facilitated homogeneous nucleation, near the liquid-air interface.

  5. Isolation and characterisation of sericin antifreeze peptides and molecular dynamics modelling of their ice-binding interaction.

    Science.gov (United States)

    Wu, Jinhong; Rong, Yuzhi; Wang, Zhengwu; Zhou, Yanfu; Wang, Shaoyun; Zhao, Bo

    2015-05-01

    This study aimed to isolate and characterise a novel sericin antifreeze peptide and investigate its ice-binding molecular mechanism. The thermal hysteresis activity of ice-binding sericin peptides (I-SP) was measured and their activity reached as high as 0.94 °C. A P4 fraction, with high hypothermia protective activity and inhibition activity of ice recrystallisation, was obtained from I-SP, and a purified sericin peptide, named SM-AFP, with the sequence of TTSPTNVSTT and a molecular weight of 1009.50 Da was then isolated from the P4 fraction. Treatment of Lactobacillus delbrueckii Subsp. bulgaricus LB340 LYO with 100 μg/ml synthetic SM-AFP led to 1.4-fold increased survival (p Sericin peptides could be developed into beneficial cryoprotectants and used in frozen food processing. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. FT-IR Spectra of Antifreeze Glycoproteins in Heavy Water and D2O Ice.

    Science.gov (United States)

    Tsvetkova, N. M.; Crowe, J. H.; Feeney, R. H.; Fink, W. H.; Yeh, Yin

    2000-03-01

    This work presents FT-IR studies on the antifreeze glycoprotein (AFGP)/heavy water (D2O) mixtures during freezing and melting. AFGP in the blood serum of polar fish are known to prevent ice crystal growth by a non-colligative mechanism. There are 8 known fractions of AFGP (1 8) that range in molecular mass from 33.7 to 2.6 kD respectively, each composed of alanine-alanine-threonine repeats, with a disaccharide attached to the threonine residue. The smallest peptide (AFGP-8) is structurally different from fractions 1-5 in that it contains proline substituting for alanine in certain positions. Substantial linewidth change of the D20 bending mode (ca. 1210 cm-1) was measured with solutions containing fractions 2-5 during both freezing and thawing cycles, suggesting significant coupling between protein and water molecules. At the same time, the Amide I band between 1620 and 1675 cm-1 shows that 310 helix and random coils are the main conformations of fractions 2-5 and fraction 8 in the presence of ice. In liquid state, b-sheet dominates the secondary structure of AFGP 8, whereas b-sheet and random coil are the main conformations of AFGP 2-5. These results are discussed in terms of the ability of AFGP 2-5 to affect the surface states of ice.

  7. Ancient climate change, antifreeze, and the evolutionary diversification of Antarctic fishes.

    Science.gov (United States)

    Near, Thomas J; Dornburg, Alex; Kuhn, Kristen L; Eastman, Joseph T; Pennington, Jillian N; Patarnello, Tomaso; Zane, Lorenzo; Fernández, Daniel A; Jones, Christopher D

    2012-02-28

    The Southern Ocean around Antarctica is among the most rapidly warming regions on Earth, but has experienced episodic climate change during the past 40 million years. It remains unclear how ancient periods of climate change have shaped Antarctic biodiversity. The origin of antifreeze glycoproteins (AFGPs) in Antarctic notothenioid fishes has become a classic example of how the evolution of a key innovation in response to climate change can drive adaptive radiation. By using a time-calibrated molecular phylogeny of notothenioids and reconstructed paleoclimate, we demonstrate that the origin of AFGP occurred between 42 and 22 Ma, which includes a period of global cooling approximately 35 Ma. However, the most species-rich lineages diversified and evolved significant ecological differences at least 10 million years after the origin of AFGPs, during a second cooling event in the Late Miocene (11.6-5.3 Ma). This pattern indicates that AFGP was not the sole trigger of the notothenioid adaptive radiation. Instead, the bulk of the species richness and ecological diversity originated during the Late Miocene and into the Early Pliocene, a time coincident with the origin of polar conditions and increased ice activity in the Southern Ocean. Our results challenge the current understanding of the evolution of Antarctic notothenioids suggesting that the ecological opportunity that underlies this adaptive radiation is not linked to a single trait, but rather to a combination of freeze avoidance offered by AFGPs and subsequent exploitation of new habitats and open niches created by increased glacial and ice sheet activity.

  8. Tribomechanical micronization and activation of whey protein ...

    Indian Academy of Sciences (India)

    Tribomechanics is a part of physics that is concerned with the study of phenomena that appear during milling under dynamic conditions. Tribomechanical micronization and activation (TMA) of whey protein concentrates (WPC) and zeolites (type clinoptilolite) were carried out. Samples of powdered WPC and zeolite were ...

  9. Antifreeze polymeric additives for fuels; Aditivos polimericos anticongelantes para combustiveis

    Energy Technology Data Exchange (ETDEWEB)

    Muniz, Aline S.; Carvalho, Agne Roani de; Sakae, George Hideki; Oliveira, Angelo R.S.; Cesar-Oliveira, Maria Aparecida F. [Universidade Federal do Parana - UFPR - Departamento de Quimica - LABPOL-Laboratorio de Polimeros Sinteticos, Centro Politecnico, Curitiba, PR (Brazil)], e-mails: mafco@ufpr.br, alinemuniz@ufpr.br

    2011-07-01

    Owing to the current interest in the reduction of environmental pollution, several researchers are seeking renewable sources of energy which can at least partially replace combustibles derived from petroleum. Diesel oil is the combustible that most seriously pollutes the environment and is thus the biodiesel that is being considered as a fuel which can be replaced by a renewable combustible; this can possibly be used in diesel engines without any modifications. However, certain problems have to be overcome with regard to the temperature at which the biodiesel should be stored and used, since there is a tendency for biodiesel to solidify at low temperatures. This suggests that there is a need for the use of anti-freeze additives. This work behind the main focus additives with only 25 ppm, were able to reduce the pour point of fuel, achieving significant results, for example, the additive M14A18 lowered the pour point (PP) of B20 to -20 degree C, showing that the use of increasing amounts of biodiesel to diesel can aggregate. The main focus of work behind the development of additives that with only 25 ppm, were able to reduce the pour point of fuel, producing significant results such as those obtained with the use of additive M14A18 which lowered the pour point of the B20 to -20 degree C, showing the possibility of using increasing amounts of biodiesel added to diesel. (author)

  10. FISH GLYCOPEPTIDE AND PEPTIDE ANTIFREEZES : THEIR INTERACTION WITH ICE AND WATER

    OpenAIRE

    Wilson, P.; Devries, A.

    1987-01-01

    Glycopeptide and peptide antifreeze agents are present in the body fluids of polar fishes and allow them to avoid freezing in ice-laden seawater. These antifreezes lower the freezing point 200 times more than predicted by colligative relations, but have little effect on the melting point of ice. They bind to ice and appear to inhibit growth by increasing the curvature of growth steps on the ice crystal surface. Such a growth would result in a substantial increase in the roughness of the surfa...

  11. Raman optical activity of proteins and glycoproteins

    International Nuclear Information System (INIS)

    Smyth, E.

    2000-03-01

    Raman optical activity (ROA), measured in this project as a small difference in the intensity of Raman scattering from chiral molecules in right- and left-circularly polarised incident laser light, offers the potential to provide more information about the structure of biological molecules in aqueous solution than conventional spectroscopic techniques. Chapter one contains a general discussion of the relative merits of different spectroscopic techniques for structure determination of biomolecules, as well as a brief introduction to ROA. In Chapter two a theoretical analysis of ROA is developed, which extends the discussion in chapter one. The spectrometer setup and sample preparation is then discussed in chapter three. Instrument and sample conditions are monitored to ensure that the best results are obtained. As with any experimental project problems occur, which may result in a degradation of the spectra obtained. The cause of these problems was explored and remedied whenever possible. Chapter four introduces a brief account of protein, glycoprotein and carbohydrate structure and function, with a particular emphasis on the structure of proteins. In the remaining chapters experimental ROA results on proteins and glycoproteins, with some carbohydrate samples, from a wide range of sources are examined. For example, in chapter five some β-sheet proteins are examined. Structural features in these proteins are examined in the extended amide III region of their ROA spectra, revealing that ROA is sensitive to the rigidity or flexibility inherent in proteins. Chapter six concentrates on a group of proteins (usually glycoproteins) known as the serine proteinase inhibitors (serpins). Medically, the serpins are one of the most important groups of proteins of current interest, with wide-ranging implications in conditions such as Down's syndrome, Alzheimer's disease, and emphysema with associated cirrhosis of the liver. With favourable samples and conditions ROA may offer the

  12. De Novo Construction of Redox Active Proteins.

    Science.gov (United States)

    Moser, C C; Sheehan, M M; Ennist, N M; Kodali, G; Bialas, C; Englander, M T; Discher, B M; Dutton, P L

    2016-01-01

    Relatively simple principles can be used to plan and construct de novo proteins that bind redox cofactors and participate in a range of electron-transfer reactions analogous to those seen in natural oxidoreductase proteins. These designed redox proteins are called maquettes. Hydrophobic/hydrophilic binary patterning of heptad repeats of amino acids linked together in a single-chain self-assemble into 4-alpha-helix bundles. These bundles form a robust and adaptable frame for uncovering the default properties of protein embedded cofactors independent of the complexities introduced by generations of natural selection and allow us to better understand what factors can be exploited by man or nature to manipulate the physical chemical properties of these cofactors. Anchoring of redox cofactors such as hemes, light active tetrapyrroles, FeS clusters, and flavins by His and Cys residues allow cofactors to be placed at positions in which electron-tunneling rates between cofactors within or between proteins can be predicted in advance. The modularity of heptad repeat designs facilitates the construction of electron-transfer chains and novel combinations of redox cofactors and new redox cofactor assisted functions. Developing de novo designs that can support cofactor incorporation upon expression in a cell is needed to support a synthetic biology advance that integrates with natural bioenergetic pathways. © 2016 Elsevier Inc. All rights reserved.

  13. Cranberry Proanthocyanidins - Protein complexes for macrophage activation.

    Science.gov (United States)

    Carballo, Sergio M; Haas, Linda; Krueger, Christian G; Reed, Jess D

    2017-09-20

    In this work we characterize the interaction of cranberry (Vaccinium macrocarpon) proanthocyanidins (PAC) with bovine serum albumin (BSA) and hen egg-white lysozyme (HEL) and determine the effects of these complexes on macrophage activation and antigen presentation. We isolated PAC from cranberry and complexed the isolated PAC with BSA and HEL. The properties of the PAC-protein complexes were studied by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), gel electrophoresis and zeta-potential. The effects of PAC-BSA complexes on macrophage activation were studied in RAW 264.7 macrophage like cells after treatment with lipopolysaccharide (LPS). Fluorescence microscopy was used to study the endocytosis of PAC-BSA complexes. The effects of the PAC complexes on macrophage antigen presentation were studied in an in vitro model of HEL antigen presentation by mouse peritoneal mononuclear cells to a T-cell hybridoma. The mass spectra of the PAC complexes with BSA and HEL differed from the spectra of the proteins alone by the presence of broad shoulders on the singly and doubly charged protein peaks. Complexation with PAC altered the electrophoretic mobility shift assay in native agarose gel and the electrophoretic mobility (ζ-potential) values. These results indicate that the PAC-protein complexes are stable and alter the protein structure without precipitating the protein. Fluorescence microscopy showed that the RAW 264.7 macrophages endocytosed BSA and PAC-BSA complexes in discrete vesicles that surrounded the nucleus. Macrophages treated with increasing amounts of PAC-BSA complexes had significantly reduced COX-2 and iNOS expression in response to treatment with lipopolysaccharide (LPS) in comparison to the controls. The PAC-HEL complexes modulated antigen uptake, processing and presentation in murine peritoneal macrophages. After 4 h of pre-incubation, only trace amounts of IL-2 were detected in the co-cultures treated with HEL

  14. [Antioxidant activity of cationic whey protein isolate].

    Science.gov (United States)

    titova, M E; Komolov, S A; Tikhomirova, N A

    2012-01-01

    The process of lipid peroxidation (LPO) in biological membranes of cells is carried out by free radical mechanism, a feature of which is the interaction of radicals with other molecules. In this work we investigated the antioxidant activity of cationic whey protein isolate, obtained by the cation-exchange chromatography on KM-cellulose from raw cow's milk, in vitro and in vivo. In biological liquids, which are milk, blood serum, fetal fluids, contains a complex of biologically active substances with a unique multifunctional properties, and which are carrying out a protective, antimicrobial, regenerating, antioxidant, immunomodulatory, regulatory and others functions. Contents of the isolate were determined electrophoretically and by its biological activity. Cationic whey protein isolate included lactoperoxidase, lactoferrin, pancreatic RNase, lysozyme and angeogenin. The given isolate significantly has an antioxidant effect in model experimental systems in vitro and therefore may be considered as a factor that can adjust the intensity of lipid oxidation. In model solutions products of lipid oxidation were obtained by oxidation of phosphatidylcholine by hydrogen peroxide in the presence of a source of iron. The composition of the reaction mixture: 0,4 mM H2O2; 50 mcM of hemin; 2 mg/ml L-alpha-phosphatidylcholine from soybean (Sigma, German). Lipid peroxidation products were formed during the incubation of the reaction mixture for two hours at 37 degrees C. In our studies rats in the adaptation period immediately after isolation from the nest obtained from food given orally native cationic whey protein isolate at the concentration three times higher than in fresh cow's milk. On the manifestation of the antioxidant activity of cationic whey protein isolate in vivo evidence decrease of lipid peroxidation products concentration in the blood of rats from the experimental group receipt whey protein isolate in dos 0,6 mg/g for more than 20% (pisolate has an antioxidant

  15. Complement activation by ceramide transporter proteins.

    Science.gov (United States)

    Bode, Gerard H; Losen, Mario; Buurman, Wim A; Veerhuis, Robert; Molenaar, Peter C; Steinbusch, Harry W M; De Baets, Marc H; Daha, Mohamed R; Martinez-Martinez, Pilar

    2014-02-01

    C1q is the initiator of the classical complement pathway and, as such, is essential for efficient opsonization and clearance of pathogens, altered self-structures, and apoptotic cells. The ceramide transporter protein (CERT) and its longer splicing isoform CERTL are known to interact with extracellular matrix components, such as type IV collagen, and with the innate immune protein serum amyloid P. In this article, we report a novel function of CERT in the innate immune response. Both CERT isoforms, when immobilized, were found to bind the globular head region of C1q and to initiate the classical complement pathway, leading to activation of C4 and C3, as well as generation of the membrane attack complex C5b-9. In addition, C1q was shown to bind to endogenous CERTL on the surface of apoptotic cells. These results demonstrate the role of CERTs in innate immunity, especially in the clearance of apoptotic cells.

  16. Oscillations and accelerations of ice crystal growth rates in microgravity in presence of antifreeze glycoprotein impurity in supercooled water

    Science.gov (United States)

    Furukawa, Yoshinori; Nagashima, Ken; Nakatsubo, Shun-Ichi; Yoshizaki, Izumi; Tamaru, Haruka; Shimaoka, Taro; Sone, Takehiko; Yokoyama, Etsuro; Zepeda, Salvador; Terasawa, Takanori; Asakawa, Harutoshi; Murata, Ken-Ichiro; Sazaki, Gen

    2017-03-01

    The free growth of ice crystals in supercooled bulk water containing an impurity of glycoprotein, a bio-macromolecule that functions as ‘antifreeze’ in living organisms in a subzero environment, was observed under microgravity conditions on the International Space Station. We observed the acceleration and oscillation of the normal growth rates as a result of the interfacial adsorption of these protein molecules, which is a newly discovered impurity effect for crystal growth. As the convection caused by gravity may mitigate or modify this effect, secure observations of this effect were first made possible by continuous measurements of normal growth rates under long-term microgravity condition realized only in the spacecraft. Our findings will lead to a better understanding of a novel kinetic process for growth oscillation in relation to growth promotion due to the adsorption of protein molecules and will shed light on the role that crystal growth kinetics has in the onset of the mysterious antifreeze effect in living organisms, namely, how this protein may prevent fish freezing.

  17. Receptor activity-modifying proteins; multifunctional G protein-coupled receptor accessory proteins.

    Science.gov (United States)

    Hay, Debbie L; Walker, Christopher S; Gingell, Joseph J; Ladds, Graham; Reynolds, Christopher A; Poyner, David R

    2016-04-15

    Receptor activity-modifying proteins (RAMPs) are single pass membrane proteins initially identified by their ability to determine the pharmacology of the calcitonin receptor-like receptor (CLR), a family B G protein-coupled receptor (GPCR). It is now known that RAMPs can interact with a much wider range of GPCRs. This review considers recent developments on the structure of the complexes formed between the extracellular domains (ECDs) of CLR and RAMP1 or RAMP2 as these provide insights as to how the RAMPs direct ligand binding. The range of RAMP interactions is also considered; RAMPs can interact with numerous family B GPCRs as well as examples of family A and family C GPCRs. They influence receptor expression at the cell surface, trafficking, ligand binding and G protein coupling. The GPCR-RAMP interface offers opportunities for drug targeting, illustrated by examples of drugs developed for migraine. © 2016 Authors; published by Portland Press Limited.

  18. Antioxidative Activity of Tobacco Leaf Protein Hydrolysates

    Directory of Open Access Journals (Sweden)

    Guohua Rao

    2007-01-01

    Full Text Available Discarded tobacco leaf protein hydrolysate (DTLPH was prepared by enzymatic hydrolysis using papain and then separated using ultrafiltration (UF membranes with molecular mass cut-off (MMCO of 10, 5, 3 and 1 kDa. Four permeate fractions including 10-K, 5-K, 3-K and 1-K (the permeate fractions from 10, 5, 3 and 1 kDa hydrolysate fractions were obtained. The 5-K hydrolysate fraction had high oxidation inhibilitory ratio (42.62 %, which was about twofold higher than the original hydrolysate and as high as that of vitamin E (α-tocopherol. The fractionated hydrolysates were superior to the original hydrolysate in the antioxidative activity tested. Moreover, these separated hydrolysates showed the enhanced functional property. The amino acid composition of 5-K hydrolysate was analyzed and the results show that the high antioxidative activity of 5-K hydrolysate was derived from high content of histidine, methionine, cystine and tryptophan.

  19. Egg yolk proteins and peptides with biological activity

    Directory of Open Access Journals (Sweden)

    Aleksandra Zambrowicz

    2014-12-01

    Full Text Available Many proteins of food reveal biological activity. In the sequence of these proteins also numerous biologically active peptides are encrypted. These peptides are released during proteolysis naturally occurring in the gastrointestinal tract, food fermentation or during designed enzymatic hydrolysis in vitro. Biopeptides may exert multiple activities, affecting the cardiovascular, endocrine, nervous and immune systems. An especially rich source of bioactive proteins and biopeptides is egg. Bioactive peptides released from egg white proteins have been well described, whereas egg yolk proteins as precursors of biopeptides are less well characterized. This manuscript describes biologically active proteins and peptides originating from egg yolk and presents their potential therapeutic role.

  20. Arabinogalactan proteins: focus on carbohydrate active enzymes

    Directory of Open Access Journals (Sweden)

    Eva eKnoch

    2014-06-01

    Full Text Available Arabinogalactan proteins (AGPs are a highly diverse class of cell surface proteoglycans that are commonly found in most plant species. AGPs play important roles in many cellular processes during plant development, such as reproduction, cell proliferation, pattern formation and growth, and in plant-microbe interaction. However, little is known about the molecular mechanisms of their function. Numerous studies using monoclonal antibodies that recognize different AGP glycan epitopes have shown the appearance of a slightly altered AGP glycan in a specific stage of development in plant cells. Therefore, it is anticipated that the biosynthesis and degradation of AGP glycan is tightly regulated during development. Until recently, however, little was known about the enzymes involved in the metabolism of AGP glycans. In this review, we summarize recent discoveries of carbohydrate active enzymes (CAZy; http://www.cazy.org/ involved in the biosynthesis and degradation of AGP glycans, and we discuss the biological role of these enzymes in plant development.

  1. Tamoxifen induces resistance to activated protein C.

    Science.gov (United States)

    Rühl, Heiko; Schröder, Lars; Müller, Jens; Fimmers, Rolf; Sukhitashvili, Shorena; Welz, Julia; Kuhn, Walther C; Oldenburg, Johannes; Rudlowski, Christian; Pötzsch, Bernd

    2014-05-01

    The estrogen antagonist tamoxifen (TAM) increases the thrombotic risk similar to estrogen containing oral contraceptives (OC). In OC users this risk is attributed to alterations of hemostasis resulting in acquired resistance to activated protein C (APC). TAM-induced APC resistance has not been reported yet. Blood samples were collected prospectively from women with breast cancer before (n=25) and monthly after start of adjuvant TAM treatment (n=75). APC resistance was evaluated on basis of the effect of APC on the endogenous thrombin generation potential. To detect increased in vivo APC generation APC plasma levels were measured using a highly sensitive oligonucleotide-based enzyme capture assay. Routine hemostasis parameters were measured additionally. APC sensitivity decreased by 41% (p=0.001) compared to baseline after one month of TAM application and remained significantly decreased during the study period. Free protein S increased (p=0.008) while other analyzed procoagulant factors, inhibitors, and activation markers of coagulation decreased or did not change significantly. In five patients the APC concentration increased to non-physiological levels but an overall significant increase of APC was not observed. This is the first study showing acquired APC resistance under TAM therapy. Acquired APC resistance might explain the increased thrombotic risk during TAM treatment. Observed changes of hemostasis parameters suggest different determinants of TAM-induced APC resistance than in OC-induced APC resistance. The presence of acquired APC resistance in TAM patients warrants further evaluation if these patients may benefit from antithrombotic prophylaxis in the presence of additional thrombotic risk factors. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Rapid kinetic BRET measurements to monitor G protein activation by GPCR and non-GPCR proteins.

    Science.gov (United States)

    Maziarz, Marcin; Garcia-Marcos, Mikel

    2017-01-01

    Heterotrimeric G proteins are central hubs of signal transduction whose activity is controlled by G protein-coupled receptors (GPCRs) as well as by a complex network of regulatory proteins. Recently, bioluminescence resonance energy transfer (BRET)-based assays have been used to monitor real-time activation of heterotrimeric G proteins in cells. Here we describe the use of a previously established BRET assay to monitor G protein activation upon GPCR stimulation and its adaptation to measure G protein activation by non-GPCR proteins, such as by cytoplasmic guanine nucleotide exchange factors (GEFs) like GIV/Girdin. The BRET assay monitors the release of free Gβγ from Gα-Gβγ heterotrimers as a readout of G protein activation, which is readily observable upon agonist stimulation of GPCRs. To control the signal input for non-GPCR activators, we describe the use of a chemically induced dimerization strategy to promote rapid membrane translocation of proteins containing the Gα-binding and -activating (GBA) motif found in some nonreceptor GEFs. The assay described here allows the kinetic measurement of G protein activation with subsecond temporal resolution and to compare the levels of activation induced by GPCR agonists vs those induced by the membrane recruitment of nonreceptor G protein signaling activators. © 2017 Elsevier Inc. All rights reserved.

  3. Synthesis and characterization of natural and modified antifreeze glycopeptides: glycosylated foldamers.

    Science.gov (United States)

    Nagel, Lilly; Plattner, Carolin; Budke, Carsten; Majer, Zsuzsanna; DeVries, Arthur L; Berkemeier, Thomas; Koop, Thomas; Sewald, Norbert

    2011-08-01

    In Arctic and Antarctic marine regions, where the temperature declines below the colligative freezing point of physiological fluids, efficient biological antifreeze agents are crucial for the survival of polar fish. One group of such agents is classified as antifreeze glycoproteins (AFGP) that usually consist of a varying number (n = 4-55) of [AAT]( n )-repeating units. The threonine side chain of each unit is glycosidically linked to β-D: -galactosyl-(1 → 3)-α-N-acetyl-D: -galactosamine. These biopolymers can be considered as biological antifreeze foldamers. A preparative route for stepwise synthesis of AFGP allows for efficient synthesis. The diglycosylated threonine building block was introduced into the peptide using microwave-enhanced solid phase synthesis. By this versatile solid phase approach, glycosylated peptides of varying sequences and lengths could be obtained. Conformational studies of the synthetic AFGP analogs were performed by circular dichroism experiments (CD). Furthermore, the foldamers were analysed microphysically according to their inhibiting effect on ice recrystallization and influence on the crystal habit.

  4. Sulfated glycopeptide nanostructures for multipotent protein activation

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Sungsoo S.; Fyrner, Timmy; Chen, Feng; Álvarez, Zaida; Sleep, Eduard; Chun, Danielle S.; Weiner, Joseph A.; Cook, Ralph W.; Freshman, Ryan D.; Schallmo, Michael S.; Katchko, Karina M.; Schneider, Andrew D.; Smith, Justin T.; Yun, Chawon; Singh, Gurmit; Hashmi, Sohaib Z.; McClendon, Mark T.; Yu, Zhilin; Stock, Stuart R.; Hsu, Wellington K.; Hsu, Erin L.; Stupp , Samuel I. (NWU)

    2017-06-19

    Biological systems have evolved to utilize numerous proteins with capacity to bind polysaccharides for the purpose of optimizing their function. A well-known subset of these proteins with binding domains for the highly diverse sulfated polysaccharides are important growth factors involved in biological development and tissue repair. We report here on supramolecular sulfated glycopeptide nanostructures, which display a trisulfated monosaccharide on their surfaces and bind five critical proteins with different polysaccharide-binding domains. Binding does not disrupt the filamentous shape of the nanostructures or their internal β-sheet backbone, but must involve accessible adaptive configurations to interact with such different proteins. The glycopeptide nanostructures amplified signalling of bone morphogenetic protein 2 significantly more than the natural sulfated polysaccharide heparin, and promoted regeneration of bone in the spine with a protein dose that is 100-fold lower than that required in the animal model. These highly bioactive nanostructures may enable many therapies in the future involving proteins.

  5. Endoplasmic reticulum stress activates transglutaminase 2 leading to protein aggregation.

    Science.gov (United States)

    Lee, Jin-Haeng; Jeong, Jaeho; Jeong, Eui Man; Cho, Sung-Yup; Kang, Jeong Wook; Lim, Jisun; Heo, Jinbeom; Kang, Hyunsook; Kim, In-Gyu; Shin, Dong-Myung

    2014-04-01

    Aberrant activation of transglutaminase 2 (TGase2) contributes to a variety of protein conformational disorders such as neurodegenerative diseases and age-related cataracts. The accumulation of improperly folded proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), which promotes either repair or degradation of the damaged proteins. Inadequate UPR results in protein aggregation that may contribute to the development of age-related degenerative diseases. TGase2 is a calcium-dependent enzyme that irreversibly modifies proteins by forming cross-linked protein aggregates. Intracellular TGase2 is activated by oxidative stress which generates large quantities of unfolded proteins. However, the relationship between TGase2 activity and UPR has not yet been established. In the present study, we demonstrated that ER stress activated TGase2 in various cell types. TGase2 activation was dependent on the ER stress-induced increase in the intracellular calcium ion concentration but not on the TGase2 protein expression level. Enzyme substrate analysis revealed that TGase2-mediated protein modification promoted protein aggregation concurrently with decreasing water solubility. Moreover, treatment with KCC009, a TGase2 inhibitor, abrogated ER stress-induced TGase2 activation and subsequent protein aggregation. However, TGase2 activation had no effect on ER stress-induced cell death. These results demonstrate that the accumulation of misfolded proteins activates TGase2, which further accelerates the formation of protein aggregates. Therefore, we suggest that inhibition of TGase2 may be a novel strategy by which to prevent the protein aggregation in age-related degenerative diseases.

  6. Synergistic inhibition of the intrinsic factor X activation by protein S and C4b-binding protein

    NARCIS (Netherlands)

    Koppelman, S. J.; van't Veer, C.; Sixma, J. J.; Bouma, B. N.

    1995-01-01

    The complement protein C4b-binding protein plays an important role in the regulation of the protein C anticoagulant pathway. C4b-binding protein can bind to protein S, thereby inhibiting the cofactor activity of protein S for activated protein C. In this report, we describe a new role for

  7. The interaction of protein S with the phospholipid surface is essential for the activated protein C-independent activity of protein S

    NARCIS (Netherlands)

    van Wijnen, M.; Stam, J. G.; van't Veer, C.; Meijers, J. C.; Reitsma, P. H.; Bertina, R. M.; Bouma, B. N.

    1996-01-01

    Protein S is a vitamin-K dependent glycoprotein involved in the regulation of the anticoagulant activity of activated protein C (APC). Recent data showed a direct anticoagulant role of protein S independent of APC, as demonstrated by the inhibition of prothrombinase and tenase activity both in

  8. Antioxidant activity of black bean (Phaseolus vulgaris L. protein hydrolysates

    Directory of Open Access Journals (Sweden)

    Jarine Amaral do EVANGELHO

    2016-01-01

    Full Text Available Abstract The objective of this work was to study the effect of enzymatic hydrolysis of black bean protein concentrate using different enzymes. Bean proteins were extracted and hydrolyzed over a period of 120 min using the enzymes pepsin or alcalase. The protein hydrolysates’ molecular weight was assayed by electrophoresis and the antioxidant activity was evaluated by the capturing methods of free radicals ABTS●+ and DPPH. Electrophoretic results showed that the bands above 50 kDa disappeared, when the beans protein was subjected to hydrolysis with pepsin. The bean protein hydrolysate obtained by hydrolysis with alcalase enzyme, showed higher antioxidant activity for inhibition of the radical ABTS●+. However, the hydrolysates obtained by hydrolysis with pepsin had higher antioxidant activity for inhibition of the radical DPPH. The use of pepsin and alcalase enzymes, under the same reaction time, produced black bean protein hydrolysates with different molecular weight profiles and superior antioxidant activity than the native bean protein.

  9. Mini G protein probes for active G protein-coupled receptors (GPCRs) in live cells.

    Science.gov (United States)

    Wan, Qingwen; Okashah, Najeah; Inoue, Asuka; Nehmé, Rony; Carpenter, Byron; Tate, Christopher G; Lambert, Nevin A

    2018-03-09

    G protein-coupled receptors (GPCRs) are key signaling proteins that regulate nearly every aspect of cell function. Studies of GPCRs have benefitted greatly from the development of molecular tools to monitor receptor activation and downstream signaling. Here we show that mini G proteins are robust probes that can be used in a variety of assay formats to report GPCR activity in living cells. Mini G (mG) proteins are engineered GTPase domains of Gα subunits that were developed for structural studies of active state GPCRs. Confocal imaging revealed that mG proteins fused to fluorescent proteins were located diffusely in the cytoplasm, and translocated to sites of receptor activation at the cell surface and at intracellular organelles. Bioluminescence resonance energy transfer (BRET) assays with mG proteins fused to either a fluorescent protein or luciferase reported agonist, superagonist and inverse agonist activities. Variants of mG proteins (mGs, mGsi, mGsq and mG12) corresponding to the four families of Gα subunits displayed appropriate coupling to their cognate GPCRs, allowing quantitative profiling of subtype-specific coupling to individual receptors. BRET between luciferase-mG fusion proteins and fluorescent markers indicated the presence of active GPCRs at the plasma membrane, Golgi apparatus and endosomes. Complementation assays with fragments of NanoLuc luciferase fused to GPCRs and mG proteins reported constitutive receptor activity and agonist-induced activation with up to 20-fold increases in luminescence. We conclude that mG proteins are versatile tools for studying GPCR activation and coupling specificity in cells, and should be useful for discovering and characterizing G protein subtype-biased ligands. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Phospholipid transfer protein activity and incident type 2 diabetes mellitus

    NARCIS (Netherlands)

    Abbasi, Ali; Dallinga-Thie, Geesje M.; Dullaart, Robin P. F.

    2015-01-01

    Background: The plasma activity of phospholipid transfer protein (PLTP), which has multifaceted functions in lipoprotein metabolism and in inflammatory responses, is elevated in insulin resistant conditions. We determined the association of plasma PLTP activity with incident type 2 diabetes mellitus

  11. Protein S binding to human endothelial cells is required for expression of cofactor activity for activated protein C

    NARCIS (Netherlands)

    Hackeng, T. M.; Hessing, M.; van 't Veer, C.; Meijer-Huizinga, F.; Meijers, J. C.; de Groot, P. G.; van Mourik, J. A.; Bouma, B. N.

    1993-01-01

    An important feedback mechanism in blood coagulation is supplied by the protein C/protein S anticoagulant pathway. In this study we demonstrate that the binding of human protein S to cultured human umbilical vein endothelial cells (HUVECs) is required for the expression of cofactor activity of

  12. Tribomechanical micronization and activation of whey protein ...

    Indian Academy of Sciences (India)

    R. Narasimhan (Krishtel eMaging) 1461 1996 Oct 15 13:05:22

    Physical and structural properties of proteins sometimes can be altered by mechanical means. ... enters the equipment through the central part of the rotor system by ventilator air streaming. The particles are ... devised equipment, striking hammers and ventilation paddles placed on rotating discs pro- duce turbulent ...

  13. Activation of fluorescent protein chromophores by encapsulation.

    Science.gov (United States)

    Baldridge, Anthony; Samanta, Shampa R; Jayaraj, Nithyanandhan; Ramamurthy, V; Tolbert, Laren M

    2010-02-10

    Chromophores related to fluorescent proteins, when sequestered into the "octaacid" capsule, recover their fluorescence. The fluorescence recovery is related to the inhibition of torsional motions within the cavity, implicating the single-bond torsion as an important contributor to internal conversion within this important class of chromophores.

  14. Complement Activation by Ceramide Transporter Proteins

    NARCIS (Netherlands)

    Bode, G.H.; Losen, M.; Buurman, W.A.; Veerhuis, R.; Molenaar, P.C.; Steinbusch, H.W.M.; De Baets, M.H.; Daha, MR; Martinez-Martinez, P.

    2014-01-01

    C1q is the initiator of the classical complement pathway and, as such, is essential for efficient opsonization and clearance of pathogens, altered self-structures, and apoptotic cells. The ceramide transporter protein (CERT) and its longer splicing isoform CERTL are known to interact with

  15. Antimicrobial activity and molecular mechanism of the CRES protein.

    Directory of Open Access Journals (Sweden)

    Li Wang

    Full Text Available Cystatin-related epididymal spermatogenic (CRES protein, a member of the cystatin superfamily of cysteine protease inhibitors (also known as CST8, exhibits highly specific, age-dependent expression in mouse testis and epididymis. The CRES protein possesses four highly conserved cysteine residues which govern the overall conformation of the cystatins through the formation of two disulfide bonds. Previous studies have revealed that other cystatin family members, such as cystatin 3 and cystatin 11, show antibacterial activity in vitro. This prompted us to investigate the potential antimicrobial activity of the CRES protein. Colony forming assays and spectrophotometry were used to investigate the effects of recombinant CRES protein on Escherichia coli (E. coli and Ureaplasma urealyticum (Uu, respectively, in vitro. After incubation of E. coli with CRES recombinant protein fused with glutathione-S-transferase (GST, a substantial decrease in colony forming units was observed, and the effect was dose and time dependent. Furthermore, it took longer for Uu to grow to plateau stage when incubated with GST-CRES recombinant protein compared with the control GST. The antibacterial and Anti-Uu activities were not impaired when the cysteine residues of CRES protein were mutated, indicating that the antimicrobial effect was not dependent on its disulfide bonds. Functional analysis of three CRES polypeptides showed that the N-terminal 30 residues (N30 had no antimicrobial activity while N60 showed similar activity as full-length CRES protein. These results suggest that the active center of CRES protein resides between amino acid residues 31 and 60 of its N-terminus. Mechanistically, E. coli membrane permeabilization was increased in a dose-dependent manner, and macromolecular synthesis was inhibited on treatment with GST-CRES. Together, our data on the antimicrobial activities of CRES protein suggest that it is a novel and innate antimicrobial protein which

  16. STUDY ON PROPERTIES OF SKID RESISTANCE ON FREEZING PAVEMENTS AND QUANTITATIVE EVALUATION METHOD OF ANTIFREEZING EFFECTS

    Science.gov (United States)

    Tanaka, Shunsuke; Takeichi, Kiyoshi; Masuyama, Yukiei; Takahashi, Naoto

    Snow and ice control in winter roads trends to be controlled by the skid friction coefficients in North America and North European countries at present, but the measurements are not necessarily easy. We studied on a simplified measurement method based on the relationship between skid friction coefficients and the bare pavement ratio (BPR) in the laboratory tests and field tests. The factors of BPR, surface textures and antifreezing materials which affect the skid friction coefficient are reviewed by a multiple linear regression analysis and a spectrum analysis, considering different freezing surfaces. These studies indicate that conclusions induced by laboratory tests could be applied to roads in service.

  17. Controlled Activation of Protein Rotational Dynamics Using Smart Hydrogel Tethering

    Energy Technology Data Exchange (ETDEWEB)

    Beech, Brenda M.; Xiong, Yijia; Boschek, Curt B.; Baird, Cheryl L.; Bigelow, Diana J.; Mcateer, Kathleen; Squier, Thomas C.

    2014-09-05

    Stimulus-responsive hydrogel materials that stabilize and control protein dynamics have the potential to enable a range of applications to take advantage of the inherent specificity and catalytic efficiencies of proteins. Here we describe the modular construction of a hydrogel using an engineered calmodulin (CaM) within a polyethylene glycol (PEG) matrix that involves the reversible tethering of proteins through an engineered CaM-binding sequence. For these measurements, maltose binding protein (MBP) was isotopically labeled with [13C] and [15N], permitting dynamic structural measurements using TROSY-HSQC NMR spectroscopy. Upon initial formation of hydrogels protein dynamics are suppressed, with concomitant increases in protein stability. Relaxation of the hydrogel matrix following transient heating results in the activation of protein dynamics and restoration of substrate-induced large-amplitude domain motions necessary for substrate binding.

  18. Mitogen-activated protein kinase signaling in plants

    DEFF Research Database (Denmark)

    Rodriguez, Maria Cristina Suarez; Petersen, Morten; Mundy, John

    2010-01-01

    Eukaryotic mitogen-activated protein kinase (MAPK) cascades have evolved to transduce environmental and developmental signals into adaptive and programmed responses. MAPK cascades relay and amplify signals via three types of reversibly phosphorylated kinases leading to the phosphorylation of subs...... the Arabidopsis thaliana MAPKs MPK3, 4, and 6 and MAP2Ks MKK1, 2, 4, and 5. Future work needs to focus on identifying substrates of MAPKs, and on understanding how specificity is achieved among MAPK signaling pathways.......Eukaryotic mitogen-activated protein kinase (MAPK) cascades have evolved to transduce environmental and developmental signals into adaptive and programmed responses. MAPK cascades relay and amplify signals via three types of reversibly phosphorylated kinases leading to the phosphorylation...... of substrate proteins, whose altered activities mediate a wide array of responses, including changes in gene expression. Cascades may share kinase components, but their signaling specificity is maintained by spaciotemporal constraints and dynamic protein-protein interactions and by mechanisms that include...

  19. Mitogen-activated protein kinases mediate Mycobacterium ...

    Indian Academy of Sciences (India)

    2012-01-19

    Jan 19, 2012 ... heat shock, UV irradiation and also to inflammatory cytokines. ERK is mainly activated by growth factors and phorbol esters. (Lewis et al. 1998; Cowan and Storey 2003). The activation of some MAPK family members by. M. tuberculosis H37Rv in human monocytes has already been reported. Song et al.

  20. Antioxidant activities of protein hydrolysates obtained from the housefly larvae.

    Science.gov (United States)

    Zhang, Huan; Wang, Pan; Zhang, Ai-Jun; Li, Xuan; Zhang, Ji-Hong; Qin, Qi-Lian; Wu, Yi-Jun

    2016-09-01

    The housefly is an important resource insect and the housefly larvae are ideal source of food additives. The housefly larvae protein hydrolysates were obtained by enzymatic hydrolysis by alcalase and neutral proteinase. Their antioxidant activities were investigated, including the superoxide and hydroxyl radicalscavenging activity, 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity, reducing power and metal chelating activity. The antioxidant activities of both hydrolysates increased with their increasing concentrations. The alcalase hydrolysate (AH) showed higher scavenging activities against hydroxyl radical and superoxide anion radical at low concentrations and higher metal-chelating activity than the neutral proteinase hydrolysate (NPH). The NPH exhibited higher scavenging activity against DPPH free radical and higher reducing power than the AH. Both hydrolysates showed more than 50% superoxide anion radical-scavenging activity at 10 μg/mL. These results indicate that both housefly larvae protein hydrolysates display high antioxidant activities and they could serve as potential natural antioxidant food additives.

  1. Modeling the SHG activities of diverse protein crystals

    International Nuclear Information System (INIS)

    Haupert, Levi M.; DeWalt, Emma L.; Simpson, Garth J.

    2012-01-01

    The origins of the diversity in the SHG signal from protein crystals are investigated and potential protein-crystal coverage by SHG microscopy is assessed. A symmetry-additive ab initio model for second-harmonic generation (SHG) activity of protein crystals was applied to assess the likely protein-crystal coverage of SHG microscopy. Calculations were performed for 250 proteins in nine point-group symmetries: a total of 2250 crystals. The model suggests that the crystal symmetry and the limit of detection of the instrument are expected to be the strongest predictors of coverage of the factors considered, which also included secondary-structural content and protein size. Much of the diversity in SHG activity is expected to arise primarily from the variability in the intrinsic protein response as well as the orientation within the crystal lattice. Two or more orders-of-magnitude variation in intensity are expected even within protein crystals of the same symmetry. SHG measurements of tetragonal lysozyme crystals confirmed detection, from which a protein coverage of ∼84% was estimated based on the proportion of proteins calculated to produce SHG responses greater than that of tetragonal lysozyme. Good agreement was observed between the measured and calculated ratios of the SHG intensity from lysozyme in tetragonal and monoclinic lattices

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

    African Journals Online (AJOL)

    Hiessu

    2016-02-24

    Feb 24, 2016 ... 2F2 showed IC50 values of 67.46 µg/ml on HeLa cells. These results suggest that protein extracts from. C. fluctifraga might be potential anticancer agents. Key words: Protein extracts, clam, antiproliferative activity, breast cancer, cervical cancer. INTRODUCTION. Cancer is a worldwide health issue; it is not ...

  3. The contact activation proteins: a structure/function overview

    NARCIS (Netherlands)

    Meijers, J. C.; McMullen, B. A.; Bouma, B. N.

    1992-01-01

    In recent years, extensive knowledge has been obtained on the structure/function relationships of blood coagulation proteins. In this overview, we present recent developments on the structure/function relationships of the contact activation proteins: factor XII, high molecular weight kininogen,

  4. Plasma Renin Activity in Children with Protein Energy Malnutrition ...

    African Journals Online (AJOL)

    renin activity was significantly increased in children with kwashiorkor; and also that the activity was significantly higher in the patients who subsequently died in hospital, than in those who survived. Increased renin activity probably contributes to the retention of water characteris- tic of protein energy malnutrition. 5. Afr. Med.

  5. Adenosine monophosphate-activated protein kinase from the mud ...

    Indian Academy of Sciences (India)

    2016-12-01

    Hochachka and Somero 2002). Therefore, some animals have to initiate anaerobic metabolism to meet part of energy needs (Costanzo et al. 2004; Colson-Proch et al. 2009). Adenosine monophosphate-activated protein kinase.

  6. Stress-induced activation of protein kinase CK2 by direct interaction with p38 mitogen-activated protein kinase

    DEFF Research Database (Denmark)

    Sayed, M; Kim, S O; Salh, B S

    2000-01-01

    Protein kinase CK2 has been implicated in the regulation of a wide range of proteins that are important in cell proliferation and differentiation. Here we demonstrate that the stress signaling agents anisomycin, arsenite, and tumor necrosis factor-alpha stimulate the specific enzyme activity of CK2...... to be an allosteric mechanism. Furthermore, we demonstrate that anisomycin- and tumor necrosis factor-alpha-induced phosphorylation of p53 at Ser-392, which is important for the transcriptional activity of this growth suppressor protein, requires p38 MAP kinase and CK2 activities....

  7. Gamma irradiation effect on soy protein modification, protein - phenolic interaction and antioxidant activity in soybean

    International Nuclear Information System (INIS)

    Kumari, Sweta; Dahuja, Anil; Vinutha, T.; Singh, Bhupinder

    2014-01-01

    Soy protein is one of the most important sources of protein to feed the world population in the future. Consumption of soybean quality protein and their texture is dependent on the protein modification. In the present study, four soybean genotypes PL5039 (black), EC 472143 (black), Pusa 9814 (yellow) and SL525 (yellow), differing in their seed coat colour were gamma irradiated at 0.5,1.0, 2.0 and 5.0 kGy and the extent of protein modification and parameters affecting it viz. free phenolics, bound phenolics, lip oxygenase and antioxidant activity were analysed. Modifications of soybean proteins were investigated by chemical analysis and electrophoresis. The irradiation dose of 1.0 kGy showed decreased turbidity, protein oxidation, surface hydrophobicity but increased solubility and sulfhydryl and disulfide contents in all the genotypes. Further, SDS PAGE profile of treated soybean seeds revealed remarkable difference in electrophoretic bands as compared to the untreated seeds. Lipoxygense activity in all the genotypes decreased with increased exposure of gamma irradiation, which produced peroxide products that changes the structural characteristics of soy protein. Free phenolics, bound phenolics and total antioxidant activity measured in terms of FRAP in all the genotypes increased significantly at a dose of 2.0 kGy and it declined at a dose of 5.0 kGy. Antioxidant potential measured in terms of 1,1-diphenyl-2- picrylhydrazyl (DPPH) scavenging activity showed an increasing trend with dose, indicating that radiation processing as a method of food preservation has a positive nutritional implication. Hence, it is suggested that, mild gamma irradiation upto 2.0 kGy may reduce the protein oxidation, enhance the antioxidant activity and improve the soybean protein quality compared to higher dose 5.0 kGy, which reduced the protein quality. (author)

  8. Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins

    Energy Technology Data Exchange (ETDEWEB)

    Nayak, Jaladhi; Gastonguay, Adam J.; Talipov, Marat R.; Vakeel, Padmanabhan; Span, Elise A.; Kalous, Kelsey S.; Kutty, Raman G.; Jensen, Davin R.; Pokkuluri, Phani Raj; Sem, Daniel S.; Rathore, Rajendra; Ramchandran, Ramani

    2014-12-01

    Background: The mitogen-activated protein kinases (MAPKs) pathway is critical for cellular signaling, and proteins such as phosphatases that regulate this pathway are important for normal tissue development. Based on our previous work on dual specificity phosphatase-5 (DUSP5), and its role in embryonic vascular development and disease, we hypothesized that mutations in DUSP5 will affect its function. Results: In this study, we tested this hypothesis by generating full-length glutathione-S-transferase-tagged DUSP5 and serine 147 proline mutant (S147P) proteins from bacteria. Light scattering analysis, circular dichroism, enzymatic assays and molecular modeling approaches have been performed to extensively characterize the protein form and function. We demonstrate that both proteins are active and, interestingly, the S147P protein is hypoactive as compared to the DUSP5 WT protein in two distinct biochemical substrate assays. Furthermore, due to the novel positioning of the S147P mutation, we utilize computational modeling to reconstruct full-length DUSP5 and S147P to predict a possible mechanism for the reduced activity of S147P. Conclusion: Taken together, this is the first evidence of the generation and characterization of an active, full-length, mutant DUSP5 protein which will facilitate future structure-function and drug development-based studies.

  9. Measurement of GPCR-G protein activity in living cells.

    Science.gov (United States)

    Ratnayake, Kasun; Kankanamge, Dinesh; Senarath, Kanishka; Siripurapu, Praneeth; Weis, Nicole; Tennakoon, Mithila; Payton, John L; Karunarathne, Ajith

    2017-01-01

    G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors in eukaryotic genomes. They control a variety of cellular and physiological processes such as hormone secretion and heart rate, and therefore are associated with a majority of pathological conditions including cancer and heart diseases. Currently established assays to measure ligand-induced activation of GPCRs and G proteins possess limitations such as being time consuming, indirect, and expensive. Thus, an efficient method to measure GPCR-G protein activation is required to identify novel pharmacological modulators to control them and gain insights about molecular underpinnings of the associated pathways. Activation of GPCRs induces dissociation of G protein heterotrimers to form GαGTP and free Gβγ. Free Gβγ subunits have been shown to translocate reversibly from the plasma membrane to internal membranes. Gβγ translocation therefore represents the GPCR-G protein activation, and thus, imaging of this process can be used to quantify the kinetics and magnitude of the pathway activation-deactivation in real time in living cells. The objective of this chapter is to elaborate the protocols of (i) generation and optimization of the required sensor constructs; (ii) development of cell culture, transient transfection, imaging, and optogenetic procedures; (iii) imaging and data analysis methods; and (iv) stable cell line generation, pertaining to this assay to measure GPCR-G protein activation. © 2017 Elsevier Inc. All rights reserved.

  10. Dissociation of activated protein C functions by elimination of protein S cofactor enhancement.

    LENUS (Irish Health Repository)

    Harmon, Shona

    2008-11-07

    Activated protein C (APC) plays a critical anticoagulant role in vivo by inactivating procoagulant factor Va and factor VIIIa and thus down-regulating thrombin generation. In addition, APC bound to the endothelial cell protein C receptor can initiate protease-activated receptor-1 (PAR-1)-mediated cytoprotective signaling. Protein S constitutes a critical cofactor for the anticoagulant function of APC but is not known to be involved in regulating APC-mediated protective PAR-1 signaling. In this study we utilized a site-directed mutagenesis strategy to characterize a putative protein S binding region within the APC Gla domain. Three single amino acid substitutions within the APC Gla domain (D35T, D36A, and A39V) were found to mildly impair protein S-dependent anticoagulant activity (<2-fold) but retained entirely normal cytoprotective activity. However, a single amino acid substitution (L38D) ablated the ability of protein S to function as a cofactor for this APC variant. Consequently, in assays of protein S-dependent factor Va proteolysis using purified proteins or in the plasma milieu, APC-L38D variant exhibited minimal residual anticoagulant activity compared with wild type APC. Despite the location of Leu-38 in the Gla domain, APC-L38D interacted normally with endothelial cell protein C receptor and retained its ability to trigger PAR-1 mediated cytoprotective signaling in a manner indistinguishable from that of wild type APC. Consequently, elimination of protein S cofactor enhancement of APC anticoagulant function represents a novel and effective strategy by which to separate the anticoagulant and cytoprotective functions of APC for potential therapeutic gain.

  11. Depletion of WRN protein causes RACK1 to activate several protein kinase C isoforms

    DEFF Research Database (Denmark)

    Massip, L; Garand, C; Labbé, A

    2010-01-01

    Werner's syndrome (WS) is a rare autosomal disease characterized by the premature onset of several age-associated pathologies. The protein defective in patients with WS (WRN) is a helicase/exonuclease involved in DNA repair, replication, transcription and telomere maintenance. In this study, we...... show that a knock down of the WRN protein in normal human fibroblasts induces phosphorylation and activation of several protein kinase C (PKC) enzymes. Using a tandem affinity purification strategy, we found that WRN physically and functionally interacts with receptor for activated C-kinase 1 (RACK1......), a highly conserved anchoring protein involved in various biological processes, such as cell growth and proliferation. RACK1 binds strongly to the RQC domain of WRN and weakly to its acidic repeat region. Purified RACK1 has no impact on the helicase activity of WRN, but selectively inhibits WRN exonuclease...

  12. New constitutive latex osmotin-like proteins lacking antifungal activity.

    Science.gov (United States)

    Freitas, Cleverson D T; Silva, Maria Z R; Bruno-Moreno, Frederico; Monteiro-Moreira, Ana C O; Moreira, Renato A; Ramos, Márcio V

    2015-11-01

    Proteins that share similar primary sequences to the protein originally described in salt-stressed tobacco cells have been named osmotins. So far, only two osmotin-like proteins were purified and characterized of latex fluids. Osmotin from Carica papaya latex is an inducible protein lacking antifungal activity, whereas the Calotropis procera latex osmotin is a constitutive antifungal protein. To get additional insights into this subject, we investigated osmotins in latex fluids of five species. Two potential osmotin-like proteins in Cryptostegia grandiflora and Plumeria rubra latex were detected by immunological cross-reactivity with polyclonal antibodies produced against the C. procera latex osmotin (CpOsm) by ELISA, Dot Blot and Western Blot assays. Osmotin-like proteins were not detected in the latex of Thevetia peruviana, Himatanthus drasticus and healthy Carica papaya fruits. Later, the two new osmotin-like proteins were purified through immunoaffinity chromatography with anti-CpOsm immobilized antibodies. Worth noting the chromatographic efficiency allowed for the purification of the osmotin-like protein belonging to H. drasticus latex, which was not detectable by immunoassays. The identification of the purified proteins was confirmed after MS/MS analyses of their tryptic digests. It is concluded that the constitutive osmotin-like proteins reported here share structural similarities to CpOsm. However, unlike CpOsm, they did not exhibit antifungal activity against Fusarium solani and Colletotrichum gloeosporioides. These results suggest that osmotins of different latex sources may be involved in distinct physiological or defensive events. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  13. Phospholipid transfer protein activity and incident type 2 diabetes mellitus

    NARCIS (Netherlands)

    Abbasi, Ali; Dallinga-Thie, Geesje M.; Dullaart, Robin P. F.

    2015-01-01

    The plasma activity of phospholipid transfer protein (PLTP), which has multifaceted functions in lipoprotein metabolism and in inflammatory responses, is elevated in insulin resistant conditions. We determined the association of plasma PLTP activity with incident type 2 diabetes mellitus (T2DM).

  14. Energy transfer at the active sites of heme proteins

    International Nuclear Information System (INIS)

    Dlott, D.D.; Hill, J.R.

    1995-01-01

    Experiments using a picosecond pump-probe apparatus at the Picosecond Free-electron Laser Center at Stanford University, were performed to investigate the relaxation of carbon monoxide bound to the active sites of heme proteins. The significance of these experiments is two-fold: (1) they provide detailed information about molecular dynamics occurring at the active sites of proteins; and (2) they provide insight into the nature of vibrational relaxation processes in condensed matter. Molecular engineering is used to construct various molecular systems which are studied with the FEL. We have studied native proteins, mainly myoglobin obtained from different species, mutant proteins produced by genetic engineering using recombinant DNA techniques, and a variety of model systems which mimic the structures of the active sites of native proteins, which are produced using molecular synthesis. Use of these different systems permits us to investigate how specific molecular structural changes affect dynamical processes occurring at the active sites. This research provides insight into the problems of how different species needs are fulfilled by heme proteins which have greatly different functionality, which is induced by rather small structural changes

  15. Activated entomopathogenic nematode infective juveniles release lethal venom proteins.

    Directory of Open Access Journals (Sweden)

    Dihong Lu

    2017-04-01

    Full Text Available Entomopathogenic nematodes (EPNs are unique parasites due to their symbiosis with entomopathogenic bacteria and their ability to kill insect hosts quickly after infection. It is widely believed that EPNs rely on their bacterial partners for killing hosts. Here we disproved this theory by demonstrating that the in vitro activated infective juveniles (IJs of Steinernema carpocapsae (a well-studied EPN species release venom proteins that are lethal to several insects including Drosophila melanogaster. We confirmed that the in vitro activation is a good approximation of the in vivo process by comparing the transcriptomes of individual in vitro and in vivo activated IJs. We further analyzed the transcriptomes of non-activated and activated IJs and revealed a dramatic shift in gene expression during IJ activation. We also analyzed the venom proteome using mass spectrometry. Among the 472 venom proteins, proteases and protease inhibitors are especially abundant, and toxin-related proteins such as Shk domain-containing proteins and fatty acid- and retinol-binding proteins are also detected, which are potential candidates for suppressing the host immune system. Many of the venom proteins have conserved orthologs in vertebrate-parasitic nematodes and are differentially expressed during IJ activation, suggesting conserved functions in nematode parasitism. In summary, our findings strongly support a new model that S. carpocapsae and likely other Steinernema EPNs have a more active role in contributing to the pathogenicity of the nematode-bacterium complex than simply relying on their symbiotic bacteria. Furthermore, we propose that EPNs are a good model system for investigating vertebrate- and human-parasitic nematodes, especially regarding the function of excretory/secretory products.

  16. Activation of repair and checkpoints by double-strand breaks of DNA. Activational cascade of protein phosphorylation

    International Nuclear Information System (INIS)

    Koltovaya, N.A.

    2007-01-01

    Molecular mechanisms of double-strand breaks repair and checkpoints include phosphorylations of repair and checkpoint-proteins by protein kinases. Chemical modification of proteins has different consequences including activation, changing of affinity to proteins and localization

  17. The functional significance of the autolysis loop in protein C and activated protein C.

    Science.gov (United States)

    Yang, Likui; Manithody, Chandrashekhara; Rezaie, Alireza R

    2005-07-01

    The autolysis loop of activated protein C (APC) is five residues longer than the autolysis loop of other vitamin K-dependent coagulation proteases. To investigate the role of this loop in the zymogenic and anticoagulant properties of the molecule, a protein C mutant was constructed in which the autolysis loop of the protein was replaced with the corresponding loop of factor X. The protein C mutant was activated by thrombin with approximately 5-fold higher rate in the presence of Ca2+. Both kinetics and direct binding studies revealed that the Ca2+ affinity of the mutant has been impaired approximately 3-fold. The result of a factor Va degradation assay revealed that the anticoagulant function of the mutant has been improved 4-5-fold in the absence but not in the presence of protein S. The improvement was due to a better recognition of both the P1-Arg506 and P1-Arg306 cleavage sites by the mutant protease. However, the plasma half-life of the mutant was markedly shortened due to faster inactivation by plasma serpins. These results suggest that the autolysis loop of protein C is critical for the Ca(2+)-dependence of activation by thrombin. Moreover, a longer autolysis loop in APC is not optimal for interaction with factor Va in the absence of protein S, but it contributes to the lack of serpin reactivity and longer half-life of the protease in plasma.

  18. Forkhead box protein A2 (FOXA2 protein stability and activity are regulated by sumoylation.

    Directory of Open Access Journals (Sweden)

    Narasimhaswamy S Belaguli

    Full Text Available The forkhead box protein A2 (FOXA2 is an important regulator of glucose and lipid metabolism and organismal energy balance. Little is known about how FOXA2 protein expression and activity are regulated by post-translational modifications. We have identified that FOXA2 is post-translationally modified by covalent attachment of a small ubiquitin related modifier-1 (SUMO-1 and mapped the sumoylation site to the amino acid lysine 6 (K6. Preventing sumoylation by mutating the SUMO acceptor K6 to arginine resulted in downregulation of FOXA2 protein but not RNA expression in INS-1E insulinoma cells. K6R mutation also downregulated FOXA2 protein levels in HepG2 hepatocellular carcinoma cells, HCT116 colon cancer cells and LNCaP and DU145 prostate cancer cells. Further, interfering with FOXA2 sumoylation through siRNA mediated knockdown of UBC9, an essential SUMO E2 conjugase, resulted in downregulation of FOXA2 protein levels. Stability of sumoylation deficient FOXA2K6R mutant protein was restored when SUMO-1 was fused in-frame. FOXA2 sumoylation and FOXA2 protein levels were increased by PIAS1 SUMO ligase but not a SUMO ligase activity deficient PIAS1 mutant. Although expressed at lower levels, sumoylation deficient FOXA2K6R mutant protein was detectable in the nucleus indicating that FOXA2 nuclear localization is independent of sumoylation. Sumoylation increased the transcriptional activity of FOXA2 on Pdx-1 area I enhancer. Together, our results show that sumoylation regulates FOXA2 protein expression and activity.

  19. Forkhead box protein A2 (FOXA2) protein stability and activity are regulated by sumoylation.

    Science.gov (United States)

    Belaguli, Narasimhaswamy S; Zhang, Mao; Brunicardi, F Charles; Berger, David H

    2012-01-01

    The forkhead box protein A2 (FOXA2) is an important regulator of glucose and lipid metabolism and organismal energy balance. Little is known about how FOXA2 protein expression and activity are regulated by post-translational modifications. We have identified that FOXA2 is post-translationally modified by covalent attachment of a small ubiquitin related modifier-1 (SUMO-1) and mapped the sumoylation site to the amino acid lysine 6 (K6). Preventing sumoylation by mutating the SUMO acceptor K6 to arginine resulted in downregulation of FOXA2 protein but not RNA expression in INS-1E insulinoma cells. K6R mutation also downregulated FOXA2 protein levels in HepG2 hepatocellular carcinoma cells, HCT116 colon cancer cells and LNCaP and DU145 prostate cancer cells. Further, interfering with FOXA2 sumoylation through siRNA mediated knockdown of UBC9, an essential SUMO E2 conjugase, resulted in downregulation of FOXA2 protein levels. Stability of sumoylation deficient FOXA2K6R mutant protein was restored when SUMO-1 was fused in-frame. FOXA2 sumoylation and FOXA2 protein levels were increased by PIAS1 SUMO ligase but not a SUMO ligase activity deficient PIAS1 mutant. Although expressed at lower levels, sumoylation deficient FOXA2K6R mutant protein was detectable in the nucleus indicating that FOXA2 nuclear localization is independent of sumoylation. Sumoylation increased the transcriptional activity of FOXA2 on Pdx-1 area I enhancer. Together, our results show that sumoylation regulates FOXA2 protein expression and activity.

  20. In vitro study of proteins surface activity by tritium probe

    International Nuclear Information System (INIS)

    Chernysheva, M.G.; Badun, G.A.

    2010-01-01

    A new technique for in vitro studies of biomacromolecules interactions, their adsorption at aqueous/organic liquid interfaces and distribution in the bulk of liquid/liquid systems was developed. The method includes (1) tritium labeling of biomolecules by tritium thermal activation method and (2) scintillation phase step with organic phase, which can be concerned as a model of cellular membrane. Two globular proteins lysozyme and human serum albumin tested. We have determined the conditions of tritium labeling when labeled by-products can be easy separated by means of dialysis and size-exclusion chromatography. Scintillation phase experiments were conducted for three types of organic liquids. Thus, the influences of the nature of organic phase on proteins adsorption and its distribution in the bulk of aqueous/organic liquid system were determined. It was found that proteins possess high surface activity at aqueous/organic liquid interface. Furthermore, values of hydrophobicity of globular proteins were found by the experiment. (author)

  1. [Activity of polygalacturonase-inhibiting protein of banana fruit tissues].

    Science.gov (United States)

    Bulantseva, E A; Thang, Nguen; Buza, N L; Krinitsyna, A A; Protsenko, M A

    2005-01-01

    The activity of polygalacturonase and the protein inhibiting this enzyme, which affected polygalacturonases of phytopathogenic fungi Verticillium dahliae and Gloesporium musarium, were detected in banana (Musa acumthata L.) fruit of cultivars Cavendish and Korolevskii. The polygalacturonase from banana fruit was inhibited by the preparations of the protein inhibitor not only from bananas but also from potato (Solanum tuberosum L.) tubers and pepper (Capsicum annuum L.) fruit.

  2. Numerical prediction of micro-channel LD heat sink operated with antifreeze based on CFD method

    Science.gov (United States)

    Liu, Gang; Liu, Yang; Wang, Chao; Wang, Wentao; Wang, Gang; Tang, Xiaojun

    2014-12-01

    To theoretically study the feasibility of antifreeze coolants applied as cooling fluids for high power LD heat sink, detailed Computational Fluid Dynamics (CFD) analysis of liquid cooled micro-channels heat sinks is presented. The performance operated with antifreeze coolant (ethylene glycol aqueous solution) compared with pure water are numerical calculated for the heat sinks with the same micro-channels structures. The maximum thermal resistance, total pressure loss (flow resistance), thermal resistance vs. flow-rate, and pressure loss vs. flow-rate etc. characteristics are numerical calculated. The results indicate that the type and temperature of coolants plays an important role on the performance of heat sinks. The whole thermal resistance and pressure loss of heat sinks increase significantly with antifreeze coolants compared with pure water mainly due to its relatively lower thermal conductivity and higher fluid viscosity. The thermal resistance and pressure loss are functions of the flow rate and operation temperature. Increasing of the coolant flow rate can reduce the thermal resistance of heat sinks; meanwhile increase the pressure loss significantly. The thermal resistance tends to a limit with increasing flow rate, while the pressure loss tends to increase exponentially with increasing flow rate. Low operation temperature chiefly increases the pressure loss rather than thermal resistance due to the remarkable increasing of fluid viscosity. The actual working point of the cooling circulation system can be determined on the basis of the pressure drop vs. flow rate curve for the micro-channel heat sink and that for the circulation system. In the same system, if the type or/and temperature of the coolant is changed, the working point is accordingly influenced, that is, working flow rate and pressure is changed simultaneously, due to which the heat sink performance is influenced. According to the numerical simulation results, if ethylene glycol aqueous

  3. Beyond the zone: protein needs of active individuals.

    Science.gov (United States)

    Lemon, P W

    2000-10-01

    There has been debate among athletes and nutritionists regarding dietary protein needs for centuries. Although contrary to traditional belief, recent scientific information collected on physically active individuals tends to indicate that regular exercise increases daily protein requirements; however, the precise details remain to be worked out. Based on laboratory measures, daily protein requirements are increased by perhaps as much as 100% vs. recommendations for sedentary individuals (1.6-1.8 vs. 0.8 g/kg). Yet even these intakes are much less than those reported by most athletes. This may mean that actual requirements are below what is needed to optimize athletic performance, and so the debate continues. Numerous interacting factors including energy intake, carbohydrate availability, exercise intensity, duration and type, dietary protein quality, training history, gender, age, timing of nutrient intake and the like make this topic extremely complex. Many questions remain to be resolved. At the present time, substantial data indicate that the current recommended protein intake should be adjusted upward for those who are physically active, especially in populations whose needs are elevated for other reasons, e.g., growing individuals, dieters, vegetarians, individuals with muscle disease-induced weakness and the elderly. For these latter groups, specific supplementation may be appropriate, but for most North Americans who consume a varied diet, including complete protein foods (meat, eggs, fish and dairy products), and sufficient energy the increased protein needs induced by a regular exercise program can be met in one's diet.

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

    Science.gov (United States)

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

    2018-03-20

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

  5. Emerging Roles of AMP-Activated Protein Kinase

    DEFF Research Database (Denmark)

    Fritzen, Andreas Mæchel

    The cellular energy sensor AMP-activated protein kinase (AMPK) is activated, when the energy balance of the cell decreases. AMPK has been proposed to regulate multiple metabolic processes. However, much of the evidence for these general effects of AMPK relies on investigations in cell systems...... exercise appears to inhibit pyruvate dehydrogenase (PDH) activity by an immediate up-regulation of pyruvate dehydrogenase kinase 4 (PDK4) protein content. Consequently, this may inhibit glucose oxidation and thereby generate conditions for increased FA oxidation and glycogen resynthesis in skeletal muscle...... be of importance for prioritising energy dissipation, inhibition of lipid storage pathways and regulation of mitochondrial and metabolic proteins, but this needs further investigations. In addition, we provide evidence that AMPK is regulating autophagic signalling in skeletal muscle. Thus, in skeletal muscle AMPK...

  6. Organizers and activators: Cytosolic Nox proteins impacting on vascular function.

    Science.gov (United States)

    Schröder, Katrin; Weissmann, Norbert; Brandes, Ralf P

    2017-08-01

    NADPH oxidases of the Nox family are important enzymatic sources of reactive oxygen species (ROS) in the cardiovascular system. Of the 7 members of the Nox family, at least three depend for their activation on specific cytosolic proteins. These are p47phox and its homologue NoxO1 and p67phox and its homologue NoxA1. Also the Rho-GTPase Rac is important but as this protein has many additional functions, it will not be covered here. The Nox1 enzyme is preferentially activated by the combination of NoxO1 with NoxA1, whereas Nox2 gains highest activity with p47phox together with p67phox. As p47phox, different to NoxO1 contains an auto inhibitory region it has to be phosphorylated prior to complex formation. In the cardio-vascular system, all cytosolic Nox proteins are expressed but the evidence for their contribution to ROS production is not well established. Most data have been collected for p47phox, whereas NoxA1 has basically not yet been studied. In this article the specific aspects of cytosolic Nox proteins in the cardiovascular system with respect to Nox activation, their expression and their importance will be reviewed. Finally, it will be discussed whether cytosolic Nox proteins are suitable pharmacological targets to tamper with vascular ROS production. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  7. Outer Membrane Protein 25 of Brucella Activates Mitogen-Activated Protein Kinase Signal Pathway in Human Trophoblast Cells

    Directory of Open Access Journals (Sweden)

    Jing Zhang

    2017-12-01

    Full Text Available Outer membrane protein 25 (OMP25, a virulence factor from Brucella, plays an important role in maintaining the structural stability of Brucella. Mitogen-activated protein kinase (MAPK signal pathway widely exists in eukaryotic cells. In this study, human trophoblast cell line HPT-8 and BALB/c mice were infected with Brucella abortus 2308 strain (S2308 and 2308ΔOmp25 mutant strain. The expression of cytokines and activation of MAPK signal pathway were detected. We found that the expressions of tumor necrosis factor-α, interleukin-1, and interleukin-10 (IL-10 were increased in HPT-8 cells infected with S2308 and 2308ΔOmp25 mutant. S2308 also activated p38 phosphorylation protein, extracellular-regulated protein kinases (ERK, and Jun-N-terminal kinase (JNK from MAPK signal pathway. 2308ΔOmp25 could not activate p38, ERK, and JNK branches. Immunohistochemistry experiments showed that S2308 was able to activate phosphorylation of p38 and ERK in BABL/c mice. However, 2308ΔOmp25 could weakly activate phosphorylation of p38 and ERK. These results suggest that Omp25 played an important role in the process of Brucella activation of the MAPK signal pathway.

  8. Chaperone activity of human small heat shock protein-GST fusion proteins.

    Science.gov (United States)

    Arbach, Hannah; Butler, Caley; McMenimen, Kathryn A

    2017-07-01

    Small heat shock proteins (sHsps) are a ubiquitous part of the machinery that maintains cellular protein homeostasis by acting as molecular chaperones. sHsps bind to and prevent the aggregation of partially folded substrate proteins in an ATP-independent manner. sHsps are dynamic, forming an ensemble of structures from dimers to large oligomers through concentration-dependent equilibrium dissociation. Based on structural studies and mutagenesis experiments, it is proposed that the dimer is the smallest active chaperone unit, while larger oligomers may act as storage depots for sHsps or play additional roles in chaperone function. The complexity and dynamic nature of their structural organization has made elucidation of their chaperone function challenging. HspB1 and HspB5 are two canonical human sHsps that vary in sequence and are expressed in a wide variety of tissues. In order to determine the role of the dimer in chaperone activity, glutathione-S-transferase (GST) was genetically linked as a fusion protein to the N-terminus regions of both HspB1 and HspB5 (also known as Hsp27 and αB-crystallin, respectively) proteins in order to constrain oligomer formation of HspB1 and HspB5, by using GST, since it readily forms a dimeric structure. We monitored the chaperone activity of these fusion proteins, which suggest they primarily form dimers and monomers and function as active molecular chaperones. Furthermore, the two different fusion proteins exhibit different chaperone activity for two model substrate proteins, citrate synthase (CS) and malate dehydrogenase (MDH). GST-HspB1 prevents more aggregation of MDH compared to GST-HspB5 and wild type HspB1. However, when CS is the substrate, both GST-HspB1 and GST-HspB5 are equally effective chaperones. Furthermore, wild type proteins do not display equal activity toward the substrates, suggesting that each sHsp exhibits different substrate specificity. Thus, substrate specificity, as described here for full-length GST

  9. Nanomaterials for efficiently lowering the freezing point of anti-freeze coolants.

    Science.gov (United States)

    Hong, Haiping; Zheng, Yingsong; Roy, Walter

    2007-09-01

    In this paper, we report, for the first time, the effect of the lowered freezing point in a 50% water/50% anti-freeze coolant (PAC) or 50% water/50% ethylene glycol (EG) solution by the addition of carbon nanotubes and other particles. The experimental results indicated that the nano materials are much more efficient (hundreds fold) in lowering the freezing point than the regular ionic materials (e.g., NaCl). The possible explanation for this interesting phenomenon is the colligative property of fluid and relative small size of nano material. It is quite certain that the carbon nanotubes and metal oxide nano particles could be a wonderful candidate for the nano coolant application because they could not only increase the thermal conductivity, but also efficiently lower the freezing point of traditional coolants.

  10. Detergent activation of the binding protein in the folate radioassay

    International Nuclear Information System (INIS)

    Hansen, S.I.; Holm, J.; Lyngbye, J.

    1982-01-01

    A minor cow's whey protein associated with β-lactoglobulin is used as binding protein in the competitive radioassay for serum and erythrocyte folate. Seeking to optimize the assay, we tested the performance of binder solutions of increasing purity. The folate binding protein was isolated from cow's whey by means of CM-Sepharose CL-6B cation-exchange chromatography, and further purified on a methotrexate-AH-Sepharose 4B affinity matrix. In contrast to β-lactoglobulin, the purified protein did not bind folate unless the detergents cetyltrimethylammonium (10 mmol/Ll) or Triton X-100 (1 g/L) were present. Such detergent activation was not needed in the presence of serum. There seems to be a striking analogy between these phenomena and the well-known reactivation of certain purified membrane-derived enzymes by surfactants

  11. Hepatic protein synthetic activity in vivo after ethanol administration

    International Nuclear Information System (INIS)

    Donohue, T.M. Jr.; Sorrell, M.F.; Tuma, D.J.

    1987-01-01

    Hepatic protein synthetic activity in vivo was measured by the incorporation of [ 3 H]puromycin into elongating nascent polypeptides of rat liver to form peptidyl-[ 3 H]puromycin. Our initial experiments showed that saturating doses of [ 3 H]puromycin were achieved at 3-6 mumol/100 g body weight, and that maximum labeling of nascent polypeptides was obtained 30 min after injection of the labeled precursor. Labeled puromycin was found to be suitable for measuring changes in the status of protein synthesis, since the formation of the peptidyl-[ 3 H]puromycin was decreased in fasted animals and was increased in rats pretreated with L-tryptophan. [ 3 H]Puromycin incorporation into polypeptides was then measured after acute ethanol administration as well as after prolonged consumption of ethanol which was administered as part of a liquid diet for 31 days. Acute alcohol treatment caused no significant change in [ 3 H]puromycin incorporation into liver polypeptides. In rats exposed to chronic ethanol feeding, peptidyl-[3H]puromycin formation, when expressed per mg of protein, was slightly lower compared to pair-fed controls, but was unchanged compared to chow-fed animals. When the data were expressed per mg of DNA or per 100 g body wt, no differences in protein synthetic activity were observed among the three groups. These findings indicate that neither acute nor chronic alcohol administration significantly affects protein synthetic activity in rat liver. They further suggest that accumulation of protein in the liver, usually seen after prolonged ethanol consumption, is apparently not reflected by an alteration of hepatic protein synthesis

  12. Auxin efflux by PIN-FORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID

    KAUST Repository

    Zourelidou, Melina

    2014-06-19

    The development and morphology of vascular plants is critically determined by synthesis and proper distribution of the phytohormone auxin. The directed cell-to-cell distribution of auxin is achieved through a system of auxin influx and efflux transporters. PIN-FORMED (PIN) proteins are proposed auxin efflux transporters, and auxin fluxes can seemingly be predicted based on the-in many cells-asymmetric plasma membrane distribution of PINs. Here, we show in a heterologous Xenopus oocyte system as well as in Arabidopsis thaliana inflorescence stems that PIN-mediated auxin transport is directly activated by D6 PROTEIN KINASE (D6PK) and PINOID (PID)/WAG kinases of the Arabidopsis AGCVIII kinase family. At the same time, we reveal that D6PKs and PID have differential phosphosite preferences. Our study suggests that PIN activation by protein kinases is a crucial component of auxin transport control that must be taken into account to understand auxin distribution within the plant.

  13. GTPase-activating protein oligophrenin 1 is a new partner of multifunctional adapter protein intersectin 1

    Directory of Open Access Journals (Sweden)

    Gasman S.

    2012-09-01

    Full Text Available Intersectin 1 (ITSN1 is a multifunctional adaptor protein which is involved in endocytosis, exocytosis and cellular signaling and it is also associated with such pathologies as Down syndrome and Alzheimer’s disease. The aim of this study was to identify new ITSN1 protein partners which are implicated in membrane trafficking. Methods. In silico analysis by Scansite online resource had identified a GTPase activating protein oligophrenin 1 (OPHN1 as a potential partner of ITSN1 SH3A domain. GST pull-down and immunoprecipitation were used to prove complex formation between ITSN1 and OPHN1. Subcellular protein localization was determined by immunofluorescence and confocal microscopy. Results. We have shown that brain-specific and ubiquitously expressed SH3A domain isoforms of ITSN1 interact with OPHN1. ITSN1 and OPHN1 form complexes in both resting and stimulated to exocytosis PC12 cell line. Conclusions. GTPase activating protein OPHN1 and adaptor protein ITSN1 interact in PC12 cell line independently of exocytosis stimulation.

  14. Increased Prevalence of Activated Protein C Resistance During ...

    African Journals Online (AJOL)

    centrifuged this time at 3000 gravitational under identical conditions. The resulting platelet poor plasma (PPP) was either tested immediately or stored in aliquots at in a -80oC freezer until testing. Protein C activity was measured using a clot-based assay.

  15. Fibroblast activation protein (FAP) as a novel metabolic target

    DEFF Research Database (Denmark)

    Sánchez-Garrido, Miguel Angel; Habegger, Kirk M; Clemmensen, Christoffer

    2016-01-01

    OBJECTIVE: Fibroblast activation protein (FAP) is a serine protease belonging to a S9B prolyl oligopeptidase subfamily. This enzyme has been implicated in cancer development and recently reported to regulate degradation of FGF21, a potent metabolic hormone. Using a known FAP inhibitor, talabostat...

  16. VHH Activators and Inhibitors for Protein Kinase C Epsilon

    NARCIS (Netherlands)

    Summanen, M.M.I.

    2012-01-01

    Protein kinase C epsilon (PKCε), which is one of the novel PKC isozymes, is widely expressed throughout the body and has important roles in the function of the nervous, cardiovascular and immune systems. In order to better understand PKCε regulated pathways, isozyme specific activity modulators are

  17. Adenosine monophosphate-activated protein kinase from the mud ...

    Indian Academy of Sciences (India)

    2016-12-01

    Dec 1, 2016 ... ... Journal of Genetics; Volume 95; Issue 4. Adenosine monophosphate-activated protein kinase from the mud crab, Scylla paramamosain: cDNA cloning and profiles under cold stress. CHENCUI HUANG KUN YU HUIYANG HUANG HAIHUI YE. RESEARCH ARTICLE Volume 95 Issue 4 December 2016 pp ...

  18. Adenosine monophosphate-activated protein kinase from the mud ...

    Indian Academy of Sciences (India)

    CHENCUI HUANG

    Adenosine monophosphate-activated protein kinase from the mud crab, Scylla paramamosain: cDNA cloning and profiles under cold stress. CHENCUI HUANG1, KUN YU1, HUIYANG HUANG1,2 and HAIHUI YE1,2∗. 1College of Ocean and Earth Sciences, Xiamen University, Xiamen 361102, People's Republic of China.

  19. Adenosine monophosphate-activated protein kinase from the mud ...

    Indian Academy of Sciences (India)

    2016-12-01

    Dec 1, 2016 ... to the understanding of the molecular mechanism of acclimation to cold hardiness in S. paramamosain. [Huang C., Yu K., Huang H. and Ye H. 2016 Adenosine monophosphate-activated protein kinase from the mud crab, Scylla paramamosain: cDNA cloning and profiles under cold stress. J. Genet.

  20. Activity-Based Protein Profiling of Rhomboid Proteases in Liposomes

    Czech Academy of Sciences Publication Activity Database

    Wolf, E. V.; Seybold, M.; Hadravová, Romana; Stříšovský, Kvido; Verhelst, S. H. L.

    2015-01-01

    Roč. 16, č. 11 (2015), s. 1616-1621 ISSN 1439-4227 R&D Projects: GA MŠk(CZ) LK11206; GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : activity-based protein profiling * chemical probes * inhibitors * intramembrane proteases * liposomes Subject RIV: CE - Biochemistry Impact factor: 2.850, year: 2015

  1. The dopamine D2 receptor can directly recruit and activate GRK2 without G protein activation.

    Science.gov (United States)

    Pack, Thomas F; Orlen, Margo I; Ray, Caroline; Peterson, Sean M; Caron, Marc G

    2018-02-27

    The dopamine D2 receptor (D2R) is a G protein-coupled receptor (GPCR) that is critical for many central nervous system functions. The D2R carries out these functions by signaling through two transducers: G proteins and β-arrestins (βarrs). Selectively engaging either the G protein or βarr pathway may be a way to improve drugs targeting GPCRs. The current model of GPCR signal transduction posits a chain of events where G protein activation ultimately leads to βarr recruitment. GPCR kinases (GRKs), which are regulated by G proteins and whose kinase action facilitates βarr recruitment, bridge these pathways. Therefore βarr recruitment appears to be intimately tied to G protein activation via GRKs. Here, we sought to understand how GRK2 action at the D2R would be disrupted when G protein activation is eliminated and the effect of this on βarr recruitment. We used two recently developed biased D2R mutants that can preferentially interact either with G proteins or βarrs as well as a βarr-biased D2R ligand, UNC9994. With these functionally selective tools, we investigated the mechanism whereby the βarr-preferring D2R achieves βarr pathway activation in the complete absence of G protein activation. We describe how direct, G protein-independent recruitment of GRK2 drives interactions at the βarr-preferring D2R and also contributes to βarr recruitment at the WT D2R. Additionally, we found an additive interaction between the βarr-preferring D2R mutant and UNC9994. These results reveal that the D2R can directly recruit GRK2 without G protein activation and that this mechanism may have relevance to achieving βarr-biased signaling. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Salt bridge integrates GPCR activation with protein trafficking.

    Science.gov (United States)

    Janovick, Jo Ann; Conn, P Michael

    2010-03-02

    G protein-coupled receptors (GPCRs) play central roles in almost all physiological functions; mutations in GPCRs are responsible for more than 30 disorders. There is a great deal of information about GPCR structure but little information that directly relates structure to protein trafficking or to activation. The gonadotropin releasing hormone receptor, because of its small size among GPCRs, is amenable to preparation of mutants and was used in this study to establish the relation among a salt bridge, protein trafficking, and receptor activation. This bridge, between residues E(90) [located in transmembrane segment (TM) 2] and K(121) (TM3), is associated with correct trafficking to the plasma membrane. Agonists, but not antagonists, interact with residue K(121), and destabilize the TM2-TM3 association of the receptor in the plasma membrane. The hGnRHR mutant E(90)K has a broken salt bridge, which also destabilizes the TM2-TM3 association and is typically retained in the endoplasmic reticulum. We show that this mutant, if rescued to the plasma membrane by either of two different means, has constitutive activity and shows modified ligand specificity, revealing a role for the salt bridge in receptor activation, ligand specificity, trafficking, and structure. The data indicate that destabilizing the TM2-TM3 relation for receptor activation, while requiring an intact salt bridge for correct trafficking, provides a mechanism that protects the cell from plasma membrane expression of constitutive activity.

  3. Cholesterol-Lowering Activity of Tartary Buckwheat Protein.

    Science.gov (United States)

    Zhang, Chengnan; Zhang, Rui; Li, Yuk Man; Liang, Ning; Zhao, Yimin; Zhu, Hanyue; He, Zouyan; Liu, Jianhui; Hao, Wangjun; Jiao, Rui; Ma, Ka Ying; Chen, Zhen-Yu

    2017-03-08

    Previous research has shown that Tartary buckwheat flour is capable of reducing plasma cholesterol. The present study was to examine the effect of rutin and Tartary buckwheat protein on plasma total cholesterol (TC) in hypercholesterolemia hamsters. In the first animal experiment, 40 male hamsters were divided into four groups fed either the control diet or one of the three experimental diets containing 8.2 mmol rutin, 8.2 mmol quercetin, or 2.5 g kg -1 cholestyramine, respectively. Results showed that only cholestyramine but not rutin and its aglycone quercetin decreased plasma TC, which suggested that rutin was not the active ingredient responsible for plasma TC-lowering activity of Tartary buckwheat flour. In the second animal experiment, 45 male hamsters were divided into five groups fed either the control diet or one of the four experimental diets containing 24% Tartary buckwheat protein, 24% rice protein, 24% wheat protein, or 5 g kg -1 cholestyramine, respectively. Tartary buckwheat protein reduced plasma TC more effectively than cholestyramine (45% versus 37%), while rice and wheat proteins only reduced plasma TC by 10-13%. Tartary buckwheat protein caused 108% increase in the fecal excretion of total neutral sterols and 263% increase in the fecal excretion of total acidic sterols. real-time polymerase chain reaction and Western blotting analyses showed that Tartary buckwheat protein affected the gene expression of intestinal Niemann-Pick C1-like protein 1 (NPC1L1), acyl CoA:cholesterol acyltransferase 2 (ACAT2), and ATP binding cassette transporters 5 and 8 (ABCG5/8) in a down trend, whereas it increased the gene expression of hepatic cholesterol-7α -hydroxylase (CYP7A1). It was concluded that Tartary buckwheat protein was at least one of the active ingredients in Tartary buckwheat flour to lower plasma TC, mainly mediated by enhancing the excretion of bile acids via up-regulation of hepatic CYP7A1 and also by inhibiting the absorption of dietary

  4. Acupuncture upregulates G protein coupled activity in SAMP8 mice.

    Science.gov (United States)

    Luo, Benhua; Zhao, Lan; Zhang, Xuezhu; Kan, Bohong; Liu, Yunhe; Jia, Yujie; Han, Jingxian; Yu, Jianchun

    2017-08-01

    Transmembrane and intracellular signal transduction of G protein is closely related to the pathophysiology of Alzheimer's disease (AD). To explore the effects of Sanjiao acupuncture on G protein signal transduction pathways in the pathogenesis of AD. 36 senescence-accelerated (SAM) prone 8 mice were divided into three groups that remained untreated (SAMP8, n=12) or received Sanjiao acupuncture (SAMP8+SA, n=12) or control acupuncture (SAMP8+CA, n=12). An additional control group of SAM resistant 1 mice was included (SAMR1 group, n=12). Morris water maze tests were used to investigate learning and memory abilities. Immunoprecipitation and Western blotting were used to study expression of G protein subunits and their activities in the cortex/hippocampus. Behavioural analysis showed that acupuncture attenuated the severe cognitive deficits observed in untreated/CA-treated SAMP8 mice. The findings of the G protein activation assays via immunoprecipitation and Western blots were that the physiologically coupled activation rate (PCAR) and maximal coupled activation rate (MCAR) of Gα s and Gα i were decreased in the cortex of SAMP8 vs SAMR1 mice. Sanjiao acupuncture induced an upregulation in the PCAR of Gα s and Gα i . In the hippocampus of untreated SAMP8 mice, the PCAR of Gα s and MCAR of both Gα s and Gα i declined, and Sanjiao acupuncture was associated with an upregulation in the MCAR of Gα s and Gα i . There were no significant differences in Gα s and Gα i expression between the groups. Sanjiao acupuncture attenuates cognitive deficits in a mouse model of AD via upregulation of G protein activity and stabilisation of the cellular signal. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  5. In vitro antithrombotic activities of peanut protein hydrolysates.

    Science.gov (United States)

    Zhang, Shao Bing

    2016-07-01

    The antithrombotic activities of peanut protein hydrolysates were investigated using a microplates assay. When peanut proteins were hydrolyzed to a limited extent by various enzymes, their thrombin inhibitory abilities were significantly enhanced. However, the resultant hydrolysates showed significantly different activities even at the same degrees of hydrolysis. The hydrolysates generated by Alcalase 2.4L displayed the best antithrombotic activities and the hydrolysis process was further optimized by response surface methodology. The antithrombotic activities were increased to 86% based on a protein concentration of 50mg/ml under the optimal conditions: pH 8.5, enzyme concentration of 5000IU/g of peanut proteins, and 2h hydrolysis time at 50°C. The Alcalase 2.4L crude hydrolysates were then fractionated successively by preparative and semi-preparative reverse-phase high-performance liquid chromatography (RP-HPLC). The peptide fraction collected inhibited thrombin-catalyzed coagulation of fibrinogen completely at a concentration of 0.4mg/ml, with an antithrombotic activity close to that of heparin at quite a low concentration (0.2mg/ml). This peptide fraction was further analyzed by online reverse-phase ultra-performance liquid chromatography (RP-UPLC) coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and three new peptides were identified as Ser-Trp-Ala-Gln-Leu, Gly-Asn-His-Glu-Ala-Gly-Glu and Cys-Phe-Asn-Glu-Tyr-Glu, respectively. This research provided an effective way to produce antithrombotic peptides from peanut proteins, and also helped to elucidate the structure-function relationships of peanut peptides. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Reassessing the Potential Activities of Plant CGI-58 Protein.

    Directory of Open Access Journals (Sweden)

    Abdallah Khatib

    Full Text Available Comparative Gene Identification-58 (CGI-58 is a widespread protein found in animals and plants. This protein has been shown to participate in lipolysis in mice and humans by activating Adipose triglyceride lipase (ATGL, the initial enzyme responsible for the triacylglycerol (TAG catabolism cascade. Human mutation of CGI-58 is the cause of Chanarin-Dorfman syndrome, an orphan disease characterized by a systemic accumulation of TAG which engenders tissue disorders. The CGI-58 protein has also been shown to participate in neutral lipid metabolism in plants and, in this case, a mutation again provokes TAG accumulation. Although its roles as an ATGL coactivator and in lipid metabolism are quite clear, the catalytic activity of CGI-58 is still in question. The acyltransferase activities of CGI-58 have been speculated about, reported or even dismissed and experimental evidence that CGI-58 expressed in E. coli possesses an unambiguous catalytic activity is still lacking. To address this problem, we developed a new set of plasmids and site-directed mutants to elucidate the in vivo effects of CGI-58 expression on lipid metabolism in E. coli. By analyzing the lipid composition in selected E. coli strains expressing CGI-58 proteins, and by reinvestigating enzymatic tests with adequate controls, we show here that recombinant plant CGI-58 has none of the proposed activities previously described. Recombinant plant and mouse CGI-58 both lack acyltransferase activity towards either lysophosphatidylglycerol or lysophosphatidic acid to form phosphatidylglycerol or phosphatidic acid and recombinant plant CGI-58 does not catalyze TAG or phospholipid hydrolysis. However, expression of recombinant plant CGI-58, but not mouse CGI-58, led to a decrease in phosphatidylglycerol in all strains of E. coli tested, and a mutation of the putative catalytic residues restored a wild-type phenotype. The potential activities of plant CGI-58 are subsequently discussed.

  7. Synthesis of Degradable Poly(vinyl alcohol) by Radical Ring-Opening Copolymerization and Ice Recrystallization Inhibition Activity.

    Science.gov (United States)

    Hedir, Guillaume; Stubbs, Christopher; Aston, Phillip; Dove, Andrew P; Gibson, Matthew I

    2017-12-19

    Poly(vinyl alcohol) (PVA) is the most active synthetic mimic of antifreeze proteins and has extremely high ice recrystallization inhibition (IRI) activity. Addition of PVA to cellular cryopreservation solutions increases the number of recovered viable cells due to its potent IRI, but it is intrinsically nondegradable in vivo . Here we report the synthesis, characterization, and IRI activity of PVA containing degradable ester linkages. Vinyl chloroacetate (VClAc) was copolymerized with 2-methylene-1,3-dioxepane (MDO) which undergoes radical ring-opening polymerization to install main-chain ester units. The use of the chloroacetate monomer enabled selective deacetylation with retention of esters within the polymer backbone. Quantitative IRI assays revealed that the MDO content had to be finely tuned to retain IRI activity, with higher loadings (24 mol %) resulting in complete loss of IRI activity. These degradable materials will help translate PVA, which is nontoxic and biocompatible, into a range of biomedical applications.

  8. Protein energy malnutrition increases arginase activity in monocytes and macrophages.

    Science.gov (United States)

    Corware, Karina; Yardley, Vanessa; Mack, Christopher; Schuster, Steffen; Al-Hassi, Hafid; Herath, Shanthi; Bergin, Philip; Modolell, Manuel; Munder, Markus; Müller, Ingrid; Kropf, Pascale

    2014-01-01

    Protein energy malnutrition is commonly associated with immune dysfunctions and is a major factor in susceptibility to infectious diseases. In this study, we evaluated the impact of protein energy malnutrition on the capacity of monocytes and macrophages to upregulate arginase, an enzyme associated with immunosuppression and increased pathogen replication. Our results show that monocytes and macrophages are significantly increased in the bone marrow and blood of mice fed on a protein low diet. No alteration in the capacity of bone marrow derived macrophages isolated from malnourished mice to phagocytose particles, to produce the microbicidal molecule nitric oxide and to kill intracellular Leishmania parasites was detected. However, macrophages and monocytes from malnourished mice express significantly more arginase both in vitro and in vivo. Using an experimental model of visceral leishmaniasis, we show that following protein energy malnutrition, the increased parasite burden measured in the spleen of these mice coincided with increased arginase activity and that macrophages provide a more permissive environment for parasite growth. Taken together, these results identify a novel mechanism in protein energy malnutrition that might contributes to increased susceptibility to infectious diseases by upregulating arginase activity in myeloid cells.

  9. Activation of mitogen-activated protein kinase by heat shock treatment in Drosophila.

    OpenAIRE

    Chen, F; Torres, M; Duncan, R F

    1995-01-01

    Heat shock treatment of Drosophila melanogaster tissue culture cells causes increased tyrosine phosphorylation of several 44 kDa proteins, which are identified as Drosophila mitogen-activated protein (MAP) kinases. Tyrosine phosphorylation occurs within 5 min, and is maintained at high levels during heat shock. It decreases to basal levels during recovery, concurrent with the repression of heat shock transcription and heat-shock-protein synthesis. The increased MAP kinase tyrosine phosphoryla...

  10. Fungal aegerolysin-like proteins: distribution, activities, and applications.

    Science.gov (United States)

    Novak, Maruša; Kraševec, Nada; Skočaj, Matej; Maček, Peter; Anderluh, Gregor; Sepčić, Kristina

    2015-01-01

    The aegerolysin protein family (from aegerolysin of the mushroom Agrocybe aegerita) comprises proteins of ∼15-20 kDa from various eukaryotic and bacterial taxa. Aegerolysins are inconsistently distributed among fungal species, and variable numbers of homologs have been reported for species within the same genus. As such noncore proteins, without a member of a protein family in each of the sequenced fungi, they can give insight into different species-specific processes. Some aegerolysins have been reported to be hemolytically active against mammalian erythrocytes. However, some function as bi-component proteins that have membrane activity in concert with another protein that contains a membrane attack complex/perforin domain. The function of most of aegerolysins is unknown, although some have been suggested to have a role in development of the organism. Potential biotechnological applications of aegerolysins are already evident, despite the limited scientific knowledge available at present. Some mushroom aegerolysins, for example, can be used as markers to detect and label specific membrane lipids. Others can be used as biomarkers of fungal exposure, where their genes can serve as targets for detection of fungi and their progression during infectious diseases. Antibodies against aegerolysins can also be raised as immuno-diagnostic tools. Aegerolysins have been shown to serve as a species determination tool for fungal phytopathogen isolates in terms of some closely related species, where commonly used internal transcribed spacer barcoding has failed. Moreover, strong promoters that regulate aegerolysin genes can promote secretion of heterologous proteins from fungi and have been successfully applied in simultaneous multi-gene expression techniques.

  11. Novel condensation products having high activity to insolubilize proteins and protein-insolubilized products

    Energy Technology Data Exchange (ETDEWEB)

    Krasnobajew, V.; Boeniger, R.

    1980-01-01

    According to the invention a substantially more active product with respect to the fixing or insolubilization pf proteins, including enzymes, is obtained when 1,3 phenylenediamine is condensed with glutardialdehyde. One application of the process is the enzymatic hydrolysis of lactose in milk products by lactase.

  12. Activity Based Protein Profiling Leads to Identification of Novel Protein Targets for Nerve Agent VX.

    Science.gov (United States)

    Carmany, Dan; Walz, Andrew J; Hsu, Fu-Lian; Benton, Bernard; Burnett, David; Gibbons, Jennifer; Noort, Daan; Glaros, Trevor; Sekowski, Jennifer W

    2017-04-17

    Organophosphorus (OP) nerve agents continue to be a threat at home and abroad during the war against terrorism. Human exposure to nerve agents such as VX results in a cascade of toxic effects relative to the exposure level including ocular miosis, excessive secretions, convulsions, seizures, and death. The primary mechanism behind these overt symptoms is the disruption of cholinergic pathways. While much is known about the primary toxicity mechanisms of nerve agents, there remains a paucity of information regarding impacts on other pathways and systemic effects. These are important for establishing a comprehensive understanding of the toxic mechanisms of OP nerve agents. To identify novel proteins that interact with VX, and that may give insight into these other mechanisms, we used activity-based protein profiling (ABPP) employing a novel VX-probe on lysates from rat heart, liver, kidney, diaphragm, and brain tissue. By making use of a biotin linked VX-probe, proteins covalently bound by the probe were isolated and enriched using streptavidin beads. The proteins were then digested, labeled with isobarically distinct tandem mass tag (TMT) labels, and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Quantitative analysis identified 132 bound proteins, with many proteins found in multiple tissues. As with previously published ABPP OP work, monoacylglycerol lipase associated proteins and fatty acid amide hydrolase (FAAH) were shown to be targets of VX. In addition to these two and other predicted neurotransmitter-related proteins, a number of proteins involved with energy metabolism were identified. Four of these enzymes, mitochondrial isocitrate dehydrogenase 2 (IDH2), isocitrate dehydrogenase 3 (IDH3), malate dehydrogenase (MDH), and succinyl CoA (SCS) ligase, were assayed for VX inhibition. Only IDH2 NADP+ activity was shown to be inhibited directly. This result is consistent with other work reporting animals exposed to OP compounds exhibit

  13. Mitogen Activated Protein kinase signal transduction pathways in the prostate

    Directory of Open Access Journals (Sweden)

    Koul Sweaty

    2004-06-01

    Full Text Available Abstract The biochemistry of the mitogen activated protein kinases ERK, JNK, and p38 have been studied in prostate physiology in an attempt to elucidate novel mechanisms and pathways for the treatment of prostatic disease. We reviewed articles examining mitogen-activated protein kinases using prostate tissue or cell lines. As with other tissue types, these signaling modules are links/transmitters for important pathways in prostate cells that can result in cellular survival or apoptosis. While the activation of the ERK pathway appears to primarily result in survival, the roles of JNK and p38 are less clear. Manipulation of these pathways could have important implications for the treatment of prostate cancer and benign prostatic hypertrophy.

  14. Pharmacokinetics of activated protein C in guinea pigs

    International Nuclear Information System (INIS)

    Berger, H. Jr.; Kirstein, C.G.; Orthner, C.L.

    1991-01-01

    Protein C is a vitamin K-dependent zymogen of the serine protease, activated protein C (APC), an important regulatory enzyme in hemostasis. In view of the potential of human APC as an anticoagulant and profibrinolytic agent, the pharmacokinetics and tissue distribution of APC were studied in guinea pigs. The plasma elimination of a trace dose of 125 I-APC was biphasic following an initial rapid elimination of approximately 15% of the injected dose within 1 to 2 minutes. This rapid removal of 125 I-APC from the circulation was found to be a result of an association with the liver regardless of the route of injection. Essentially identical results were obtained with active site-blocked forms of APC generated with either diisopropylfluorophosphate or D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone, which indicates that the active site was not essential for the liver association. Accumulation of all three forms of APC in the liver peaked at 30 minutes and then declined as increasing amounts of degraded radiolabeled material appeared in the gastrointestinal tract and urine. Removal of the gamma-carboxyglutamic acid (gla) domain of diisopropylphosphoryl-APC resulted in a 50% reduction in the association with liver and an accumulation in the kidneys. Protein C and protein S were cleared from the circulation at rates approximately one-half and one-fourth, respectively, that of APC. Both in vitro and in vivo, APC was found to form complexes with protease inhibitors present in guinea pig plasma. Complex formation resulted in a more rapid disappearance of the enzymatic activity of APC than elimination of the protein moiety. These findings indicate two distinct mechanisms for the elimination of APC. One mechanism involves reaction with plasma protease inhibitors and subsequent elimination by specific hepatic receptors. (Abstract Truncated)

  15. Strategies for the recovery of active proteins through refolding of bacterial inclusion body proteins

    Directory of Open Access Journals (Sweden)

    Rinas Ursula

    2004-09-01

    Full Text Available Abstract Recent advances in generating active proteins through refolding of bacterial inclusion body proteins are summarized in conjunction with a short overview on inclusion body isolation and solubilization procedures. In particular, the pros and cons of well-established robust refolding techniques such as direct dilution as well as less common ones such as diafiltration or chromatographic processes including size exclusion chromatography, matrix- or affinity-based techniques and hydrophobic interaction chromatography are discussed. Moreover, the effect of physical variables (temperature and pressure as well as the presence of buffer additives on the refolding process is elucidated. In particular, the impact of protein stabilizing or destabilizing low- and high-molecular weight additives as well as micellar and liposomal systems on protein refolding is illustrated. Also, techniques mimicking the principles encountered during in vivo folding such as processes based on natural and artificial chaperones and propeptide-assisted protein refolding are presented. Moreover, the special requirements for the generation of disulfide bonded proteins and the specific problems and solutions, which arise during process integration are discussed. Finally, the different strategies are examined regarding their applicability for large-scale production processes or high-throughput screening procedures.

  16. Stimulation of DNA Glycosylase Activities by XPC Protein Complex: Roles of Protein-Protein Interactions

    Directory of Open Access Journals (Sweden)

    Yuichiro Shimizu

    2010-01-01

    Full Text Available We showed that XPC complex, which is a DNA damage detector for nucleotide excision repair, stimulates activity of thymine DNA glycosylase (TDG that initiates base excision repair. XPC appeared to facilitate the enzymatic turnover of TDG by promoting displacement from its own product abasic site, although the precise mechanism underlying this stimulation has not been clarified. Here we show that XPC has only marginal effects on the activity of E. coli TDG homolog (EcMUG, which remains bound to the abasic site like human TDG but does not significantly interacts with XPC. On the contrary, XPC significantly stimulates the activities of sumoylated TDG and SMUG1, both of which exhibit quite different enzymatic kinetics from unmodified TDG but interact with XPC. These results point to importance of physical interactions for stimulation of DNA glycosylases by XPC and have implications in the molecular mechanisms underlying mutagenesis and carcinogenesis in XP-C patients.

  17. Activated protein C induces the release of microparticle-associated endothelial protein C receptor.

    Science.gov (United States)

    Pérez-Casal, Margarita; Downey, Colin; Fukudome, Kenji; Marx, Gernot; Toh, Cheng Hock

    2005-02-15

    Activated protein C (APC) treatment is now used for patients with severe sepsis. We investigated its effect in vitro on primary, physiologically relevant cells and demonstrate a novel mechanism of endothelial protein C receptor (EPCR) release that is not inhibited by metalloproteinase inhibitors. Exposure of human umbilical vein endothelial cells or monocytes to APC (6.25-100 nM) results in the release of EPCR-containing microparticles, as demonstrated by confocal microscopy and characterized through flow cytometry, enzyme-linked immunosorbent assay quantitation of isolated microparticles, and Western blotting. The phenomenon is time- and concentration-dependent and requires the APC active site, EPCR, and protease activated receptor 1 (PAR1) on endothelial cells. Neither protein C nor boiled or D-Phe-Pro-Arg-chloromethylketone-blocked APC can induce microparticle formation and antibody blockade of EPCR or PAR1 cleavage and activation abrogates this APC action. Coincubation with hirudin does not alter the APC effect. The released microparticle bound is full-length EPCR (49 kDa) and APC retains factor V-inactivating activity. Although tumor necrosis factor-alpha (10 ng/mL) can also induce microparticle-associated EPCR release to a similar extent as APC (100 nM), it is only APC-induced microparticles that contain bound APC. This novel observation could provide new insights into the consequences of APC therapy in the septic patient.

  18. Detecting seasonal variation of antifreeze protein distribution in Rhagium mordax using immunofluorescence and high resolution microscopy

    DEFF Research Database (Denmark)

    Buch, Johannes Lørup; Ramløv, Hans

    2017-01-01

    with UV reflected light microscopy. An automated software analysis method was developed in order to discard autofluorescence, and quantify fluorescence from bound antibodies. The results show that R. mordax cuticle and gut exhibit a higher degree of fluorophore-bound fluorescence during summer, than...

  19. Forkhead Box Protein A2 (FOXA2) Protein Stability and Activity Are Regulated by Sumoylation

    OpenAIRE

    Belaguli, Narasimhaswamy S.; Zhang, Mao; Brunicardi, F. Charles; Berger, David H.

    2012-01-01

    The forkhead box protein A2 (FOXA2) is an important regulator of glucose and lipid metabolism and organismal energy balance. Little is known about how FOXA2 protein expression and activity are regulated by post-translational modifications. We have identified that FOXA2 is post-translationally modified by covalent attachment of a small ubiquitin related modifier-1 (SUMO-1) and mapped the sumoylation site to the amino acid lysine 6 (K6). Preventing sumoylation by mutating the SUMO acceptor K6 t...

  20. Calcium/phospholipid-dependent protein kinase (protein kinase C) phosphorylates and activates tyrosine hydroxylase.

    OpenAIRE

    Albert, K A; Helmer-Matyjek, E; Nairn, A C; Müller, T H; Haycock, J W; Greene, L A; Goldstein, M; Greengard, P

    1984-01-01

    Protein kinase C, purified to homogeneity, was found to phosphorylate and activate tyrosine hydroxylase that had been partially purified from pheochromocytoma PC 12 cells. These actions of protein kinase C required the presence of calcium and phospholipid. This phosphorylation of tyrosine hydroxylase reduced the Km for the cofactor 6-methyltetrahydropterine from 0.45 mM to 0.11 mM, increased the Ki for dopamine from 4.2 microM to 47.5 microM, and produced no change in the Km for tyrosine. Lit...

  1. C4b-binding protein inhibits the factor V-dependent but not the factor V-independent cofactor activity of protein S in the activated protein C-mediated inactivation of factor VIIIa

    NARCIS (Netherlands)

    van de Poel, R. H.; Meijers, J. C.; Bouma, B. N.

    2001-01-01

    Activated protein C (APC) is an important inactivator of coagulation factors Va and VIIIa. In the inactivation of factors Va and VIIIa, protein S serves as a cofactor to APC. Protein S can bind to C4b-binding protein (C4BP), and thereby loses its cofactor activity to APC. By modulating free protein

  2. Activation of G Proteins by Guanine Nucleotide Exchange Factors Relies on GTPase Activity.

    Science.gov (United States)

    Stanley, Rob J; Thomas, Geraint M H

    2016-01-01

    G proteins are an important family of signalling molecules controlled by guanine nucleotide exchange and GTPase activity in what is commonly called an 'activation/inactivation cycle'. The molecular mechanism by which guanine nucleotide exchange factors (GEFs) catalyse the activation of monomeric G proteins is well-established, however the complete reversibility of this mechanism is often overlooked. Here, we use a theoretical approach to prove that GEFs are unable to positively control G protein systems at steady-state in the absence of GTPase activity. Instead, positive regulation of G proteins must be seen as a product of the competition between guanine nucleotide exchange and GTPase activity--emphasising a central role for GTPase activity beyond merely signal termination. We conclude that a more accurate description of the regulation of G proteins via these processes is as a 'balance/imbalance' mechanism. This result has implications for the understanding of intracellular signalling processes, and for experimental strategies that rely on modulating G protein systems.

  3. Contractions activate hormone-sensitive lipase in rat muscle by protein kinase C and mitogen-activated protein kinase

    DEFF Research Database (Denmark)

    Donsmark, Morten; Langfort, Jozef; Holm, Cecilia

    2003-01-01

    Intramuscular triacylglycerol is an important energy store and is also related to insulin resistance. The mobilization of fatty acids from this pool is probably regulated by hormone-sensitive lipase (HSL), which has recently been shown to exist in muscle and to be activated by both adrenaline...... and contractions. Adrenaline acts via cAMP-dependent protein kinase (PKA). The signalling mediating the effect of contractions is unknown and was explored in this study. Incubated soleus muscles from 70 g male rats were electrically stimulated to perform repeated tetanic contractions for 5 min. The contraction...... of the inhibitors reduced adrenaline-induced HSL activation in soleus muscle. Both phorbol-12-myristate-13-acetate (PMA), which activates PKC and, in turn, ERK, and caffeine, which increases intracellular Ca2+ without eliciting contraction, increased HSL activity. Activated ERK increased HSL activity in supernatant...

  4. Peptidoglycan recognition proteins with amidase activity in early deuterostomes (Echinodermata).

    Science.gov (United States)

    Coteur, Geoffroy; Mellroth, Peter; De Lefortery, Coline; Gillan, David; Dubois, Philippe; Communi, David; Steiner, Håkan

    2007-01-01

    Despite the ecological and evolutionary importance of echinoderms, very little is known about the immune mechanisms in this group especially regarding humoral immunity. In this paper, we screened for proteins putatively involved in immunity in the common European seastar Asterias rubens using a mass spectrometry-based proteomic approach. Two proteins showed striking sequence similarities with peptidoglycan recognition proteins (PGRPs). The two seastar proteins were identified as a single protein, termed PGRP-S1a, occurring in two forms in the coelomic plasma, one of 20kDa and another of 22kDa. We also cloned and sequenced a second member of the PGRP family, termed PGRP-S2a. It has a calculated molecular mass of 21.3kDa and is expressed in circulating phagocytes. Both the S1a-cDNA from coelomic epithelium RNA and the S2a-cDNA from phagocytes code for the amino acid residues necessary for peptidoglycan degradation. PGRP-S1a did not affect the phagocytic activity of seastar immune cells towards Micrococcus luteus but inhibited their production of reactive oxygen species (ROS). A recombinant, His-tagged, PGRP-S2a degrades peptidoglycan and increases the phagocytosis of M. luteus cells by seastar phagocytes.

  5. Protective effects of soybean protein and egg white protein on the antibacterial activity of nisin in the presence of trypsin.

    Science.gov (United States)

    Pan, Dan; Zhang, Dong; Hao, Limin; Lin, Songyang; Kang, Qiaozhen; Liu, Xin; Lu, Laizheng; Lu, Jike

    2018-01-15

    The using of nisin to prevent foodborne pathogens (Staphylococcus aureus and Listeria monocytogenes) from contamination has received broad attentions during meat processing. However, the application of nisin has been limited because its antibacterial activity may be inhibited by trypsin. In this study, the protective effects of soybean protein and egg white protein on antibacterial activity of nisin were evaluated. It could be concluded that exogenous trypsin decreased the antibacterial activity of nisin, soybean protein and egg white protein could keep the nisin activity from enzymolysis of trypsin. Trypsin inhibitors in soybean protein and egg white protein could protect the antibacterial activity of nisin. Nisin with soybean protein or egg white protein in cooked meat product presented better quality preservation effects than nisin alone in the presence of trypsin. The total viable counts (TVC) and total volatile basic nitrogen (TVB-N) of nisin-treated group were significantly higher than these in nisin-soybean protein-treated and nisin-egg white protein-treated groups with trypsin. This study showed the potential of using soybean protein and egg white protein to stabilize the antibacterial activity of nisin under high trypsin conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Detection of expansin proteins and activity during tomato fruit ontogeny.

    Science.gov (United States)

    Rose, J K; Cosgrove, D J; Albersheim, P; Darvill, A G; Bennett, A B

    2000-08-01

    Expansins are plant proteins that have the capacity to induce extension in isolated cell walls and are thought to mediate pH-dependent cell expansion. J.K.C. Rose, H.H. Lee, and A.B. Bennett ([1997] Proc Natl Acad Sci USA 94: 5955-5960) reported the identification of an expansin gene (LeExp1) that is specifically expressed in ripening tomato (Lycopersicon esculentum) fruit where cell wall disassembly, but not cell expansion, is prominent. Expansin expression during fruit ontogeny was examined using antibodies raised to recombinant LeExp1 or a cell elongation-related expansin from cucumber (CsExp1). The LeExp1 antiserum detected expansins in extracts from ripe, but not preripe tomato fruit, in agreement with the pattern of LeExp1 mRNA accumulation. In contrast, antibodies to CsExp1 cross-reacted with expansins in early fruit development and the onset of ripening, but not at a later ripening stage. These data suggest that ripening-related and expansion-related expansin proteins have distinct antigenic epitopes despite overall high sequence identity. Expansin proteins were detected in a range of fruit species and showed considerable variation in abundance; however, appreciable levels of expansin were not present in fruit of the rin or Nr tomato mutants that exhibit delayed and reduced softening. LeExp1 protein accumulation was ethylene-regulated and matched the previously described expression of mRNA, suggesting that expression is not regulated at the level of translation. We report the first detection of expansin activity in several stages of fruit development and while characteristic creep activity was detected in young and developing tomato fruit and in ripe pear, avocado, and pepper, creep activity in ripe tomato showed qualitative differences, suggesting both hydrolytic and expansin activities.

  7. Mitogen-activated protein kinases in the acute diabetic myocardium

    Czech Academy of Sciences Publication Activity Database

    Strnisková, M.; Barančík, M.; Neckář, Jan; Ravingerová, T.

    2003-01-01

    Roč. 249, 1-2 (2003), s. 59-65 ISSN 0300-8177 R&D Projects: GA MŠk LN00A069 Grant - others:VEGA(SK) 2/2063/22 Institutional research plan: CEZ:AV0Z5011922 Keywords : experimental diabetes * ischemia * mitogen-activated protein kinases (MAPK) Subject RIV: ED - Physiology Impact factor: 1.763, year: 2003

  8. Activation of the Unfolded Protein Response Contributes toward the Antitumor Activity of Vorinostat

    Directory of Open Access Journals (Sweden)

    Soumen Kahali

    2010-01-01

    Full Text Available Histone deacetylase (HDAC inhibitors represent an emerging class of anticancer agents progressing through clinical trials. Although their primary target is thought to involve acetylation of core histones, several nonhistone substrates have been identified, including heat shock protein (HSP 90, which may contribute towards their antitumor activity. Glucose-regulated protein 78 (GRP78 is a member of the HSP family of molecular chaperones and plays a central role in regulating the unfolded protein response (UPR. Emerging data suggest that GRP78 is critical in cellular adaptation and survival associated with oncogenesis and may serve as a cancer-specific therapeutic target. On the basis of shared homology with HSP family proteins, we sought to determine whether GRP78 could serve as a molecular target of the HDAC inhibitor vorinostat. Vorinostat treatment led to GRP78 acetylation, dissociation, and subsequent activation of its client protein double-stranded RNA-activated protein-like endoplasmic reticulum kinase (PERK. Investigations in a panel of cancer cell lines identified that UPR activation after vorinostat exposure is specific to certain lines. Mass spectrometry performed on immunoprecipitated GRP78 identified lysine-585 as a specific vorinostat-induced acetylation site of GRP78. Downstream activation of the UPR was confirmed, including eukaryotic initiating factor 2α phosphorylation and increase in ATF4 and C/EBP homologous protein expression. To determine the biologic relevance of UPR activation after vorinostat, RNA interference of PERK was performed, demonstrating significantly decreased sensitivity to vorinostat-induced cytotoxicity. Collectively, these findings indicate that GRP78 is a biologic target of vorinostat, and activation of the UPR through PERK phosphorylation contributes toward its antitumor activity.

  9. Study on antibacterial activity of hydrogel from irradiated silk protein

    Energy Technology Data Exchange (ETDEWEB)

    Bunnak, J.; Chaisupakitsin, M. [King Mongkut' s Institute of Technology Lardkrabang, Bangkok (Thailand)

    2001-03-01

    Hydrogels for biomedical application were prepared from solution blends of 3% silk protein and 3%, 10% poly (vinyl alcohol) (PVA) and followed with irradiation. Mixture of hydrogels were gamma irradiated at 10, 20, 30, 40 and 50 kGy under N{sub 2} atmosphere. To clarify anti-bacterial activity of hydrogels, modified of the Agar disk diffusion method and American Association of Textile Chemists and Colorists, AATCC Test Method 90-1977, were carried out. The four kinds of bacteria such as Escherichia coli, Bacillus subtilis, Staphylococcus aureus and Staphylococcus epidermidis, were used. It was found that a 1:3 volume ratio of 3% silk protein and 3% PVA respectively, at 50 kGy irradiation, is suitable conditions for preparation hydrogels and trend to indicate the highest of an antibacterial activity against E. coli, B. subtilis and S. aureus. However the antibacterial activity of hydrogels against S. epidermidis was not clearly. These results are very useful to expand the application of hydrogel from irradiated silk protein to the medical products. (author)

  10. Study on antibacterial activity of hydrogel from irradiated silk protein

    International Nuclear Information System (INIS)

    Bunnak, J.; Chaisupakitsin, M.

    2001-01-01

    Hydrogels for biomedical application were prepared from solution blends of 3% silk protein and 3%, 10% poly (vinyl alcohol) (PVA) and followed with irradiation. Mixture of hydrogels were gamma irradiated at 10, 20, 30, 40 and 50 kGy under N 2 atmosphere. To clarify anti-bacterial activity of hydrogels, modified of the Agar disk diffusion method and American Association of Textile Chemists and Colorists, AATCC Test Method 90-1977, were carried out. The four kinds of bacteria such as Escherichia coli, Bacillus subtilis, Staphylococcus aureus and Staphylococcus epidermidis, were used. It was found that a 1:3 volume ratio of 3% silk protein and 3% PVA respectively, at 50 kGy irradiation, is suitable conditions for preparation hydrogels and trend to indicate the highest of an antibacterial activity against E. coli, B. subtilis and S. aureus. However the antibacterial activity of hydrogels against S. epidermidis was not clearly. These results are very useful to expand the application of hydrogel from irradiated silk protein to the medical products. (author)

  11. Dual regulation of G proteins and the G-protein-activated K+ channels by lithium.

    Science.gov (United States)

    Farhy Tselnicker, Isabella; Tsemakhovich, Vladimir; Rishal, Ida; Kahanovitch, Uri; Dessauer, Carmen W; Dascal, Nathan

    2014-04-01

    Lithium (Li(+)) is widely used to treat bipolar disorder (BPD). Cellular targets of Li(+), such as glycogen synthase kinase 3β (GSK3β) and G proteins, have long been implicated in BPD etiology; however, recent genetic studies link BPD to other proteins, particularly ion channels. Li(+) affects neuronal excitability, but the underlying mechanisms and the relevance to putative BPD targets are unknown. We discovered a dual regulation of G protein-gated K(+) (GIRK) channels by Li(+), and identified the underlying molecular mechanisms. In hippocampal neurons, therapeutic doses of Li(+) (1-2 mM) increased GIRK basal current (Ibasal) but attenuated neurotransmitter-evoked GIRK currents (Ievoked) mediated by Gi/o-coupled G-protein-coupled receptors (GPCRs). Molecular mechanisms of these regulations were studied with heterologously expressed GIRK1/2. In excised membrane patches, Li(+) increased Ibasal but reduced GPCR-induced GIRK currents. Both regulations were membrane-delimited and G protein-dependent, requiring both Gα and Gβγ subunits. Li(+) did not impair direct activation of GIRK channels by Gβγ, suggesting that inhibition of Ievoked results from an action of Li(+) on Gα, probably through inhibition of GTP-GDP exchange. In direct binding studies, Li(+) promoted GPCR-independent dissociation of Gαi(GDP) from Gβγ by a Mg(2+)-independent mechanism. This previously unknown Li(+) action on G proteins explains the second effect of Li(+), the enhancement of GIRK's Ibasal. The dual effect of Li(+) on GIRK may profoundly regulate the inhibitory effects of neurotransmitters acting via GIRK channels. Our findings link between Li(+), neuronal excitability, and both cellular and genetic targets of BPD: GPCRs, G proteins, and ion channels.

  12. Activated protein synthesis and suppressed protein breakdown signaling in skeletal muscle of critically ill patients.

    Directory of Open Access Journals (Sweden)

    Jakob G Jespersen

    Full Text Available BACKGROUND: Skeletal muscle mass is controlled by myostatin and Akt-dependent signaling on mammalian target of rapamycin (mTOR, glycogen synthase kinase 3β (GSK3β and forkhead box O (FoxO pathways, but it is unknown how these pathways are regulated in critically ill human muscle. To describe factors involved in muscle mass regulation, we investigated the phosphorylation and expression of key factors in these protein synthesis and breakdown signaling pathways in thigh skeletal muscle of critically ill intensive care unit (ICU patients compared with healthy controls. METHODOLOGY/PRINCIPAL FINDINGS: ICU patients were systemically inflamed, moderately hyperglycemic, received insulin therapy, and showed a tendency to lower plasma branched chain amino acids compared with controls. Using Western blotting we measured Akt, GSK3β, mTOR, ribosomal protein S6 kinase (S6k, eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1, and muscle ring finger protein 1 (MuRF1; and by RT-PCR we determined mRNA expression of, among others, insulin-like growth factor 1 (IGF-1, FoxO 1, 3 and 4, atrogin1, MuRF1, interleukin-6 (IL-6, tumor necrosis factor α (TNF-α and myostatin. Unexpectedly, in critically ill ICU patients Akt-mTOR-S6k signaling was substantially higher compared with controls. FoxO1 mRNA was higher in patients, whereas FoxO3, atrogin1 and myostatin mRNAs and MuRF1 protein were lower compared with controls. A moderate correlation (r2=0.36, p<0.05 between insulin infusion dose and phosphorylated Akt was demonstrated. CONCLUSIONS/SIGNIFICANCE: We present for the first time muscle protein turnover signaling in critically ill ICU patients, and we show signaling pathway activity towards a stimulation of muscle protein synthesis and a somewhat inhibited proteolysis.

  13. FAS activation induces dephosphorylation of SR proteins - Dependence on the de novo generation of ceramide and activation of protein phosphatase 1

    NARCIS (Netherlands)

    Chalfant, CE; Ogretmen, B; Galadari, S; Kroesen, BJ; Pettus, BJ; Hannun, YA

    2001-01-01

    The search for potential targets for ceramide action led to the identification of ceramide-activated protein phosphatases (CAPP). To date, two serine/threonine protein phosphatases, protein phosphatase 2A (PP2A) and protein phosphatase 1 (PP1), have been demonstrated to function as

  14. Platelet factor 4 impairs the anticoagulant activity of activated protein C.

    LENUS (Irish Health Repository)

    Preston, Roger J S

    2009-02-27

    Platelet factor 4 (PF4) is an abundant platelet alpha-granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the gamma-carboxyglutamic acid (Gla) domain of protein C, thereby enhancing activated protein C (APC) generation by the thrombin-thrombomodulin complex. However, the protein C Gla domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. In this study we demonstrate that PF4 significantly inhibits APC anti-coagulant activity. PF4 inhibited both protein S-dependent APC anticoagulant function in plasma and protein S-dependent factor Va (FVa) proteolysis 3- to 5-fold, demonstrating that PF4 impairs protein S cofactor enhancement of APC anticoagulant function. Using recombinant factor Va variants FVa-R506Q\\/R679Q and FVa-R306Q\\/R679Q, PF4 was shown to impair APC proteolysis of FVa at position Arg(306) by 3-fold both in the presence and absence of protein S. These data suggest that PF4 contributes to the poorly understood APC resistance phenotype associated with activated platelets. Finally, despite PF4 binding to the APC Gla domain, we show that APC in the presence of PF4 retains its ability to initiate PAR-1-mediated cytoprotective signaling. In summary, we propose that PF4 acts as a critical regulator of APC generation, but also differentially targets APC toward cytoprotective, rather than anticoagulant function at sites of vascular injury with concurrent platelet activation.

  15. Platelet factor 4 impairs the anticoagulant activity of activated protein C.

    LENUS (Irish Health Repository)

    Preston, Roger J S

    2012-02-01

    Platelet factor 4 (PF4) is an abundant platelet alpha-granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the gamma-carboxyglutamic acid (Gla) domain of protein C, thereby enhancing activated protein C (APC) generation by the thrombin-thrombomodulin complex. However, the protein C Gla domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. In this study we demonstrate that PF4 significantly inhibits APC anti-coagulant activity. PF4 inhibited both protein S-dependent APC anticoagulant function in plasma and protein S-dependent factor Va (FVa) proteolysis 3- to 5-fold, demonstrating that PF4 impairs protein S cofactor enhancement of APC anticoagulant function. Using recombinant factor Va variants FVa-R506Q\\/R679Q and FVa-R306Q\\/R679Q, PF4 was shown to impair APC proteolysis of FVa at position Arg(306) by 3-fold both in the presence and absence of protein S. These data suggest that PF4 contributes to the poorly understood APC resistance phenotype associated with activated platelets. Finally, despite PF4 binding to the APC Gla domain, we show that APC in the presence of PF4 retains its ability to initiate PAR-1-mediated cytoprotective signaling. In summary, we propose that PF4 acts as a critical regulator of APC generation, but also differentially targets APC toward cytoprotective, rather than anticoagulant function at sites of vascular injury with concurrent platelet activation.

  16. Delineation of protein structure classes from multivariate analysis of protein Raman optical activity data.

    Science.gov (United States)

    Zhu, Fujiang; Tranter, George E; Isaacs, Neil W; Hecht, Lutz; Barron, Laurence D

    2006-10-13

    Vibrational Raman optical activity (ROA), measured as a small difference in the intensity of Raman scattering from chiral molecules in right and left-circularly polarized incident light, or as the intensity of a small circularly polarized component in the scattered light, is a powerful probe of the aqueous solution structure of proteins. On account of the large number of structure-sensitive bands in protein ROA spectra, multivariate analysis techniques such as non-linear mapping (NLM) are especially favourable for determining structural relationships between different proteins. Here NLM is used to map a dataset of 80 polypeptide, protein and virus ROA spectra, considered as points in a multidimensional space with axes representing the digitized wavenumbers, into readily visualizable two and three-dimensional spaces in which points close to or distant from each other, respectively, represent similar or dissimilar structures. Discrete clusters are observed which correspond to the seven structure classes all alpha, mainly alpha, alphabeta, mainly beta, all beta, mainly disordered/irregular and all disordered/irregular. The average standardised ROA spectra of the proteins falling within each structure class have distinct features characteristic of each class. A distinct cluster containing the wheat protein A-gliadin and the plant viruses potato virus X, narcissus mosaic virus, papaya mosaic virus and tobacco rattle virus, all of which appear in the mainly alpha cluster in the two-dimensional representation, becomes clearly separated in the direction of increasing disorder in the three-dimensional representation. This suggests that the corresponding five proteins, none of which to date has yielded high-resolution X-ray structures, consist mainly of alpha-helix and disordered structure with little or no beta-sheet. This combination of structural elements may have functional significance, such as facilitating disorder-to-order transitions (and vice versa) and suppressing

  17. Plasma Protein Turnover Rates in Rats Using Stable Isotope Labeling, Global Proteomics, and Activity-Based Protein Profiling

    Energy Technology Data Exchange (ETDEWEB)

    Smith, Jordan N.; Tyrrell, Kimberly J.; Hansen, Joshua R.; Thomas, Dennis G.; Murphree, Taylor A.; Shukla, Anil K.; Luders, Teresa; Madden, James M.; Li, Yunying; Wright, Aaron T.; Piehowski, Paul D.

    2017-12-06

    Protein turnover is important for general health on cellular and organism scales providing a strategy to replace old, damaged, or dysfunctional proteins. Protein turnover also informs of biomarker kinetics, as a better understanding of synthesis and degradation of proteins increases the clinical utility of biomarkers. Here, turnover rates of plasma proteins in rats were measured in vivo using a pulse-chase stable isotope labeling experiment. During the pulse, rats (n=5) were fed 13C6-labeled lysine (“heavy”) feed for 23 days to label proteins. During the chase, feed was changed to an unlabeled equivalent feed (“light”), and blood was repeatedly sampled from rats over 10 time points for 28 days. Plasma samples were digested with trypsin, and analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). MaxQuant was used to identify peptides and proteins, and quantify heavy:light lysine ratios. A system of ordinary differential equations was used to calculate protein turnover rates. Using this approach, 273 proteins were identified, and turnover rates were quantified for 157 plasma proteins with half-lives ranging 0.3-103 days. For the ~70 most abundant proteins, variability in turnover rates among rats was low (median coefficient of variation: 0.09). Activity-based protein profiling was applied to pooled plasma samples to enrich serine hydrolases using a fluorophosphonate (FP2) activity-based probe. This enrichment resulted in turnover rates for an additional 17 proteins. This study is the first to measure global plasma protein turnover rates in rats in vivo, measure variability of protein turnover rates in any animal model, and utilize activity-based protein profiling for enhancing measurements of targeted, low-abundant proteins, such as those commonly used as biomarkers. Measured protein turnover rates will be important for understanding of the role of protein turnover in cellular and organism health as well as increasing the utility of protein

  18. Fast calcium sensor proteins for monitoring neural activity.

    Science.gov (United States)

    Badura, Aleksandra; Sun, Xiaonan Richard; Giovannucci, Andrea; Lynch, Laura A; Wang, Samuel S-H

    2014-10-01

    A major goal of the BRAIN Initiative is the development of technologies to monitor neuronal network activity during active information processing. Toward this goal, genetically encoded calcium indicator proteins have become widely used for reporting activity in preparations ranging from invertebrates to awake mammals. However, slow response times, the narrow sensitivity range of Ca 2+ and in some cases, poor signal-to-noise ratio still limit their usefulness. Here, we review recent improvements in the field of neural activity-sensitive probe design with a focus on the GCaMP family of calcium indicator proteins. In this context, we present our newly developed Fast-GCaMPs, which have up to 4-fold accelerated off-responses compared with the next-fastest GCaMP, GCaMP6f. Fast-GCaMPs were designed by destabilizing the association of the hydrophobic pocket of calcium-bound calmodulin with the RS20 binding domain, an intramolecular interaction that protects the green fluorescent protein chromophore. Fast-GCaMP6f-RS06 and Fast-GCaMP6f-RS09 have rapid off-responses in stopped-flow fluorimetry, in neocortical brain slices, and in the intact cerebellum in vivo . Fast-GCaMP6f variants should be useful for tracking action potentials closely spaced in time, and for following neural activity in fast-changing compartments, such as axons and dendrites. Finally, we discuss strategies that may allow tracking of a wider range of neuronal firing rates and improve spike detection.

  19. When Heterotrimeric G Proteins Are Not Activated by G Protein-Coupled Receptors: Structural Insights and Evolutionary Conservation.

    Science.gov (United States)

    DiGiacomo, Vincent; Marivin, Arthur; Garcia-Marcos, Mikel

    2018-01-23

    Heterotrimeric G proteins are signal-transducing switches conserved across eukaryotes. In humans, they work as critical mediators of intercellular communication in the context of virtually any physiological process. While G protein regulation by G protein-coupled receptors (GPCRs) is well-established and has received much attention, it has become recently evident that heterotrimeric G proteins can also be activated by cytoplasmic proteins. However, this alternative mechanism of G protein regulation remains far less studied than GPCR-mediated signaling. This Viewpoint focuses on recent advances in the characterization of a group of nonreceptor proteins that contain a sequence dubbed the "Gα-binding and -activating (GBA) motif". So far, four proteins present in mammals [GIV (also known as Girdin), DAPLE, CALNUC, and NUCB2] and one protein in Caenorhabditis elegans (GBAS-1) have been described as possessing a functional GBA motif. The GBA motif confers guanine nucleotide exchange factor activity on Gαi subunits in vitro and activates G protein signaling in cells. The importance of this mechanism of signal transduction is highlighted by the fact that its dysregulation underlies human diseases, such as cancer, which has made the proteins attractive new candidates for therapeutic intervention. Here we discuss recent discoveries on the structural basis of GBA-mediated activation of G proteins and its evolutionary conservation and compare them with the better-studied mechanism mediated by GPCRs.

  20. Rapamycin Induces Mitogen-activated Protein (MAP) Kinase Phosphatase-1 (MKP-1) Expression through Activation of Protein Kinase B and Mitogen-activated Protein Kinase Kinase Pathways*

    Science.gov (United States)

    Rastogi, Ruchi; Jiang, Zhongliang; Ahmad, Nisar; Rosati, Rita; Liu, Yusen; Beuret, Laurent; Monks, Robert; Charron, Jean; Birnbaum, Morris J.; Samavati, Lobelia

    2013-01-01

    Mitogen-activated protein kinase phosphatase-1 (MKP-1), also known as dual specificity phosphatase-1 (DUSP-1), plays a crucial role in the deactivation of MAPKs. Several drugs with immune-suppressive properties modulate MKP-1 expression as part of their mechanism of action. We investigated the effect of mTOR inhibition through rapamycin and a dual mTOR inhibitor (AZD2014) on MKP-1 expression. Low dose rapamycin led to a rapid activation of both AKT and ERK pathways with a subsequent increase in MKP-1 expression. Rapamycin treatment led to phosphorylation of CREB, transcription factor 1 (ATF1), and ATF2, three transcription factors that bind to the cyclic AMP-responsive elements on the Mkp-1 promoter. Inhibition of either the MEK/ERK or the AKT pathway attenuated rapamycin-mediated MKP-1 induction. AZD2014 did not activate AKT but activated the ERK pathway, leading to a moderate MKP-1 induction. Using bone marrow-derived macrophages (BMDMs) derived from wild-type (WT) mice or mice deficient in AKT1 and AKT2 isoforms or BMDM from targeted deficiency in MEK1 and MEK2, we show that rapamycin treatment led to an increased MKP1 expression in BMDM from WT but failed to do so in BMDMs lacking the AKT1 isoform or MEK1 and MEK2. Importantly, rapamycin pretreatment inhibited LPS-mediated p38 activation and decreased nitric oxide and IL-6 production. Our work provides a conceptual framework for the observed immune modulatory effect of mTOR inhibition. PMID:24126911

  1. Fusion expression of Helicobacter pylori neutrophil-activating protein in E.coli

    OpenAIRE

    Kang, Qiao-Zhen; Duan, Guang-Cai; Fan, Qing-Tang; Xi, Yuan-Lin

    2005-01-01

    AIM: To produce a recombinant protein rMBP-NAP, which was fusionally expressed by Helicobacter pylori (H pylori) neutrophil-activating protein (NAP) and E. coli maltose-binding protein (MBP) and to evaluate its immunoreactivity and immunogenicity.

  2. Refolding techniques for recovering biologically active recombinant proteins from inclusion bodies.

    Science.gov (United States)

    Yamaguchi, Hiroshi; Miyazaki, Masaya

    2014-02-20

    Biologically active proteins are useful for studying the biological functions of genes and for the development of therapeutic drugs and biomaterials in a biotechnology industry. Overexpression of recombinant proteins in bacteria, such as Escherichia coli, often results in the formation of inclusion bodies, which are protein aggregates with non-native conformations. As inclusion bodies contain relatively pure and intact proteins, protein refolding is an important process to obtain active recombinant proteins from inclusion bodies. However, conventional refolding methods, such as dialysis and dilution, are time consuming and, often, recovered yields of active proteins are low, and a trial-and-error process is required to achieve success. Recently, several approaches have been reported to refold these aggregated proteins into an active form. The strategies largely aim at reducing protein aggregation during the refolding procedure. This review focuses on protein refolding techniques using chemical additives and laminar flow in microfluidic chips for the efficient recovery of active proteins from inclusion bodies.

  3. Cyclic AMP activates the mitogen-activated protein kinase cascade in PC12 cells

    DEFF Research Database (Denmark)

    Frödin, M; Peraldi, P; Van Obberghen, E

    1994-01-01

    Mitogen-activated protein (MAP) kinases are activated in response to a large variety of extracellular signals, including growth factors, hormones, and neurotransmitters, which activate distinct intracellular signaling pathways. Their activation by the cAMP-dependent pathway, however, has not been...... reported. In rat pheochromocytoma PC12 cells, we demonstrate here a stimulation of the MAP kinase isozyme extracellular signal-regulated kinase 1 (ERK1) following elevation of intracellular cAMP after exposure of the cells to isobutylmethylxanthine, cholera toxin, forskolin, or cAMP-analogues. cAMP acted...... synergistically with phorbol ester, an activator of protein kinase C, in the stimulation of ERK1. In accordance with this observation, the peptide neurotransmitter pituitary adenylate cyclase-activating polypeptide 38 (PACAP38), which stimulates cAMP production as well as phosphatidylinositol breakdown in PC12...

  4. Protein C inhibitor acts as a procoagulant by inhibiting the thrombomodulin-induced activation of protein C in human plasma

    NARCIS (Netherlands)

    Elisen, M. G.; von dem Borne, P. A.; Bouma, B. N.; Meijers, J. C.

    1998-01-01

    Protein C inhibitor (PCI), which was originally identified as an inhibitor of activated protein C, also efficiently inhibits coagulation factors such as factor Xa and thrombin. Recently it was found, using purified proteins, that the anticoagulant thrombin-thrombomodulin complex was also inhibited

  5. Plasma cholesteryl ester transfer protein mass and phospholipid transfer protein activity are associated with leptin in type 2 diabetes mellitus

    NARCIS (Netherlands)

    Dullaart, R. P. F.; de Vries, R.; Dallinga-Thie, G. M.; van Tol, A.; Sluiter, W. J.

    Adipose tissue contributes to plasma levels of lipid transfer proteins and is also the major source of plasma adipokines. We hypothesized that plasma cholesteryl ester transfer protein (CETP) mass, phospholipid transfer protein (PLTP) activity and cholesteryl ester transfer (CET, a measure of CETP

  6. Metals in the active site of native protein phosphatase-1.

    Science.gov (United States)

    Heroes, Ewald; Rip, Jens; Beullens, Monique; Van Meervelt, Luc; De Gendt, Stefan; Bollen, Mathieu

    2015-08-01

    Protein phosphatase-1 (PP1) is a major protein Ser/Thr phosphatase in eukaryotic cells. Its activity depends on two metal ions in the catalytic site, which were identified as manganese in the bacterially expressed phosphatase. However, the identity of the metal ions in native PP1 is unknown. In this study, total reflection X-ray fluorescence (TXRF) was used to detect iron and zinc in PP1 that was purified from rabbit skeletal muscle. Metal exchange experiments confirmed that the distinct substrate specificity of recombinant and native PP1 is determined by the nature of their associated metals. We also found that the iron level associated with native PP1 is decreased by incubation with inhibitor-2, consistent with a function of inhibitor-2 as a PP1 chaperone. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Annotating activation/inhibition relationships to protein-protein interactions using gene ontology relations.

    Science.gov (United States)

    Yim, Soorin; Yu, Hasun; Jang, Dongjin; Lee, Doheon

    2018-04-11

    Signaling pathways can be reconstructed by identifying 'effect types' (i.e. activation/inhibition) of protein-protein interactions (PPIs). Effect types are composed of 'directions' (i.e. upstream/downstream) and 'signs' (i.e. positive/negative), thereby requiring directions as well as signs of PPIs to predict signaling events from PPI networks. Here, we propose a computational method for systemically annotating effect types to PPIs using relations between functional information of proteins. We used regulates, positively regulates, and negatively regulates relations in Gene Ontology (GO) to predict directions and signs of PPIs. These relations indicate both directions and signs between GO terms so that we can project directions and signs between relevant GO terms to PPIs. Independent test results showed that our method is effective for predicting both directions and signs of PPIs. Moreover, our method outperformed a previous GO-based method that did not consider the relations between GO terms. We annotated effect types to human PPIs and validated several highly confident effect types against literature. The annotated human PPIs are available in Additional file 2 to aid signaling pathway reconstruction and network biology research. We annotated effect types to PPIs by using regulates, positively regulates, and negatively regulates relations in GO. We demonstrated that those relations are effective for predicting not only signs, but also directions of PPIs. The usefulness of those relations suggests their potential applications to other types of interactions such as protein-DNA interactions.

  8. Steric effects in peptide and protein exchange with activated disulfides.

    Science.gov (United States)

    Kerr, Jason; Schlosser, Jessica L; Griffin, Donald R; Wong, Darice Y; Kasko, Andrea M

    2013-08-12

    Disulfide exchange is an important bioconjugation tool, enabling chemical modification of peptides and proteins containing free cysteines. We previously reported the synthesis of a macromer bearing an activated disulfide and its incorporation into hydrogels. Despite their ability to diffuse freely into hydrogels, larger proteins were unable to undergo in-gel disulfide exchange. In order to understand this phenomenon, we synthesized four different activated disulfide-bearing model compounds (Mn = 300 Da to 10 kDa) and quantified their rate of disulfide exchange with a small peptide (glutathione), a moderate-sized protein (β-lactoglobulin), and a large protein (bovine serum albumin) in four different pH solutions (6.0, 7.0, 7.4, and 8.0) to mimic biological systems. Rate constants of exchange depend significantly on the size and accessibility of the thiolate. pH also significantly affects the rate of reaction, with the faster reactions occurring at higher pH. Surprisingly, little difference in exchange rates is seen between macromolecular disulfides of varying size (Mn = 2 kDa - 10 kDa), although all undergo exchange more slowly than their small molecule analogue (MW = 300 g/mol). The maximum exchange efficiencies (% disulfides exchanged after 24 h) are not siginificantly affected by thiol size or pH, but somewhat affected by disulfide size. Therefore, while all three factors investigated (pH, disulfide size, and thiolate size) can influence the exchange kinetics and extent of reaction, the size of the thiolate and its accessibility plays the most significant role.

  9. Keap1-Independent Regulation of Nrf2 Activity by Protein Acetylation and a BET Bromodomain Protein.

    Science.gov (United States)

    Chatterjee, Nirmalya; Tian, Min; Spirohn, Kerstin; Boutros, Michael; Bohmann, Dirk

    2016-05-01

    Mammalian BET proteins comprise a family of bromodomain-containing epigenetic regulators with complex functions in chromatin organization and gene regulation. We identified the sole member of the BET protein family in Drosophila, Fs(1)h, as an inhibitor of the stress responsive transcription factor CncC, the fly ortholog of Nrf2. Fs(1)h physically interacts with CncC in a manner that requires the function of its bromodomains and the acetylation of CncC. Treatment of cultured Drosophila cells or adult flies with fs(1)h RNAi or with the BET protein inhibitor JQ1 de-represses CncC transcriptional activity and engages protective gene expression programs. The mechanism by which Fs(1)h inhibits CncC function is distinct from the canonical mechanism that stimulates Nrf2 function by abrogating Keap1-dependent proteasomal degradation. Consistent with the independent modes of CncC regulation by Keap1 and Fs(1)h, combinations of drugs that can specifically target these pathways cause a strong synergistic and specific activation of protective CncC- dependent gene expression and boosts oxidative stress resistance. This synergism might be exploitable for the design of combinatorial therapies to target diseases associated with oxidative stress or inflammation.

  10. A recyclable protein resource derived from cauliflower by-products: Potential biological activities of protein hydrolysates.

    Science.gov (United States)

    Xu, Yang; Li, Yuting; Bao, Tao; Zheng, Xiaodong; Chen, Wei; Wang, Jianxu

    2017-04-15

    Cauliflower by-products (CBP) are rich in leaf protein. Every year tons of CBP will lead to environmental pollution. Therefore, this study was conducted to extract leaf protein from CBP and investigate its biological activities. Our results showed that the optimal extraction parameters were: a liquid to solid ratio of 4mL/g, a pH of 11, an ultrasonic extraction lasting 15min, and at an applied power of 175W. Under these optimized conditions, 12.066g of soluble leaf protein (SLP) was obtained from 1000g of CBP and its extraction yield was 53.07%. The obtained SLP was further hydrolysed by Alcalase and the SLP hydrolysate (SLPH) showed a potent angiotensin I-converting enzyme (ACE) inhibitory activity with an IC 50 value of 138.545μg/mL in vitro. In addition, SLPH promoted the glucose consumption and enhanced the glycogen content in HepG2 cells. Overall, our results suggested that CBP may be recycled for designing future functional foods. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Structural analyses of a constitutively active mutant of exchange protein directly activated by cAMP.

    Science.gov (United States)

    White, Mark A; Li, Sheng; Tsalkova, Tamara; Mei, Fang C; Liu, Tong; Woods, Virgil L; Cheng, Xiaodong

    2012-01-01

    Exchange proteins directly activated by cAMP (EPACs) are important allosteric regulators of cAMP-mediated signal transduction pathways. To understand the molecular mechanism of EPAC activation, we have combined site-directed mutagenesis, X-ray crystallography, and peptide amide hydrogen/deuterium exchange mass spectrometry (DXMS) to probe the structural and conformational dynamics of EPAC2-F435G, a constitutively active EPAC2 mutant. Our study demonstrates that conformational dynamics plays a critical role in cAMP-induced EPAC activation. A glycine mutation at 435 position shifts the equilibrium of conformational dynamics towards the extended active conformation.

  12. Structural analyses of a constitutively active mutant of exchange protein directly activated by cAMP.

    Directory of Open Access Journals (Sweden)

    Mark A White

    Full Text Available Exchange proteins directly activated by cAMP (EPACs are important allosteric regulators of cAMP-mediated signal transduction pathways. To understand the molecular mechanism of EPAC activation, we have combined site-directed mutagenesis, X-ray crystallography, and peptide amide hydrogen/deuterium exchange mass spectrometry (DXMS to probe the structural and conformational dynamics of EPAC2-F435G, a constitutively active EPAC2 mutant. Our study demonstrates that conformational dynamics plays a critical role in cAMP-induced EPAC activation. A glycine mutation at 435 position shifts the equilibrium of conformational dynamics towards the extended active conformation.

  13. Inhibition of the intrinsic factor X activating complex by protein S: evidence for a specific binding of protein S to factor VIII

    NARCIS (Netherlands)

    Koppelman, S.J.

    1995-01-01

    Protein S is a vitamin K-dependent nonenzymatic anticoagulant protein that acts as a cofactor to activated protein C. Recently it was shown that protein S inhibits the prothrombinase reaction independent of activated protein C. In this study, we show that protein S can also inhibit the intrinsic

  14. Immersion freezing of ice nucleation active protein complexes

    Directory of Open Access Journals (Sweden)

    S. Hartmann

    2013-06-01

    Full Text Available Utilising the Leipzig Aerosol Cloud Interaction Simulator (LACIS, the immersion freezing behaviour of droplet ensembles containing monodisperse particles, generated from a Snomax™ solution/suspension, was investigated. Thereto ice fractions were measured in the temperature range between −5 °C to −38 °C. Snomax™ is an industrial product applied for artificial snow production and contains Pseudomonas syringae} bacteria which have long been used as model organism for atmospheric relevant ice nucleation active (INA bacteria. The ice nucleation activity of such bacteria is controlled by INA protein complexes in their outer membrane. In our experiments, ice fractions increased steeply in the temperature range from about −6 °C to about −10 °C and then levelled off at ice fractions smaller than one. The plateau implies that not all examined droplets contained an INA protein complex. Assuming the INA protein complexes to be Poisson distributed over the investigated droplet populations, we developed the CHESS model (stoCHastic modEl of similar and poiSSon distributed ice nuclei which allows for the calculation of ice fractions as function of temperature and time for a given nucleation rate. Matching calculated and measured ice fractions, we determined and parameterised the nucleation rate of INA protein complexes exhibiting class III ice nucleation behaviour. Utilising the CHESS model, together with the determined nucleation rate, we compared predictions from the model to experimental data from the literature and found good agreement. We found that (a the heterogeneous ice nucleation rate expression quantifying the ice nucleation behaviour of the INA protein complex is capable of describing the ice nucleation behaviour observed in various experiments for both, Snomax™ and P. syringae bacteria, (b the ice nucleation rate, and its temperature dependence, seem to be very similar regardless of whether the INA protein complexes inducing ice

  15. Biological activity and dimerization state of modified phytochrome A proteins.

    Directory of Open Access Journals (Sweden)

    Peng Liu

    Full Text Available To assess potential physical interactions of type I phyA with the type II phyB-phyE phytochromes in vivo, transgenes expressing fusion gene forms of phyA were introduced into the Arabidopsis phyA mutant background. When a single c-Myc (myc epitope is added to either the N- or C-terminus of phyA, the constructs completely complement phyA mutant phenotypes. However, addition of larger tags, such as six consecutive myc epitopes or the yellow fluorescent protein sequence, result in fusion proteins that show reduced activity. All the tagged phyA proteins migrate as dimers on native gels and co-immunoprecipitation reveals no binding interaction of phyA to any of the type II phys in the dark or under continuous far-red light. Dimers of the phyA 1-615 amino acid N-terminal photosensory domain (NphyA, generated in vivo with a yeast GAL4 dimerization domain and attached to a constitutive nuclear localization sequence, are expressed at a low level and, although they cause a cop phenotype in darkness and mediate a very low fluence response to pulses of FR, have no activity under continuous FR. It is concluded that type I phyA in its Pr form is present in plants predominantly or exclusively as a homodimer and does not stably interact with type II phys in a dimer-to-dimer manner. In addition, its activity in mediating response to continuous FR is sensitive to modification of its N- or C-terminus.

  16. Activated protein C resistance testing for factor V Leiden.

    Science.gov (United States)

    Kadauke, Stephan; Khor, Bernard; Van Cott, Elizabeth M

    2014-12-01

    Activated protein C resistance assays can detect factor V Leiden with high accuracy, depending on the method used. Factor Xa inhibitors such as rivaroxaban and direct thrombin inhibitors including dabigatran, argatroban, and bivalirudin can cause falsely normal results. Lupus anticoagulants can cause incorrect results in most current assays. Assays that include dilution into factor V-deficient plasma are needed to avoid interference from factor deficiencies or elevations, which can arise from a wide variety of conditions such as warfarin, liver dysfunction, or pregnancy. The pros and cons of the currently available assays are discussed. © 2014 Wiley Periodicals, Inc.

  17. Redox regulation of the AMP-activated protein kinase.

    Directory of Open Access Journals (Sweden)

    Yingying Han

    2010-11-01

    Full Text Available Redox state is a critical determinant of cell function, and any major imbalances can cause severe damage or death.The aim of this study is to determine if AMP-activated protein kinase (AMPK, a cellular energy sensor, is activated by oxidants generated by Berberine in endothelial cells (EC.Bovine aortic endothelial cells (BAEC were exposed to Berberine. AMPK activity and reactive oxygen species were monitored after the incubation.In BAEC, Berberine caused a dose- and time-dependent increase in the phosphorylation of AMPK at Thr172 and acetyl CoA carboxylase (ACC at Ser79, a well characterized downstream target of AMPK. Concomitantly, Berberine increased peroxynitrite, a potent oxidant formed by simultaneous generation of superoxide and nitric oxide. Pre-incubation of BAEC with anti-oxidants markedly attenuated Berberine-enhanced phosphorylation of both AMPK and ACC. Consistently, adenoviral expression of superoxide dismutase and pretreatment of L-N(G-Nitroarginine methyl ester (L-NAME; a non-selective NOS inhibitor blunted Berberine-induced phosphorylation of AMPK. Furthermore, mitochondria-targeted tempol (mito-tempol pretreatment or expression of uncoupling protein attenuated AMPK activation caused by Berberine. Depletion of mitochondria abolished the effects of Berberine on AMPK in EC. Finally, Berberine significantly increased the phosphorylation of LKB1 at Ser307 and gene silencing of LKB1 attenuated Berberine-enhanced AMPK Thr172 phosphorylation in BAEC.Our results suggest that mitochondria-derived superoxide anions and peroxynitrite are required for Berberine-induced AMPK activation in endothelial cells.

  18. PENURUNAN KADAR PROTEIN LIMBAH CAIR TAHU DENGAN PEMANFAATAN KARBON BAGASSE TERAKTIVASI (Protein Reduction of Tofu Wastewater Using Activated Carbon Bagasse

    Directory of Open Access Journals (Sweden)

    Candra Purnawan

    2014-10-01

    Full Text Available ABSTRAK Penurunan kadar protein limbah tahu telah dilakukan dengan pemanfaatan karbon Bagasse teraktivasi. Tujuan dari penelitian ini adalah untuk mengetahui kondisi optimum dari karbon teraktivasi NaOH dan H2SO4 dalam menurunkan kadar protein limbah cair tahu dan mengetahui jenis isoterm adsorpsi dari karbon aktif yang digunakan untuk menyerap protein limbah cair tahu. Hasil penelitian menunjukkan konsentrasi NaOH yang optimum untuk aktivasi karbon aktif 15%, massa optimum karbon bagasse teraktivasi NaOH adalah 2 g dan penurunan kadar proteinnya 71,95%, sedangkan massa optimum karbon bagasse teraktivasi H2SO4 adalah 1 g dengan penurunan kadar protein sebesar 38,19%. Waktu kontak optimum karbon bagasse teraktivasi  NaOH dan H2SO4 adalah 12 jam. Adsorpsi protein oleh karbon bagasse teraktivasi NaOH mengikuti isoterm adsorpsi Langmuir dan Freundlich sedangkan karbon bagasse teraktivasi H2SO4 dominan mengikuti isoterm Freundlich.   ABSTRACT The protein reduction of tofu wastewater using activated carbon from bagasse  had been conducted. The purposes of this research were to analysis optimum condition of activated carbon bagsse using NaOH and H2SO4 for reduction protein in tofu wastewater, and analysis adsorption isotherm of activated carbon with protein. The result showed that optimum mass of carbon bagasse activated NaOH was  2 g with 71.95% protein reduction, while carbon bagasse activated H2SO4 has 1 g with 38.19% protein reduction. The optimum contact time between protein and activated carbon (with NaOH and H2SO4 was happened in 12 hours. Adsorption protein with carbon bagasse activated NaOH had followed Langmuir and Freundlich adsorption isotherm, while adsorption with carbon bagasse activated H2SO4 dominantlyhad followed Freundlich adsorption isotherm

  19. Protein tyrosine kinase but not protein kinase C inhibition blocks receptor induced alveolar macrophage activation

    Directory of Open Access Journals (Sweden)

    K. Pollock

    1993-01-01

    Full Text Available The selective enzyme inhibitors genistein and Ro 31-8220 were used to assess the importance of protein tyrosine kinase (PTK and protein kinase C (PKC, respectively, in N-formyl-methionyl-leucyl-phenylalanine (FMLP induced generation of superoxide anion and thromboxane B2 (TXB2 in guinea-pig alveolar macrophages (AM. Genistein (3–100 μM dose dependently inhibited FMLP (3 nM induced superoxide generation in non-primed AM and TXB2 release in non-primed or in lipopolysaccharide (LPS (10 ng/ml primed AM to a level > 80% but had litle effect up to 100 μM on phorbol myristate acetate (PMA (10 nM induced superoxide release. Ro 31-8220 inhibited PMA induced superoxide generation (IC50 0.21 ± 0.10 μM but had no effect on or potentiated (at 3 and 10 μM FMLP responses in non-primed AM. In contrast, when present during LPS priming as well as during FMLP challenge Ro 31-8220 (10 μM inhibited primed TXB2 release by > 80%. The results indicate that PTK activation is required for the generation of these inflammatory mediators by FMLP in AM. PKC activation appears to be required for LPS priming but not for transducing the FMLP signal; rather, PKC activation may modulate the signal by a negative feedback mechanism.

  20. Small Molecule Inhibitors Targeting Activator Protein 1 (AP-1)

    Science.gov (United States)

    2015-01-01

    Activator protein 1 (AP-1) is a pivotal transcription factor that regulates a wide range of cellular processes including proliferation, apoptosis, differentiation, survival, cell migration, and transformation. Accumulating evidence supports that AP-1 plays an important role in several severe disorders including cancer, fibrosis, and organ injury, as well as inflammatory disorders such as asthma, psoriasis, and rheumatoid arthritis. AP-1 has emerged as an actively pursued drug discovery target over the past decade. Excitingly, a selective AP-1 inhibitor T-5224 (51) has been investigated in phase II human clinical trials. Nevertheless, no effective AP-1 inhibitors have yet been approved for clinical use. Despite significant advances achieved in understanding AP-1 biology and function, as well as the identification of small molecules modulating AP-1 associated signaling pathways, medicinal chemistry efforts remain an urgent need to yield selective and efficacious AP-1 inhibitors as a viable therapeutic strategy for human diseases. PMID:24831826

  1. Antistaphylococcal activity of bacteriophage derived chimeric protein P128

    Directory of Open Access Journals (Sweden)

    Vipra Aradhana A

    2012-03-01

    Full Text Available Abstract Background Bacterial drug resistance is one of the most significant challenges to human health today. In particular, effective antibacterial agents against methicillin-resistant Staphylococcus aureus (MRSA are urgently needed. A causal relationship between nasal commensal S. aureus and infection has been reported. Accordingly, elimination of nasal S. aureus reduces the risk of infection. Enzymes that degrade bacterial cell walls show promise as antibacterial agents. Bacteriophage-encoded bacterial cell wall-degrading enzymes exhibit intrinsic bactericidal activity. P128 is a chimeric protein that combines the lethal activity of the phage tail-associated muralytic enzyme of Phage K and the staphylococcal cell wall targeting-domain (SH3b of lysostaphin. Here we report results of in vitro studies evaluating the susceptibility of staphylococcal strains to this novel protein. Results Using the broth microdilution method adapted for lysostaphin, we found that P128 is effective against S. aureus clinical strains including MRSA, methicillin-sensitive S. aureus (MSSA, and a mupirocin-resistant S. aureus. Minimum bactericidal concentrations and minimum inhibitory concentrations of P128 (1-64 μg/mL were similar across the 32 S. aureus strains tested, demonstrating its bactericidal nature. In time-kill assays, P128 reduced colony-forming units by 99.99% within 1 h and inhibited growth up to 24 h. In an assay simulating topical application of P128 to skin or other biological surfaces, P128 hydrogel was efficacious when layered on cells seeded on solid media. P128 hydrogel was lethal to Staphylococci recovered from nares of healthy people and treated without any processing or culturing steps, indicating its in situ efficacy. This methodology used for in vitro assessment of P128 as an agent for eradicating nasal carriage is unique. Conclusions The novel chimeric protein P128 is a staphylococcal cell wall-degrading enzyme under development for

  2. Rivaroxaban Causes Missed Diagnosis of Protein S Deficiency but Not of Activated Protein C Resistance (Factor V Leiden).

    Science.gov (United States)

    Maryamchik, Elena; Rosenbaum, Matthew W; Van Cott, Elizabeth M

    2018-01-01

    - Rivaroxaban causes a false increase in activated protein C resistance (APCR) ratios and protein S activity. - To investigate whether this increase masks a diagnosis of factor V Leiden (FVL) or protein S deficiency in a "real-world" population of patients undergoing rivaroxaban treatment and hypercoagulation testing. - During a 2.5-year period, we compared 4 groups of patients (n = 60): FVL heterozygous (FVL-HET)/taking rivaroxaban, wild-type/taking rivaroxaban, FVL-HET/no rivaroxaban, and normal APCR/no rivaroxaban. Patients taking rivaroxaban were tested for protein S functional activity and free antigen (n = 32). - The FVL-HET patients taking rivaroxaban had lower APCR ratios than wild-type patients ( P < .001). For FVL-HET patients taking rivaroxaban, mean APCR was 1.75 ± 0.12, versus 1.64 ± 0.3 in FVL-HET patients not taking rivaroxaban ( P = .005). Activated protein C resistance in FVL-HET patients fell more than 3 SDs below the cutoff of 2.2 at which the laboratory reflexes FVL DNA testing. No cases of FVL were missed despite rivaroxaban. In contrast, rivaroxaban falsely elevated functional protein S activity, regardless of the presence or absence of FVL ( P < .001). A total of 4 of 32 patients (12.5%) had low free protein S antigen (range, 58%-67%), whereas their functional protein S activity appeared normal (range 75%-130%). Rivaroxaban would have caused a missed diagnosis of all cases of protein S deficiency during the study if testing relied on the protein S activity assay alone. - Despite rivaroxaban treatment, APCR testing can distinguish FVL-HET from normal patients, rendering indiscriminate FVL DNA testing of all patients on rivaroxaban unnecessary. Free protein S should be tested in patients taking rivaroxaban to exclude hereditary protein S deficiency.

  3. Development of antimicrobial active packaging materials based on gluten proteins.

    Science.gov (United States)

    Gómez-Heincke, Diana; Martínez, Inmaculada; Partal, Pedro; Guerrero, Antonio; Gallegos, Críspulo

    2016-08-01

    The incorporation of natural biocide agents into protein-based bioplastics, a source of biodegradable polymeric materials, manufactured by a thermo-mechanical method is a way to contribute to a sustainable food packaging industry. This study assesses the antimicrobial activity of 10 different biocides incorporated into wheat gluten-based bioplastics. The effect that formulation, processing, and further thermal treatments exert on the thermo-mechanical properties, water absorption characteristics and rheological behaviour of these materials is also studied. Bioplastics containing six of the 10 examined bioactive agents have demonstrated suitable antimicrobial activity at 37 °C after their incorporation into the bioplastic. Moreover, the essential oils are able to create an antimicrobial atmosphere within a Petri dish. Depending on the selected biocide, its addition may alter the bioplastics protein network in a different extent, which leads to materials exhibiting less water uptake and different rheological and thermo-mechanical behaviours. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

  4. Stromal serine protein kinase activity in spinach chloroplasts

    International Nuclear Information System (INIS)

    Cortez, N.; Lucero, H.A.; Vallejos, R.H.

    1987-01-01

    At least twelve 32 P-labeled stromal proteins were detected by electrophoresis under denaturing conditions when intact chloroplasts were incubated with 32 Pi, in the light but only three were detected in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) or in the dark. Incubation of isolated stroma with [gamma- 32 P]ATP resulted in the preferential phosphorylation of one of them, a 70-kDa polypeptide, in serine residues. Thylakoid membranes in the dark promoted the phosphorylation of two additional stromal polypeptides of 55 and 40 kDa. Illumination during the phosphorylation of stroma in the presence of thylakoids stimulated severalfold the labeling of the 40-kDa polypeptide but not when DCMU was added. The protein kinase activity present in isolated stroma phosphorylated exogenous substrates like histone III, phosvitin, histone II, and casein with specific activities of 3, 1.8, 0.7, and 0.2 pmol X mg-1 X min-1. Histone III polypeptides were phosphorylated differently by stroma and by thylakoids in the dark. Moreover, histone III phosphorylated by thylakoids in the dark yielded a pattern of phosphopeptides after V8 protease treatment that was different from the pattern obtained when histone III was phosphorylated by stroma

  5. Modeling of human factor Va inactivation by activated protein C

    Directory of Open Access Journals (Sweden)

    Bravo Maria

    2012-05-01

    Full Text Available Abstract Background Because understanding of the inventory, connectivity and dynamics of the components characterizing the process of coagulation is relatively mature, it has become an attractive target for physiochemical modeling. Such models can potentially improve the design of therapeutics. The prothrombinase complex (composed of the protease factor (FXa and its cofactor FVa plays a central role in this network as the main producer of thrombin, which catalyses both the activation of platelets and the conversion of fibrinogen to fibrin, the main substances of a clot. A key negative feedback loop that prevents clot propagation beyond the site of injury is the thrombin-dependent generation of activated protein C (APC, an enzyme that inactivates FVa, thus neutralizing the prothrombinase complex. APC inactivation of FVa is complex, involving the production of partially active intermediates and “protection” of FVa from APC by both FXa and prothrombin. An empirically validated mathematical model of this process would be useful in advancing the predictive capacity of comprehensive models of coagulation. Results A model of human APC inactivation of prothrombinase was constructed in a stepwise fashion by analyzing time courses of FVa inactivation in empirical reaction systems with increasing number of interacting components and generating corresponding model constructs of each reaction system. Reaction mechanisms, rate constants and equilibrium constants informing these model constructs were initially derived from various research groups reporting on APC inactivation of FVa in isolation, or in the presence of FXa or prothrombin. Model predictions were assessed against empirical data measuring the appearance and disappearance of multiple FVa degradation intermediates as well as prothrombinase activity changes, with plasma proteins derived from multiple preparations. Our work integrates previously published findings and through the cooperative

  6. Regulation of mitogen-activated protein kinase 3/1 activity during meiosis resumption in mammals

    Czech Academy of Sciences Publication Activity Database

    Procházka, Radek; Blaha, Milan

    2015-01-01

    Roč. 61, č. 6 (2015), s. 495-502 ISSN 0916-8818 R&D Projects: GA MZe(CZ) QJ1510138 Institutional support: RVO:67985904 Keywords : cumulus oocyte complexes * meiosis resumption * mitogen-activated protein kinase 3/1 (MAPK3/1) Subject RIV: GI - Animal Husbandry ; Breeding Impact factor: 1.453, year: 2015

  7. Exercise in rats does not alter hypothalamic AMP-activated protein kinase activity

    DEFF Research Database (Denmark)

    Andersson, Ulrika; Treebak, Jonas Thue; Nielsen, Jakob Nis

    2005-01-01

    Recent studies have demonstrated that AMP-activated protein kinase (AMPK) in the hypothalamus is involved in the regulation of food intake. Because exercise is known to influence appetite and cause substrate depletion, it may also influence AMPK in the hypothalamus. Male rats that either rested o...

  8. The RecX protein interacts with the RecA protein and modulates its activity in Herbaspirillum seropedicae

    Directory of Open Access Journals (Sweden)

    C.W. Galvão

    2012-12-01

    Full Text Available DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecX Hs can interact with the H. seropedicaeRecA protein (RecA Hs and that RecA Hs possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecX Hs inhibited 90% of the RecA Hs DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecA Hs. RecA Hs ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecX Hs was present before the formation of RecA filaments (RecA-ssDNA, inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecX Hs protein negatively modulates the RecA Hs activities by protein-protein interactions and also by DNA-protein interactions.

  9. The RecX protein interacts with the RecA protein and modulates its activity in Herbaspirillum seropedicae

    International Nuclear Information System (INIS)

    Galvão, C.W.; Souza, E.M.; Etto, R.M.; Pedrosa, F.O.; Chubatsu, L.S.; Yates, M.G.; Schumacher, J.; Buck, M.; Steffens, M.B.R.

    2012-01-01

    DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecX Hs ) can interact with the H. seropedicae RecA protein (RecA Hs ) and that RecA Hs possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecX Hs inhibited 90% of the RecA Hs DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecA Hs . RecA Hs ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecX Hs was present before the formation of RecA filaments (RecA-ssDNA), inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecX Hs protein negatively modulates the RecA Hs activities by protein-protein interactions and also by DNA-protein interactions

  10. The RecX protein interacts with the RecA protein and modulates its activity in Herbaspirillum seropedicae

    Energy Technology Data Exchange (ETDEWEB)

    Galvão, C.W. [Departamento de Biologia Estrutural, Molecular e Genética, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR (Brazil); Souza, E.M. [Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR (Brazil); Etto, R.M. [Departamento de Biologia Estrutural, Molecular e Genética, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR (Brazil); Pedrosa, F.O.; Chubatsu, L.S.; Yates, M.G. [Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR (Brazil); Schumacher, J.; Buck, M. [Department of Life Sciences, Imperial College London, London (United Kingdom); Steffens, M.B.R. [Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR (Brazil)

    2012-10-15

    DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecX{sub Hs}) can interact with the H. seropedicae RecA protein (RecA{sub Hs}) and that RecA{sub Hs} possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecX{sub Hs} inhibited 90% of the RecA{sub Hs} DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecA{sub Hs}. RecA{sub Hs} ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecX{sub Hs} was present before the formation of RecA filaments (RecA-ssDNA), inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecX{sub Hs} protein negatively modulates the RecA{sub Hs} activities by protein-protein interactions and also by DNA-protein interactions.

  11. The potent, indirect adenosine monophosphate-activated protein kinase activator R419 attenuates mitogen-activated protein kinase signaling, inhibits nociceptor excitability, and reduces pain hypersensitivity in mice

    Directory of Open Access Journals (Sweden)

    Galo L. Mejia

    2016-07-01

    Full Text Available Abstract. There is a great need for new therapeutics for the treatment of pain. A possible avenue to development of such therapeutics is to interfere with signaling pathways engaged in peripheral nociceptors that cause these neurons to become hyperexcitable. There is strong evidence that mitogen-activated protein kinases and phosphoinositide 3-kinase (PI3K/mechanistic target of rapamycin signaling pathways are key modulators of nociceptor excitability in vitro and in vivo. Activation of adenosine monophosphate-activated protein kinase (AMPK can inhibit signaling in both of these pathways, and AMPK activators have been shown to inhibit nociceptor excitability and pain hypersensitivity in rodents. R419 is one of, if not the most potent AMPK activator described to date. We tested whether R419 activates AMPK in dorsal root ganglion (DRG neurons and if this leads to decreased pain hypersensitivity in mice. We find that R419 activates AMPK in DRG neurons resulting in decreased mitogen-activated protein kinase signaling, decreased nascent protein synthesis, and enhanced P body formation. R419 attenuates nerve growth factor (NGF-induced changes in excitability in DRG neurons and blocks NGF-induced mechanical pain amplification in vivo. Moreover, locally applied R419 attenuates pain hypersensitivity in a model of postsurgical pain and blocks the development of hyperalgesic priming in response to both NGF and incision. We conclude that R419 is a promising lead candidate compound for the development of potent and specific AMPK activation to inhibit pain hypersensitivity as a result of injury.

  12. Characterization of type IV antifreeze gene in Nile tilapia (Oreochromis niloticus) and influence of cold and hot weather on its expression and some immune-related genes.

    Science.gov (United States)

    Ammar, Asmma Y; El Nahas, Abeer F; Mahmoud, Shawky; Barakat, Mohamed E; Hassan, Asmaa M

    2018-04-01

    The aim of this work is to study the effect of the thermal stress of ambient temperature during winter and summer on the expression of type IV antifreeze gene (ANF IV) in different tissues of Nile tilapia (Oreochromis niloticus) as well as some immune-related genes. At first, genomic ANF IV gene was characterized from one fish; 124 amino acids were identified with 92.7% similarity with that on the gene bank. Expression of ANF IV and immune-related genes were done twice, once at the end of December (winter sample, temperature 14 °C) and the other at August (summer sample, temperature 36 °C). Assessment of ANF IV gene expression in different organs of fish was done; splenic mRNA was used for assessment of immune-related gene transcripts (CXCl2 chemokine, cc-chemokine, INF-3A, and MHC IIβ). Winter expression analysis of AFP IV in O. niloticus revealed significant upregulation of mRNA transcript levels in the intestine, gills, skin, spleen, liver, and brain with 324.03-, 170.06-, 107.63-, 97.61-, 94.35-, and 27.85-folds, respectively. Furthermore, upregulation in the gene was observed in some organs during summer: in the liver, gills, skin, intestine, and brain with lower levels compared with winter. The level of expression of immune-related genes in winter is significantly higher than summer in all assessed genes. Cc-chemokine gene expression was the most affected in both winter and summer. Variable expression profile of ANF IV in different organs and in different seasons together with its amino acid similarity of N-terminal and C-terminal with apolipoprotein (lipid binder) and form of high-density lipoprotein (HDL) suggests a different role for this protein which may be related to lipid metabolism.

  13. Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Ines eLassowskat

    2014-10-01

    Full Text Available Mitogen-activated protein kinases (MAPKs target a variety of protein substrates to regulate cellular signaling processes in eukaryotes. In plants, the number of identified MAPK substrates that control plant defense responses is still limited. Here, we generated transgenic Arabidopsis thaliana plants with an inducible system to simulate in vivo activation of two stress-activated MAPKs, MPK3 and MPK6. Metabolome analysis revealed that this artificial MPK3/6 activation (without any exposure to pathogens or other stresses is sufficient to drive the production of major defense-related metabolites, including various camalexin, indole glucosinolate and agmatine derivatives. An accompanying (phosphoproteome analysis led to detection of hundreds of potential phosphoproteins downstream of MPK3/6 activation. Besides known MAPK substrates, many candidates on this list possess typical MAPK-targeted phosphosites and in many cases, the corresponding phosphopeptides were detected by mass spectrometry. Notably, several of these putative phosphoproteins have been reported to be associated with the biosynthesis of antimicrobial defense substances (e.g. WRKY transcription factors and proteins encoded by the genes from the PEN pathway required for penetration resistance to filamentous pathogens. Thus, this work provides an inventory of candidate phosphoproteins, including putative direct MAPK substrates, for future analysis of MAPK-mediated defense control. (Proteomics data are available with the identifier PXD001252 via ProteomeXchange, http://proteomecentral.proteomexchange.org.

  14. Strategies for the photo-control of endogenous protein activity.

    Science.gov (United States)

    Brechun, Katherine E; Arndt, Katja M; Woolley, G Andrew

    2017-08-01

    Photo-controlled or 'optogenetic' effectors interfacing with endogenous protein machinery allow the roles of endogenous proteins to be probed. There are two main approaches being used to develop optogenetic effectors: (i) caging strategies using photo-controlled conformational changes, and (ii) protein relocalization strategies using photo-controlled protein-protein interactions. Numerous specific examples of these approaches have been reported and efforts to develop general methods for photo-control of endogenous proteins are a current focus. The development of improved screening and selection methods for photo-switchable proteins would advance the field. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Exploring the active site structure of photoreceptor proteins by Raman optical activity

    Science.gov (United States)

    Unno, Masashi

    2015-03-01

    Understanding protein function at the atomic level is a major challenge in a field of biophysics and requires the combined efforts of structural and functional methods. We use photoreceptor proteins as a model system to understand in atomic detail how a chromophore and a protein interact to sense light and send a biological signal. A potential technique for investigating molecular structures is Raman optical activity (ROA), which is a spectroscopic method with a high sensitivity to the structural details of chiral molecules. However, its application to photoreceptor proteins has not been reported. Thus we have constructed ROA spectrometer using near-infrared (NIR) laser excitation at 785 nm. The NIR excitation enables us to measure ROA spectra for a variety of biological samples, including photoreceptor proteins, without fluorescence from the samples. In the present study, we have applied the NIR-ROA to bacteriorhodopsin (BR) and photoactive yellow protein (PYP). BR is a light-driven proton pump and contains a protonated Schiff base of retinal as a chromophore. PYP is a blue light receptor, and this protein has the 4-hydroxycinnamyl chromophore, which is covalently linked to Cys69 through a thiolester bond. We have successfully obtained the ROA spectra of the chromophore within a protein environment. Furthermore, calculations of the ROA spectra utilizing density functional theory provide detailed structural information, such as data on out-of-plane distortions of the chromophore. The structural information obtained from the ROA spectra includes the positions of hydrogen atoms, which are usually not detected in the crystal structures of biological samples.

  16. Anticoagulant activity of a natural protein purified from Hypomesus olidus.

    Science.gov (United States)

    Gou, Mengxing; Wang, Liyan; Liu, Xuejun

    2017-05-01

    A novel anticoagulant protein (E-II-1) was separated and purified from Hypomesus olidus, a unique freshwater fish in northern China. E-II-1 had a molecular mass of approximately 40 kDa with no subunits. The high content of hydrophobic amino acids and negatively charged amino acids in E-II-1 demonstrated that the amino acid compositions might contribute to the anticoagulant activity. E-II-1 contained α-helices 16.75%, β-sheets 42.67%, β-turn 25.58% and random coil 15.00%. In vitro blood coagulation time assay, E-II-1 significantly prolonged the activated partial thrombin time in a dose-dependent manner. Results indicated that E-II-1 acted as anticoagulants through the endogenous pathway with an inhibition of FXa. The specific activity of E-II-1 was 103.50 U/mg at a concentration of 1.00 mg/mL. Therefore, E-II-1 might be one of the promising anticoagulants originated from natural food sources with more safety and less side effects.

  17. Modulation of protein C activation by histones, platelet factor 4, and heparinoids: new insights into activated protein C formation.

    Science.gov (United States)

    Kowalska, M Anna; Zhao, Guohua; Zhai, Li; David, George; Marcus, Stephen; Krishnaswamy, Sriram; Poncz, Mortimer

    2014-01-01

    Histones are detrimental in late sepsis. Both activated protein C (aPC) and heparin can reverse their effect. Here, we investigated whether histones can modulate aPC generation in a manner similar to another positively charged molecule, platelet factor 4, and how heparinoids (unfractionated heparin or oxygen-desulfated unfractionated heparin with marked decrease anticoagulant activity) may modulate this effect. We measured in vitro and in vivo effects of histones, platelet factor 4, and heparinoids on aPC formation, activated partial thromboplastin time, and murine survival. In vitro, histones and platelet factor 4 both affect thrombin/thrombomodulin aPC generation following a bell-shaped curve, with a peak of >5-fold enhancement. Heparinoids shift these curves rightward. Murine aPC generation studies after infusions of histones, platelet factor 4, and heparinoids supported the in vitro data. Importantly, although unfractionated heparin and 2-O, 3-O desulfated heparin both reversed the lethality of high-dose histone infusions, only mice treated with 2-O, 3-O desulfated heparin demonstrated corrected activated partial thromboplastin times and had significant levels of aPC. Our data provide a new contextual model of how histones affect aPC generation, and how heparinoid therapy may be beneficial in sepsis. These studies provide new insights into the complex interactions controlling aPC formation and suggest a novel therapeutic interventional strategy.

  18. Ice growth in supercooled solutions of a biological "antifreeze", AFGP 1-5: an explanation in terms of adsorption rate for the concentration dependence of the freezing point.

    Science.gov (United States)

    Knight, C A; DeVries, A L

    2009-07-21

    It is widely accepted, and we agree, that the lowering of the temperature at which ice can grow in a water solution of one of the biological antifreezes is a result of adsorption of the antifreeze molecules at the ice surface. However, how this can produce a well-defined "freezing point" that varies with the solution concentration has remained problematical. The results of a series of measurements of ice growing in supercooled solutions of an effective antifreeze are reported and interpreted in terms of this fundamental problem. It seemed that the solution of the problem would have to rely upon adsorption rate, because that appeared to be the only way for the concentration in solution to be so important. The crystal growth results are most unusual, and appear to confirm this. The growth rates over a wide range of antifreeze concentration in solution (about 0.05 to 9 mg ml(-1)) are zero from the thermodynamic freezing point down to the "non-equilibrium" freezing point, where there is a very sudden increase to a plateau value that then remains about constant as the supercooling is increased by about 2 degrees C. The plateau values of growth rate are faster than those from pure water at the lower-supercooling ends of the plateaus, but slower at higher supercooling, until the growth rate starts rising toward that from pure water. These plateau values of growth rate increase markedly with increasing concentration of the antifreeze in solution. Along with these changes there are complex changes in the growth orientations, from c-axis spicules in the plateaus to those more characteristic of growth from pure water at greater supercooling. We conclude that the non-equilibrium freezing point is determined by the adsorption rate. It is the warmest temperature at which the ice growth rate on the basal plane (where the antifreeze does not adsorb) is fast enough to prevent the area of basal face on a growing ice crystal from becoming too small to grow, which is determined in

  19. Thermal hysteresis proteins.

    Science.gov (United States)

    Barrett, J

    2001-02-01

    Extreme environments present a wealth of biochemical adaptations. Thermal hysteresis proteins (THPs) have been found in vertebrates, invertebrates, plants, bacteria and fungi and are able to depress the freezing point of water (in the presence of ice crystals) in a non-colligative manner by binding to the surface of nascent ice crystals. The THPs comprise a disparate group of proteins with a variety of tertiary structures and often no common sequence similarities or structural motifs. Different THPs bind to different faces of the ice crystal, and no single mechanism has been proposed to account for THP ice binding affinity and specificity. Experimentally THPs have been used in the cryopreservation of tissues and cells and to induce cold tolerance in freeze susceptible organisms. THPs represent a remarkable example of parallel and convergent evolution with different proteins being adapted for an anti-freeze role.

  20. Effects of anti-freeze concentration in the engine coolant on the cavitation temperature of a water pump

    International Nuclear Information System (INIS)

    David Huang, K.; Tzeng, S.-C.; Ma Weiping

    2004-01-01

    Improvements in engine-manufacturing technology have gradually increased the thermal efficiencies of engines as well as the burning temperature and pressure of fuels within the cylinders. Accordingly, greater heat dissipation are required. However, the volume of the radiators is constrained by the configuration of the engines, leading to excessive internal resistance in the engine-cooling system. Therefore, water pumps in engines are prone to cavitation, and air bubbles are likely to permeate into the anti-freeze, thereby severely reducing the performance, reliability and service life of the engines. Ethylene glycol (EG) is added to the radiator of some vehicles in cold areas to reduce the solidification point of the coolant and prevent freezing. This study probes the effects of the percentage of anti-freeze added to the cooling water in a water pump in an engine on the water-supply capability and cavitation temperature, whether air or burnt gas is present in the system. The results of this study have revealed that engines have a higher tolerance to air bubbles at lower rates of rotation. At a given fixed rotational speed, the tolerable cavitation temperature of an engine's water pump will fall slowly as the amount of air bubbles increases

  1. New Activity of a Protein from Canavalia ensiformis

    Directory of Open Access Journals (Sweden)

    Vanya Petkova BOGOEVA

    2014-06-01

    Full Text Available Concanavalin A is a legume lectin which preferentially agglutinates transformed cells and shows antitumor effects on human breast carcinoma cells in vitro and in vivo. It is considered as a new potential antineoplastic agent targeting apoptosis, autophagy, and anti-angiogenesis in preclinical or clinical trials for cancer therapeutics, which has recently become the object of intensive study. In the present investigation, we show the capacity of the lectin to bind manganese, gold, iron, and zinc porphyrins: all potential anticancer agents. The interaction of the legume lectin with the studied compounds has been investigated by tryptophan fluorescence, showing conformational changes within the quaternary and tertiary structures of the protein. The binding of Con A with manganese, gold, and iron porphyrins, as well as adenine, was studied by fluorescence quenching. In contrast, the interaction of Con A with zinc porphyrin caused an increase in Trp fluorescence and a red shift of 10 nm of the emission maximum position. However, the binding of Con A to iron porphyrin was accompanied by a 5 nm blue shift of the emission maximum, and a kD of 0.95 ± 0.13 μM was calculated, respectively. The sigmoidal shape of the curve showed cooperative interactions, which indicated the presence of more than one class of binding site within the Con A molecule for iron porphyrin, confirmed by the Hill slope (h = 1.89±0.46. We have found that the legume lectin interacts with porphyrins and adenine with an affinity (0.14–1.89 μM similar to that of the non-legume lectin, wheat germ agglutinin. In conclusion, the protein Con A shows new binding activity towards porphyrins with anticancer activities and could find prospective application as a drug delivery molecule that specifically targets cancer cells.

  2. Engineering Fluorogen Activating Proteins into Self-Assembling Materials

    Science.gov (United States)

    Saunders, Matthew J.; Liu, Wen; Szent-Gyorgyi, Christopher; Wen, Yi; Drennen, Zachary; Waggoner, Alan S.; Meng, Wilson S.

    2013-01-01

    We present herein characteristics of a conjugate in which dL5, a fluorogen-activating protein (FAP) and AEAEAKAK, an amphiphilic peptide are combined to form a solid-phase fluorescence-detection platform. The FAP dL5 is a covalently linked dimer of two identical light chain variable fragments which activates the fluorescence of the fluorogen malachite green (MG). The amphiphilic peptide of sequence AEAEAKAK is a building block of stimuli-responsive materials that undergoes sol-gel phase transition at high ionic strengths. We hypothesize that the novel bi-functional protein containing both the FAP and the amphiphile, termed dL5_EAK, co-assembles with the self-assembling peptide [AEAEAKAK]2 (EAK16-II) to form an insoluble membrane composite whereby the fluorescence enhancement function of the FAP domain remains intact. Denaturing polyacrylamide electrophoresis indicated that greater than 78% of dL5_EAK incorporates into the EAK16-II membrane. Conversely, less than 32% of dL5 without the EAK sequence associates with the insoluble fraction of EAK16-II in buffers. Membranes containing dL5_EAK and EAK16-II exhibited at least 4-fold higher fluorescence intensity compared to mixtures containing dL5 and EAK16-II. Scanning electron microscopy revealed the presence of particulates, presumably FAPs, scattering on the membrane fibrils. The evidence suggests a system of materials that can be developed into in situ-forming local sensors by immobilizing dL5 into coacervate, on which MG can be detected. It is envisioned that dL5 membranes can be established in diseased locales to monitor infiltration and migration of inflammatory cells marked with antibodies conjugated to MG. PMID:23573960

  3. Activation of AMP-activated protein kinase by tributyltin induces neuronal cell death

    International Nuclear Information System (INIS)

    Nakatsu, Yusuke; Kotake, Yaichiro; Hino, Atsuko; Ohta, Shigeru

    2008-01-01

    AMP-activated protein kinase (AMPK), a member of the metabolite-sensing protein kinase family, is activated by energy deficiency and is abundantly expressed in neurons. The environmental pollutant, tributyltin chloride (TBT), is a neurotoxin, and has been reported to decrease cellular ATP in some types of cells. Therefore, we investigated whether TBT activates AMPK, and whether its activation contributes to neuronal cell death, using primary cultures of cortical neurons. Cellular ATP levels were decreased 0.5 h after exposure to 500 nM TBT, and the reduction was time-dependent. It was confirmed that most neurons in our culture system express AMPK, and that TBT induced phosphorylation of AMPK. Compound C, an AMPK inhibitor, reduced the neurotoxicity of TBT, suggesting that AMPK is involved in TBT-induced cell death. Next, the downstream target of AMPK activation was investigated. Nitric oxide synthase, p38 phosphorylation and Akt dephosphorylation were not downstream of TBT-induced AMPK activation because these factors were not affected by compound C, but glutamate release was suggested to be controlled by AMPK. Our results suggest that activation of AMPK by TBT causes neuronal death through mediating glutamate release

  4. Protein implicated in nonsyndromic mental retardation regulates protein kinase A (PKA) activity

    KAUST Repository

    Altawashi, Azza

    2012-02-28

    Mutation of the coiled-coil and C2 domain-containing 1A (CC2D1A) gene, which encodes a C2 domain and DM14 domain-containing protein, has been linked to severe autosomal recessive nonsyndromic mental retardation. Using a mouse model that produces a truncated form of CC2D1A that lacks the C2 domain and three of the four DM14 domains, we show that CC2D1A is important for neuronal differentiation and brain development. CC2D1A mutant neurons are hypersensitive to stress and have a reduced capacitytoformdendritesandsynapsesinculture. Atthebiochemical level,CC2D1Atransduces signals to the cyclic adenosine 3?,5?-monophosphate (cAMP)-protein kinase A (PKA) pathway during neuronal cell differentiation. PKA activity is compromised, and the translocation of its catalytic subunit to the nucleus is also defective in CC2D1A mutant cells. Consistently, phosphorylation of the PKA target cAMP-responsive element-binding protein, at serine 133, is nearly abolished in CC2D1A mutant cells. The defects in cAMP/PKA signaling were observed in fibroblast, macrophage, and neuronal primary cells derived from the CC2D1A KO mice. CC2D1A associates with the cAMP-PKA complex following forskolin treatment and accumulates in vesicles or on the plasma membrane in wild-type cells, suggesting that CC2D1A may recruit the PKA complex to the membrane to facilitate signal transduction. Together, our data show that CC2D1A is an important regulator of the cAMP/PKA signaling pathway, which may be the underlying cause for impaired mental function in nonsyndromic mental retardation patients with CC2D1A mutation. 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Cyclic nucleotides and mitogen-activated protein kinases: regulation of simvastatin in platelet activation

    Directory of Open Access Journals (Sweden)

    Hou Ssu-Yu

    2010-06-01

    Full Text Available Abstract Background 3-Hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA reductase inhibitors (statins have been widely used to reduce cardiovascular risk. These statins (i.e., simvastatin may exert other effects besides from their cholesterol-lowering actions, including inhibition of platelet activation. Platelet activation is relevant to a variety of coronary heart diseases. Although the inhibitory effect of simvastatin in platelet activation has been studied; the detailed signal transductions by which simvastatin inhibit platelet activation has not yet been completely resolved. Methods The aim of this study was to systematically examine the detailed mechanisms of simvastatin in preventing platelet activation. Platelet aggregation, flow cytometric analysis, immunoblotting, and electron spin resonance studies were used to assess the antiplatelet activity of simvastatin. Results Simvastatin (20-50 μM exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen than other agonists (i.e., thrombin. Simvastatin inhibited collagen-stimulated platelet activation accompanied by [Ca2+]i mobilization, thromboxane A2 (TxA2 formation, and phospholipase C (PLCγ2, protein kinase C (PKC, and mitogen-activated protein kinases (i.e., p38 MAPK, JNKs phosphorylation in washed platelets. Simvastatin obviously increased both cyclic AMP and cyclic GMP levels. Simvastatin markedly increased NO release, vasodilator-stimulated phosphoprotein (VASP phosphorylation, and endothelial nitric oxide synthase (eNOS expression. SQ22536, an inhibitor of adenylate cyclase, markedly reversed the simvastatin-mediated inhibitory effects on platelet aggregation, PLCγ2 and p38 MAPK phosphorylation, and simvastatin-mediated stimulatory effects on VASP and eNOS phosphorylation. Conclusion The most important findings of this study demonstrate for the first time that inhibitory effect of simvastatin in platelet activation may involve activation of the cyclic AMP

  6. Xylazine Activates Adenosine Monophosphate-Activated Protein Kinase Pathway in the Central Nervous System of Rats.

    Directory of Open Access Journals (Sweden)

    Xing-Xing Shi

    Full Text Available Xylazine is a potent analgesic extensively used in veterinary and animal experimentation. Evidence exists that the analgesic effect can be inhibited using adenosine 5'-monophosphate activated protein kinase (AMPK inhibitors. Considering this idea, the aim of this study was to investigate whether the AMPK signaling pathway is involved in the central analgesic mechanism of xylazine in the rat. Xylazine was administrated via the intraperitoneal route. Sprague-Dawley rats were sacrificed and the cerebral cortex, cerebellum, hippocampus, thalamus and brainstem were collected for determination of liver kinase B1 (LKB1 and AMPKα mRNA expression using quantitative real-time polymerase chain reaction (qPCR, and phosphorylated LKB1 and AMPKα levels using western blot. The results of our study showed that compared with the control group, xylazine induced significant increases in AMPK activity in the cerebral cortex, hippocampus, thalamus and cerebellum after rats received xylazine (P < 0.01. Increased AMPK activities were accompanied with increased phosphorylation levels of LKB1 in corresponding regions of rats. The protein levels of phosphorylated LKB1 and AMPKα in these regions returned or tended to return to control group levels. However, in the brainstem, phosphorylated LKB1 and AMPKα protein levels were decreased by xylazine compared with the control (P < 0.05. In conclusion, our data indicates that xylazine alters the activities of LKB1 and AMPK in the central nervous system of rats, which suggests that xylazine affects the regulatory signaling pathway of the analgesic mechanism in the rat brain.

  7. Scaffold protein enigma homolog activates CREB whereas a short splice variant prevents CREB activation in cardiomyocytes.

    Science.gov (United States)

    Ito, Jumpei; Iijima, Masumi; Yoshimoto, Nobuo; Niimi, Tomoaki; Kuroda, Shun'ichi; Maturana, Andrés D

    2015-12-01

    Enigma Homolog (ENH1 or Pdlim5) is a scaffold protein composed of an N-terminal PDZ domain and three LIM domains at the C-terminal end. The enh gene encodes for several splice variants with opposing functions. ENH1 promotes cardiomyocytes hypertrophy whereas ENH splice variants lacking LIM domains prevent it. ENH1 interacts with various Protein Kinase C (PKC) isozymes and Protein Kinase D1 (PKD1). In addition, the binding of ENH1's LIM domains to PKC is sufficient to activate the kinase without stimulation. The downstream events of the ENH1-PKC/PKD1 complex remain unknown. PKC and PKD1 are known to phosphorylate the transcription factor cAMP-response element binding protein (CREB). We tested whether ENH1 could play a role in the activation of CREB. We found that, in neonatal rat ventricular cardiomyocytes, ENH1 interacts with CREB, is necessary for the phosphorylation of CREB at ser133, and the activation of CREB-dependent transcription. On the contrary, the overexpression of ENH3, a LIM-less splice variant, inhibited the phosphorylation of CREB. ENH3 overexpression or shRNA knockdown of ENH1 prevented the CREB-dependent transcription. Our results thus suggest that ENH1 plays an essential role in CREB's activation and dependent transcription in cardiomyocytes. At the opposite, ENH3 prevents the CREB transcriptional activity. In conclusion, these results provide a first molecular explanation to the opposing functions of ENH splice variants. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. PIAS proteins are involved in the SUMO-1 modification, intracellular translocation and transcriptional repressive activity of RET finger protein

    International Nuclear Information System (INIS)

    Matsuura, Tetsuo; Shimono, Yohei; Kawai, Kumi; Murakami, Hideki; Urano, Takeshi; Niwa, Yasumasa; Goto, Hidemi; Takahashi, Masahide

    2005-01-01

    Ret finger protein (RFP) is a nuclear protein that is highly expressed in testis and in various tumor cell lines. RFP functions as a transcriptional repressor and associates with Enhancer of Polycomb 1 (EPC1), a member of the Polycomb group proteins, and Mi-2β, a main component of the nucleosome remodeling and deacetylase (NuRD) complex. We show that RFP binds with PIAS (protein inhibitor of activated STAT) proteins, PIAS1, PIAS3, PIASxα and PIASy at their carboxyl-terminal region and is covalently modified by SUMO-1 (sumoylation). PIAS proteins enhance the sumoylation of RFP in a dose-dependent manner and induce the translocation of RFP into nuclear bodies reminiscent of the PML bodies. In addition, co-expression of PIAS proteins or SUMO-1 strengthened the transcriptional repressive activity of RFP. Finally, our immunohistochemical results show that RFP, SUMO-1 and PIASy localize in a characteristic nuclear structure juxtaposed with the inner nuclear membrane (XY body) of primary spermatocytes in mouse testis. These results demonstrate that the intracellular location and the transcriptional activity of RFP are modified by PIAS proteins which possess SUMO E3 ligase activities and suggest that they may play a co-operative role in spermatogenesis

  9. Site-specific incorporation of redox active amino acids into proteins

    Energy Technology Data Exchange (ETDEWEB)

    Alfonta, Lital; Schultz, Peter G.; Zhang, Zhiwen

    2017-10-10

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

  10. Site-specific incorporation of redox active amino acids into proteins

    Science.gov (United States)

    Alfonta, Lital [San Diego, CA; Schultz, Peter G [La Jolla, CA; Zhang, Zhiwen [San Diego, CA

    2009-02-24

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

  11. Metformin activates type I interferon signaling against HCV via activation of adenosine monophosphate-activated protein kinase.

    Science.gov (United States)

    Tsai, Wei-Lun; Chang, Tsung-Hsien; Sun, Wei-Chi; Chan, Hoi-Hung; Wu, Chun-Ching; Hsu, Ping-I; Cheng, Jin-Shiung; Yu, Ming-Lung

    2017-11-03

    Activation of the type I interferon (IFN) signaling pathway is essential for the eradication of hepatitis C virus (HCV). Metformin can activate adenosine monophosphate-activated protein kinase (AMPK) to reduce insulin resistance. Cross talks between AMPK and IFN signaling remain unclear. To understand the influence of metformin on the type I IFN signaling pathway and HCV infection, the full-length HCV replicon OR6 cells and the infectious HCV clones JFH1 were used to assess the anti-HCV effect of the insulin sensitizers, metformin and pioglitazone. Immunofluorescence staining and the immunoblotting of HCV viral protein demonstrated that metformin, but not pioglitazone, inhibited HCV replication in OR-6 and JFH-1-infected Huh 7.5.1 cells. Immunoblotting data showed that metformin activated the phosphorylation of STAT-1 and STAT-2 in OR-6 and JFH-1 infected Huh 7.5.1 cells. Metformin enhanced the phosphorylation of AMPK, and the metformin-activated IFN signaling was down-regulated by AMPK inhibitor. After treatment of AMPK inhibitor, the level of HCV core protein decreased by metformin can be rescued. In conclusion, metformin activates type I interferon signaling and inhibits the replication of HCV via activation of AMPK.

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    OBJECTIVE During energy stress, AMP-activated protein kinase (AMPK) promotes glucose transport and glycolysis for ATP production, while it is thought to inhibit anabolic glycogen synthesis by suppressing the activity of glycogen synthase (GS) to maintain the energy balance in muscle. Paradoxically......, chronic activation of AMPK causes an increase in glycogen accumulation in skeletal and cardiac muscles, which in some cases is associated with cardiac dysfunction. The aim of this study was to elucidate the molecular mechanism by which AMPK activation promotes muscle glycogen accumulation. RESEARCH DESIGN...... caused a modest inactivation of GS, it stimulated muscle glycogen synthesis that was accompanied by increases in glucose transport and intracellular [G6P]. These effects of AICAR required the catalytic activity of AMPK. Strikingly, AICAR-induced glycogen synthesis was completely abolished in G6P...

  13. Stimulation of Leishmania tropica protein kinase CK2 activities by platelet-activating factor (PAF).

    Science.gov (United States)

    Dutra, Patricia M L; Vieira, Danielle P; Meyer-Fernandes, Jose R; Silva-Neto, Mario A C; Lopes, Angela H

    2009-09-01

    Leishmania tropica is one of the causative agents of cutaneous leishmaniasis. Platelet-activating factor (PAF) is a phospholipid mediator in diverse biological and pathophysiological processes. Here we show that PAF promoted a three-fold increase on ecto-protein kinase and a three-fold increase on the secreted kinase activity of L. tropica live promastigotes. When casein was added to the reaction medium, along with PAF, there was a four-fold increase on the ecto-kinase activity. When live L. tropica promastigotes were pre-incubated for 30 min in the presence of PAF-plus casein, a six-fold increase on the secreted kinase activity was observed. Also, a protein released from L. tropica promastigotes reacted with polyclonal antibodies for the mammalian CK2 alpha catalytic subunit. Furthermore, in vitro mouse macrophage infection by L. tropica was doubled when promastigotes were pre-treated for 2 h with PAF. Similar results were obtained when the interaction was performed in the presence of purified CK2 or casein. TBB and DRB, CK2 inhibitors, reversed PAF enhancement of macrophage infection by L. tropica. WEB 2086, a competitive PAF antagonist, reversed all PAF effects here described. This study shows for the first time that PAF promotes the activation of two isoforms of CK2, secreted and membrane-bound, correlating these activities to infection of mouse macrophages.

  14. Protein Inhibitors of Activated STAT (Pias1 and Piasy) Differentially Regulate Pituitary Homeobox 2 (PITX2) Transcriptional Activity*

    Science.gov (United States)

    Wang, Jianbo; Sun, Zhao; Zhang, Zichao; Saadi, Irfan; Wang, Jun; Li, Xiao; Gao, Shan; Engle, Jamison J.; Kuburas, Adisa; Fu, Xueyao; Yu, Wenjie; Klein, William H.; Russo, Andrew F.; Amendt, Brad A.

    2013-01-01

    Protein inhibitors of activated STAT (Pias) proteins can act independent of sumoylation to modulate the activity of transcription factors and Pias proteins interacting with transcription factors can either activate or repress their activity. Pias proteins are expressed in many tissues and cells during development and we asked if Pias proteins regulated the pituitary homeobox 2 (PITX2) homeodomain protein, which modulates developmental gene expression. Piasy and Pias1 proteins are expressed during craniofacial/tooth development and directly interact and differentially regulate PITX2 transcriptional activity. Piasy and Pias1 are co-expressed in craniofacial tissues with PITX2. Yeast two-hybrid, co-immunoprecipitation and pulldown experiments demonstrate Piasy and Pias1 interactions with the PITX2 protein. Piasy interacts with the PITX2 C-terminal tail to attenuate its transcriptional activity. In contrast, Pias1 interacts with the PITX2 C-terminal tail to increase PITX2 transcriptional activity. The E3 ligase activity associated with the RING domain in Piasy is not required for the attenuation of PITX2 activity, however, the RING domain of Pias1 is required for enhanced PITX2 transcriptional activity. Bimolecular fluorescence complementation assays reveal PITX2 interactions with Piasy and Pias1 in the nucleus. Piasy represses the synergistic activation of PITX2 with interacting co-factors and Piasy represses Pias1 activation of PITX2 transcriptional activity. In contrast, Pias1 did not affect the synergistic interaction of PITX2 with transcriptional co-factors. Last, we demonstrate that Pias proteins form a complex with PITX2 and Lef-1, and PITX2 and β-catenin. Lef-1, β-catenin, and Pias interactions with PITX2 provide new molecular mechanisms for the regulation of PITX2 transcriptional activity and the activity of Pias proteins. PMID:23515314

  15. Protein inhibitors of activated STAT (Pias1 and Piasy) differentially regulate pituitary homeobox 2 (PITX2) transcriptional activity.

    Science.gov (United States)

    Wang, Jianbo; Sun, Zhao; Zhang, Zichao; Saadi, Irfan; Wang, Jun; Li, Xiao; Gao, Shan; Engle, Jamison J; Kuburas, Adisa; Fu, Xueyao; Yu, Wenjie; Klein, William H; Russo, Andrew F; Amendt, Brad A

    2013-05-03

    Protein inhibitors of activated STAT (Pias) proteins can act independent of sumoylation to modulate the activity of transcription factors and Pias proteins interacting with transcription factors can either activate or repress their activity. Pias proteins are expressed in many tissues and cells during development and we asked if Pias proteins regulated the pituitary homeobox 2 (PITX2) homeodomain protein, which modulates developmental gene expression. Piasy and Pias1 proteins are expressed during craniofacial/tooth development and directly interact and differentially regulate PITX2 transcriptional activity. Piasy and Pias1 are co-expressed in craniofacial tissues with PITX2. Yeast two-hybrid, co-immunoprecipitation and pulldown experiments demonstrate Piasy and Pias1 interactions with the PITX2 protein. Piasy interacts with the PITX2 C-terminal tail to attenuate its transcriptional activity. In contrast, Pias1 interacts with the PITX2 C-terminal tail to increase PITX2 transcriptional activity. The E3 ligase activity associated with the RING domain in Piasy is not required for the attenuation of PITX2 activity, however, the RING domain of Pias1 is required for enhanced PITX2 transcriptional activity. Bimolecular fluorescence complementation assays reveal PITX2 interactions with Piasy and Pias1 in the nucleus. Piasy represses the synergistic activation of PITX2 with interacting co-factors and Piasy represses Pias1 activation of PITX2 transcriptional activity. In contrast, Pias1 did not affect the synergistic interaction of PITX2 with transcriptional co-factors. Last, we demonstrate that Pias proteins form a complex with PITX2 and Lef-1, and PITX2 and β-catenin. Lef-1, β-catenin, and Pias interactions with PITX2 provide new molecular mechanisms for the regulation of PITX2 transcriptional activity and the activity of Pias proteins.

  16. Chemical synthesis of biologically active monoglycosylated GM2-activator protein analogue using N-sulfanylethylanilide peptide.

    Science.gov (United States)

    Sato, Kohei; Shigenaga, Akira; Kitakaze, Keisuke; Sakamoto, Ken; Tsuji, Daisuke; Itoh, Kohji; Otaka, Akira

    2013-07-22

    Going to SEA(lide): Total chemical synthesis of a 162-residue glycoprotein analogue of the monoglycosylated human GM2-activator protein (GM2AP) was achieved. Key steps were the use of N-sulfanylethylanilide (SEAlide) peptides in the kinetic chemical ligation synthesis of a large peptide fragment, and a convergent native chemical ligation for final fragment assembly. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Antioxidant activity of black bean (Phaseolus vulgaris L.) protein hydrolysates

    Science.gov (United States)

    The objective of this work was to study the effect of enzymatic hydrolysis of black bean protein concentrate using different enzymes. Bean proteins were extracted and hydrolyzed over a period of 120 min using the enzymes pepsin or alcalase. The protein hydrolysates’ molecular weight was assayed by e...

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

    African Journals Online (AJOL)

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

  19. Expression of the GM2 activator protein in mouse testis.

    Science.gov (United States)

    Li, Yu-Teh; Li, Su-Chen; Chen, I-Li

    2017-12-01

    The GM2-activator protein (GM2-AP), revealed by Li et al. in 1973 in human liver, was initially identified as a protein cofactor that stimulated β-hexosaminidase A to hydrolyze N-acetylgalactosamine from GM2 ganglioside. This cofactor was found to be missing in human variant AB Tay-Sachs disease. Over the years, the GM2-AP has also been shown to be involved in kidney vesicular transport, lipid presentation by CD1 molecule to T-cells, and interaction of human sperm with zona pellucida. Since the expression of the GM2-AP via mRNA detection in mouse tissues was found to be the highest in testis, we became interested in the localization of the GM2-AP at cellular level in mouse testis during spermatogenesis. Using immunohistochemical analysis and electron microscopy, we found that the GM2-AP was predominantly localized in the basal cytoplasm and the attenuated processes of Sertoli cells. The stained structure appeared to be lysosomes. The most interesting finding was the association of the GM2-AP with the acrosomal apparatus in early spermatids. A modest to intense staining was observed in some acrosomal granules and acrosomal caps. The GM2-AP seemed to disappear from acrosomal caps in the later stage of spermatids, in which the nucleus became elongated and condensed. These results suggest that the GM2-AP may be involved in the normal functions of Sertoli cells and play important roles during the development of acrosomal caps in the early spermatids. Copyright © 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  20. Heat Shock Protein 70 Modulates Influenza A Virus Polymerase Activity*

    Science.gov (United States)

    Manzoor, Rashid; Kuroda, Kazumichi; Yoshida, Reiko; Tsuda, Yoshimi; Fujikura, Daisuke; Miyamoto, Hiroko; Kajihara, Masahiro; Kida, Hiroshi; Takada, Ayato

    2014-01-01

    The role of heat shock protein 70 (Hsp70) in virus replication has been discussed for many viruses. The known suppressive role of Hsp70 in influenza virus replication is based on studies conducted in cells with various Hsp70 expression levels. In this study, we determined the role of Hsp70 in influenza virus replication in HeLa and HEK293T cells, which express Hsp70 constitutively. Co-immunoprecipitation and immunofluorescence studies revealed that Hsp70 interacted with PB2 or PB1 monomers and PB2/PB1 heterodimer but not with the PB1/PA heterodimer or PB2/PB1/PA heterotrimer and translocated into the nucleus with PB2 monomers or PB2/PB1 heterodimers. Knocking down Hsp70 resulted in reduced virus transcription and replication activities. Reporter gene assay, immunofluorescence assay, and Western blot analysis of nuclear and cytoplasmic fractions from infected cells demonstrated that the increase in viral polymerase activity during the heat shock phase was accompanied with an increase in Hsp70 and viral polymerases levels in the nuclei, where influenza virus replication takes place, whereas a reduction in viral polymerase activity was accompanied with an increase in cytoplasmic relocation of Hsp70 along with viral polymerases. Moreover, significantly higher levels of viral genomic RNA (vRNA) were observed during the heat shock phase than during the recovery phase. Overall, for the first time, these findings suggest that Hsp70 may act as a chaperone for influenza virus polymerase, and the modulatory effect of Hsp70 appears to be a sequel of shuttling of Hsp70 between nuclear and cytoplasmic compartments. PMID:24474693

  1. Rheb Inhibits Protein Synthesis by Activating the PERK-eIF2α Signaling Cascade

    Directory of Open Access Journals (Sweden)

    Richa Tyagi

    2015-02-01

    Full Text Available Rheb, a ubiquitous small GTPase, is well known to bind and activate mTOR, which augments protein synthesis. Inhibition of protein synthesis is also physiologically regulated. Thus, with cell stress, the unfolded protein response system leads to phosphorylation of the initiation factor eIF2α and arrest of protein synthesis. We now demonstrate a major role for Rheb in inhibiting protein synthesis by enhancing the phosphorylation of eIF2α by protein kinase-like ER kinase (PERK. Interplay between the stimulatory and inhibitory roles of Rheb may enable cells to modulate protein synthesis in response to varying environmental stresses.

  2. Background on international activities on protein quality assessment of foods.

    Science.gov (United States)

    Gilani, G Sarwar

    2012-08-01

    The subject of protein quality assessment of foods and diets was addressed at the Codex Committee on Vegetable Proteins (1982-1989), FAO/WHO (1989, 2001) and WHO/FAO (2002) expert reviews. These international developments are summarized in this manuscript. In 1989, a Joint FAO/WHO Expert Consultation on Protein Quality Evaluation reviewed knowledge of protein quality assessment of foods, and specifically evaluated amino acid score corrected for protein digestibility, the method recommended by the Codex Committee on Vegetable Proteins. The report of the Consultation published in 1991 concluded that the Protein Digestibility-corrected Amino Acid Score (PDCAAS) method was the most suitable approach for routine evaluation of protein quality for humans. The Consultation recognized that the amino acid scoring pattern proposed by FAO/WHO/UNU (1985) for preschool children was at that time the most suitable pattern for calculating PDCAAS for all ages except infants in which case the amino acid composition of human milk was recommended to be the basis of the scoring pattern. The rat balance method was considered as the most suitable practical method for predicting protein digestibility by humans. Since its adoption by FAO/WHO (1991), the PDCAAS method has been criticised for a number of reasons. The FAO/WHO (2001) Working Group on analytical issues related to protein quality assessed the validity of criticisms of the PDCAAS method. While recognizing a distinct regulatory use of protein quality data, the Working Group recommended that the PDCAAS method may be inappropriate for the routine prediction of protein quality of novel and sole source foods which contain high levels of anti nutritional factors; and that for regulatory purposes, the method should be revised to permit values of >100 for high quality proteins. In evaluating the recommendations of the Working Group, the WHO/FAO (2002) Expert Consultation on Protein and Amino Acid Requirements endorsed the PDCAAS method

  3. Protein Conformational Gating of Enzymatic Activity in Xanthine Oxidoreductase

    Energy Technology Data Exchange (ETDEWEB)

    Ishikita, Hiroshi; Eger, Bryan T.; Okamoto, Ken; Nishino, Takeshi; Pai, Emil F. (Toronto); (Kyoto)

    2012-05-24

    In mammals, xanthine oxidoreductase can exist as xanthine dehydrogenase (XDH) and xanthine oxidase (XO). The two enzymes possess common redox active cofactors, which form an electron transfer (ET) pathway terminated by a flavin cofactor. In spite of identical protein primary structures, the redox potential difference between XDH and XO for the flavin semiquinone/hydroquinone pair (E{sub sq/hq}) is {approx}170 mV, a striking difference. The former greatly prefers NAD{sup +} as ultimate substrate for ET from the iron-sulfur cluster FeS-II via flavin while the latter only accepts dioxygen. In XDH (without NAD{sup +}), however, the redox potential of the electron donor FeS-II is 180 mV higher than that for the acceptor flavin, yielding an energetically uphill ET. On the basis of new 1.65, 2.3, 1.9, and 2.2 {angstrom} resolution crystal structures for XDH, XO, the NAD{sup +}- and NADH-complexed XDH, E{sub sq/hq} were calculated to better understand how the enzyme activates an ET from FeS-II to flavin. The majority of the E{sub sq/hq} difference between XDH and XO originates from a conformational change in the loop at positions 423-433 near the flavin binding site, causing the differences in stability of the semiquinone state. There was no large conformational change observed in response to NAD{sup +} binding at XDH. Instead, the positive charge of the NAD{sup +} ring, deprotonation of Asp429, and capping of the bulk surface of the flavin by the NAD{sup +} molecule all contribute to altering E{sub sq/hq} upon NAD{sup +} binding to XDH.

  4. [Activated protein C resistance and factor V Leiden: clinical interest].

    Science.gov (United States)

    Guermazi, S; Znazen, R

    2011-10-01

    Activated protein C resistance (APCR) is a coagulation abnormality often linked to FV Leiden mutation, a single nucleotide G1691A substitution resulting in arginine 506→glutamine missense factor V mutation. FV Leiden has a frequency of 20 to 30% in groups of patients with venous thrombosis while it is of 4 to 10% in normal subjects. FV Leiden is considered as a weak risk factor of thrombosis except in homozygote. FV Leiden is implicated in deep venous thrombosis occurrence. Duration of oral anticoagulant treatment is six months in patients developing a first venous thrombosis except in patients with combined defects or a clinical context suggesting a high risk of severe relapse. Detection of APCR by coagulation methods is often used in first intention with a high specificity if plasmas tested are diluted in factor V deficient plasma. Genotyping study is essential to establish the heterozygote or homozygote statute and certain teams perform it directly. Nevertheless, APCR not related to FV Leiden could be an independent thrombosis risk factor. APCR and FV Leiden are included in laboratory investigations of thrombophilic markers in patients less than 50 years with venous thrombosis. In arterial thrombosis, FV Leiden implication is weak or absent. FV Leiden increases the risk of thrombosis in other situations as in patients with cancer. An association with recurrent miscarriages and other vasculoplacental complications is also reported in many studies but the data concerning the efficacy of antithrombotic treatment to prevent recurrence are currently insufficient. Copyright © 2009 Elsevier Masson SAS. All rights reserved.

  5. Protective features of resveratrol on human spermatozoa cryopreservation may be mediated through 5' AMP-activated protein kinase activation.

    Science.gov (United States)

    Shabani Nashtaei, M; Amidi, F; Sedighi Gilani, M A; Aleyasin, A; Bakhshalizadeh, Sh; Naji, M; Nekoonam, S

    2017-03-01

    Biochemical and physical modifications during the freeze-thaw process adversely influence the restoration of energy-dependent sperm functions required for fertilization. Resveratrol, a phytoalexin, has been introduced to activate 5' AMP-activated protein kinase which is a cell energy sensor and a cell metabolism regulator. The cryoprotection of resveratrol on sperm cryoinjury via activation of AMP-activated protein kinase also remains to be elucidated. Our aim, thus, was to investigate: (i) the presence and intracellular localization of AMP-activated protein kinase protein; (ii) whether resveratrol may exert a protective effect on certain functional properties of fresh and post-thaw human spermatozoa through modulation of AMP-activated protein kinase. Spermatozoa from normozoospermic men were incubated with or without different concentrations of Compound C as an AMP-activated protein kinase inhibitor or resveratrol as an AMP-activated protein kinase activator for different lengths of time and were then cryopreserved. AMP-activated protein kinase is expressed essentially in the entire flagellum and the post-equatorial region. Viability of fresh spermatozoa was not significantly affected by the presence of Compound C or resveratrol. However, although Compound C caused a potent inhibition of spermatozoa motility parameters, resveratrol did not induce negative effect, except a significant reduction in motility at 25 μm for 1 h. Furthermore, resveratrol significantly increased AMP-activated protein kinase phosphorylation and mitochondrial membrane potential and decreased reactive oxygen species and apoptosis-like changes in frozen-thawed spermatozoa. Nevertheless, it was not able to compensate decreased sperm viability and motility parameters following cryopreservation. In contrast, Compound C showed opposite effects to resveratrol on AMP-activated protein kinase phosphorylation, reactive oxygen species, apoptosis-like changes, mitochondrial membrane potential, and

  6. Mitogen-activated protein kinases interacting kinases are autoinhibited by a reprogrammed activation segment.

    Science.gov (United States)

    Jauch, Ralf; Cho, Min-Kyu; Jäkel, Stefan; Netter, Catharina; Schreiter, Kay; Aicher, Babette; Zweckstetter, Markus; Jäckle, Herbert; Wahl, Markus C

    2006-09-06

    Autoinhibition is a recurring mode of protein kinase regulation and can be based on diverse molecular mechanisms. Here, we show by crystal structure analysis, nuclear magnetic resonance (NMR)-based nucleotide affinity studies and rational mutagenesis that nonphosphorylated mitogen-activated protein (MAP) kinases interacting kinase (Mnk) 1 is autoinhibited by conversion of the activation segment into an autoinhibitory module. In a Mnk1 crystal structure, the activation segment is repositioned via a Mnk-specific sequence insertion at the N-terminal lobe with the following consequences: (i) the peptide substrate binding site is deconstructed, (ii) the interlobal cleft is narrowed, (iii) an essential Lys-Glu pair is disrupted and (iv) the magnesium-binding loop is locked into an ATP-competitive conformation. Consistently, deletion of the Mnk-specific insertion or removal of a conserved phenylalanine side chain, which induces a blockade of the ATP pocket, increase the ATP affinity of Mnk1. Structural rearrangements required for the activation of Mnks are apparent from the cocrystal structure of a Mnk2 D228G -staurosporine complex and can be modeled on the basis of crystal packing interactions. Our data suggest a novel regulatory mechanism specific for the Mnk subfamily.

  7. Function of the activated protein C (APC) autolysis loop in activated FVIII inactivation.

    Science.gov (United States)

    Cramer, Thomas J; Gale, Andrew J

    2011-06-01

    Activated protein C (APC) binds to its substrates activated factor V (FVa) and activated factor VIII (FVIIIa) with a basic exosite that consists of loops 37, 60, 70 and the autolysis loop. These loops have a high density of basic residues, resulting in a positive charge on the surface of APC. Many of these residues are important in the interaction of APC with FVa and FVIIIa. The current study focused on the function of the autolysis loop in the interaction with FVIIIa. This loop was previously shown to interact with FVa, and it inhibits APC inactivation by plasma serpins. Charged residues of the autolysis loop were individually mutated to alanine and the activity of these mutants was assessed in functional FVIIIa inactivation assays. The autolysis loop was functionally important for FVIIIa inactivation. Mutation of R306, K311 and R314 each resulted in significantly reduced FVIIIa inactivation. The inactivating cleavages of FVIIIa at R336 and R562 were affected equally by the mutations. Protein S and FV stimulated cleavage at R562 more than cleavage at R336, independent of mutations in the autolysis loop. Together, these results confirmed that the autolysis loop plays a significant role as part of the basic exosite on APC in the interaction with FVIIIa. © 2011 Blackwell Publishing Ltd.

  8. An activated medium with high durability and low nonspecific adsorption: application to protein A chromatography.

    Science.gov (United States)

    Maeno, Katsuyuki; Hirayama, Aya; Sakuma, Kenichi; Miyazawa, Kazuyuki

    2011-02-01

    Activated media allow the user to easily synthesize a variety of affinity media. We have developed a novel activated medium based on porous silica modified with phosphorylcholine (PC) and N-hydroxysuccinimide (NHS) groups for the purpose of high-throughput purification and reducing nonspecific protein adsorption. The PC groups function as suppressors of nonspecific protein adsorption, whereas the NHS groups are able to covalently bind to the primary amino groups of ligands. Because protein A affinity medium is the most frequently used affinity medium, we prepared protein A media in which a recombinant protein A was bound to the NHS groups of the activated media and evaluated its utility. After optimizing various factors in the synthetic process, the resultant protein A medium showed improved durability at a high flow rate over 300 purification cycles and reduced nonspecific protein adsorption compared with commercially available protein A media. Copyright © 2010 Elsevier Inc. All rights reserved.

  9. Unc-51 controls active zone density and protein composition by downregulating ERK signaling

    OpenAIRE

    Wairkar, Yogesh P.; Toda, Hirofumi; Mochizuki, Hiroaki; Furukubo-Tokunaga, Katsuo; Tomoda, Toshifumi; DiAntonio, Aaron

    2009-01-01

    Efficient synaptic transmission requires the apposition of neurotransmitter release sites opposite clusters of postsynaptic neurotransmitter receptors. Transmitter is released at active zones, which are composed of a large complex of proteins necessary for synaptic development and function. Many active zone proteins have been identified, but little is known of the mechanisms that ensure that each active zone receives the proper complement of proteins. Here we use a genetic analysis in Drosoph...

  10. Acid-denatured Green Fluorescent Protein (GFP) as model substrate to study the chaperone activity of protein disulfide isomerase.

    Science.gov (United States)

    Mares, Rosa E; Meléndez-López, Samuel G; Ramos, Marco A

    2011-01-01

    Green fluorescent protein (GFP) has been widely used in several molecular and cellular biology applications, since it is remarkably stable in vitro and in vivo. Interestingly, native GFP is resistant to the most common chemical denaturants; however, a low fluorescence signal has been observed after acid-induced denaturation. Furthermore, this acid-denatured GFP has been used as substrate in studies of the folding activity of some bacterial chaperones and other chaperone-like molecules. Protein disulfide isomerase enzymes, a family of eukaryotic oxidoreductases that catalyze the oxidation and isomerization of disulfide bonds in nascent polypeptides, play a key role in protein folding and it could display chaperone activity. However, contrasting results have been reported using different proteins as model substrates. Here, we report the further application of GFP as a model substrate to study the chaperone activity of protein disulfide isomerase (PDI) enzymes. Since refolding of acid-denatured GFP can be easily and directly monitored, a simple micro-assay was used to study the effect of the molecular participants in protein refolding assisted by PDI. Additionally, the effect of a well-known inhibitor of PDI chaperone activity was also analyzed. Because of the diversity their functional activities, PDI enzymes are potentially interesting drug targets. Since PDI may be implicated in the protection of cells against ER stress, including cancer cells, inhibitors of PDI might be able to enhance the efficacy of cancer chemotherapy; furthermore, it has been demonstrated that blocking the reductive cleavage of disulfide bonds of proteins associated with the cell surface markedly reduces the infectivity of the human immunodeficiency virus. Although several high-throughput screening (HTS) assays to test PDI reductase activity have been described, we report here a novel and simple micro-assay to test the chaperone activity of PDI enzymes, which is amenable for HTS of PDI

  11. Purification of recombinant tissue plasminogen activator (rtPA) protein from transplastomic tobacco plants.

    Science.gov (United States)

    Abdoli Nasab, Maryam; Jalali Javaran, Mokhtar; Cusido, Rosa M; Palazon, Javier

    2016-11-01

    Plants are low cost platforms for the production of recombinant proteins, but their complexity renders the purification of plant recombinant proteins more difficult than proteins expressed in yeast or bacteria. Plastid transformation enables high-level expression of foreign genes and the accumulation of recombinant proteins in plastid organelles. Histidine (His) tags are widely used for affinity purification of recombinant proteins in a nickel column. The human tissue-type plasminogen activator (tPA) is one of the most important pharmaceutical recombinant proteins involved in the breakdown of blood clots in different parts of the body. The truncated form of the tissue plasminogen activator (K2S) has a longer plasma half-life, better diffusion into the clot, and higher fibrinolytic activity. In a construct designed to insert the K2S gene in the tobacco chloroplast, the sequence of six histidines and a factor Xa protease site was fused to the C-terminus of the K2S protein. The presence and amount of tPA recombinant protein in transplastomic tobacco plants was estimated by ELISA analysis using a specific antibody. The protein was purified from total soluble protein, insoluble protein aggregates and the protein was extracted from the isolated chloroplast using nickel resin and a chromatography column. After digestion of the purified protein with factor Xa, the presence of the purified tPA protein was confirmed by western blot analysis. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  12. Beyond AICA riboside: in search of new specific AMP-activated protein kinase activators.

    Science.gov (United States)

    Guigas, Bruno; Sakamoto, Kei; Taleux, Nellie; Reyna, Sara M; Musi, Nicolas; Viollet, Benoit; Hue, Louis

    2009-01-01

    5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICA riboside) has been extensively used in vitro and in vivo to activate the AMP-activated protein kinase (AMPK), a metabolic sensor involved in both cellular and whole body energy homeostasis. However, it has been recently highlighted that AICA riboside also exerts AMPK-independent effects, mainly on AMP-regulated enzymes and mitochondrial oxidative phosphorylation (OXPHOS), leading to the conclusion that new compounds with reduced off target effects are needed to specifically activate AMPK. Here, we review recent findings on newly discovered AMPK activators, notably on A-769662, a nonnucleoside compound from the thienopyridone family. We also report that A-769662 is able to activate AMPK and stimulate glucose uptake in both L6 cells and primary myotubes derived from human satellite cells. In addition, A-769662 increases AMPK activity and phosphorylation of its main downstream targets in primary cultured rat hepatocytes but, by contrast with AICA riboside, does neither affect mitochondrial OXPHOS nor change cellular AMP:ATP ratio. We conclude that A-769662 could be one of the new promising chemical agents to activate AMPK with limited AMPK-independent side effects.

  13. Construction and genetic selection of small transmembrane proteins that activate the human erythropoietin receptor.

    Science.gov (United States)

    Cammett, Tobin J; Jun, Susan J; Cohen, Emily B; Barrera, Francisco N; Engelman, Donald M; Dimaio, Daniel

    2010-02-23

    This work describes a genetic approach to isolate small, artificial transmembrane (TM) proteins with biological activity. The bovine papillomavirus E5 protein is a dimeric, 44-amino acid TM protein that transforms cells by specifically binding and activating the platelet-derived growth factor beta receptor (PDGFbetaR). We used the E5 protein as a scaffold to construct a retrovirus library expressing approximately 500,000 unique 44-amino acid proteins with randomized TM domains. We screened this library to select small, dimeric TM proteins that were structurally unrelated to erythropoietin (EPO), but specifically activated the human EPO receptor (hEPOR). These proteins did not activate the murine EPOR or the PDGFbetaR. Genetic studies with one of these activators suggested that it interacted with the TM domain of the hEPOR. Furthermore, this TM activator supported erythroid differentiation of primary human hematopoietic progenitor cells in vitro in the absence of EPO. Thus, we have changed the specificity of a protein so that it no longer recognizes its natural target but, instead, modulates an entirely different protein. This represents a novel strategy to isolate small artificial proteins that affect diverse membrane proteins. We suggest the word "traptamer" for these transmembrane aptamers.

  14. Intracellular protein delivery activity of peptides derived from insulin-like growth factor binding proteins 3 and 5

    International Nuclear Information System (INIS)

    Goda, Natsuko; Tenno, Takeshi; Inomata, Kosuke; Shirakawa, Masahiro; Tanaka, Toshiki; Hiroaki, Hidekazu

    2008-01-01

    Insulin-like growth factor binding proteins (IGFBPs) have various IGF-independent cellular activities, including receptor-independent cellular uptake followed by transcriptional regulation, although mechanisms of cellular entry remain unclear. Herein, we focused on their receptor-independent cellular entry mechanism in terms of protein transduction domain (PTD) activity, which is an emerging technique useful for clinical applications. The peptides of 18 amino acid residues derived from IGFBP-3 and IGFBP-5, which involve heparin-binding regions, mediated cellular delivery of an exogenous protein into NIH3T3 and HeLa cells. Relative protein delivery activities of IGFBP-3/5-derived peptides were approximately 20-150% compared to that of the HIV-Tat peptide, a potent PTD. Heparin inhibited the uptake of the fusion proteins with IGFBP-3 and IGFBP-5, indicating that the delivery pathway is heparin-dependent endocytosis, similar to that of HIV-Tat. The delivery of GST fused to HIV-Tat was competed by either IGFBP-3 or IGFBP-5-derived synthetic peptides. Therefore, the entry pathways of the three PTDs are shared. Our data has shown a new approach for designing protein delivery systems using IGFBP-3/5 derived peptides based on the molecular mechanisms of IGF-independent activities of IGFBPs

  15. Cheese from ultrafiltered milk : whey proteins and chymosin activity

    NARCIS (Netherlands)

    Buijsse, C.A.P.

    1999-01-01

    The manufacture of (semi-)hard cheese from ultrafiltered milk (UF-cheese) enables the partial incorporation of whey proteins in the cheese, thereby increasing its yield. The transfer of whey proteins in curd from (UF-)milk was studied in relation to the degree of ultrafiltration of the milk

  16. A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein

    DEFF Research Database (Denmark)

    Whorton, Matthew R; Bokoch, Michael P; Rasmussen, Søren Gøgsig Faarup

    2007-01-01

    G protein-coupled receptors (GPCRs) respond to a diverse array of ligands, mediating cellular responses to hormones and neurotransmitters, as well as the senses of smell and taste. The structures of the GPCR rhodopsin and several G proteins have been determined by x-ray crystallography, yet...... the organization of the signaling complex between GPCRs and G proteins is poorly understood. The observations that some GPCRs are obligate heterodimers, and that many GPCRs form both homo- and heterodimers, has led to speculation that GPCR dimers may be required for efficient activation of G proteins. However......, technical limitations have precluded a definitive analysis of G protein coupling to monomeric GPCRs in a biochemically defined and membrane-bound system. Here we demonstrate that a prototypical GPCR, the beta2-adrenergic receptor (beta2AR), can be incorporated into a reconstituted high-density lipoprotein...

  17. Regulation of protease-activated receptor 1 signaling by the adaptor protein complex 2 and R4 subfamily of regulator of G protein signaling proteins.

    Science.gov (United States)

    Chen, Buxin; Siderovski, David P; Neubig, Richard R; Lawson, Mark A; Trejo, Joann

    2014-01-17

    The G protein-coupled protease-activated receptor 1 (PAR1) is irreversibly proteolytically activated by thrombin. Hence, the precise regulation of PAR1 signaling is important for proper cellular responses. In addition to desensitization, internalization and lysosomal sorting of activated PAR1 are critical for the termination of signaling. Unlike most G protein-coupled receptors, PAR1 internalization is mediated by the clathrin adaptor protein complex 2 (AP-2) and epsin-1, rather than β-arrestins. However, the function of AP-2 and epsin-1 in the regulation of PAR1 signaling is not known. Here, we report that AP-2, and not epsin-1, regulates activated PAR1-stimulated phosphoinositide hydrolysis via two different mechanisms that involve, in part, a subset of R4 subfamily of "regulator of G protein signaling" (RGS) proteins. A significantly greater increase in activated PAR1 signaling was observed in cells depleted of AP-2 using siRNA or in cells expressing a PAR1 (420)AKKAA(424) mutant with defective AP-2 binding. This effect was attributed to AP-2 modulation of PAR1 surface expression and efficiency of G protein coupling. We further found that ectopic expression of R4 subfamily members RGS2, RGS3, RGS4, and RGS5 reduced activated PAR1 wild-type signaling, whereas signaling by the PAR1 AKKAA mutant was minimally affected. Intriguingly, siRNA-mediated depletion analysis revealed a function for RGS5 in the regulation of signaling by the PAR1 wild type but not the AKKAA mutant. Moreover, activation of the PAR1 wild type, and not the AKKAA mutant, induced Gαq association with RGS3 via an AP-2-dependent mechanism. Thus, AP-2 regulates activated PAR1 signaling by altering receptor surface expression and through recruitment of RGS proteins.

  18. Effects of gamma irradiation on chickpea seeds vis-a-vis total seed storage proteins, antioxidant activity and protein profiling.

    Science.gov (United States)

    Bhagyawant, S S; Gupta, N; Shrivastava, N

    2015-10-23

    The present work describes radiation—induced effects on seed composition vis—à—vis total seed proteins, antioxidant levels and protein profiling employing two dimensional gel electrophoresis (2D—GE) in kabuli and desi chickpea varities. Seeds were exposed to the radiation doses of 1,2,3,4 and 5 kGy. The total protein concentrations decreased and antioxidant levels were increased with increasing dose compared to control seed samples. Radiation induced effects were dose dependent to these seed parameters while it showed tolerance to 1 kGy dose. Increase in the dose was complimented with increase in antioxidant levels, like 5 kGy enhanced % scavenging activities in all the seed extracts. Precisely, the investigations reflected that the dose range from 2 to 5 kGy was effective for total seed storage proteins, as depicted quantitatively and qualitative 2D—GE means enhance antioxidant activities in vitro.

  19. Ohmyungsamycins promote antimicrobial responses through autophagy activation via AMP-activated protein kinase pathway.

    Science.gov (United States)

    Kim, Tae Sung; Shin, Yern-Hyerk; Lee, Hye-Mi; Kim, Jin Kyung; Choe, Jin Ho; Jang, Ji-Chan; Um, Soohyun; Jin, Hyo Sun; Komatsu, Masaaki; Cha, Guang-Ho; Chae, Han-Jung; Oh, Dong-Chan; Jo, Eun-Kyeong

    2017-06-13

    The induction of host cell autophagy by various autophagy inducers contributes to the antimicrobial host defense against Mycobacterium tuberculosis (Mtb), a major pathogenic strain that causes human tuberculosis. In this study, we present a role for the newly identified cyclic peptides ohmyungsamycins (OMS) A and B in the antimicrobial responses against Mtb infections by activating autophagy in murine bone marrow-derived macrophages (BMDMs). OMS robustly activated autophagy, which was essentially required for the colocalization of LC3 autophagosomes with bacterial phagosomes and antimicrobial responses against Mtb in BMDMs. Using a Drosophila melanogaster-Mycobacterium marinum infection model, we showed that OMS-A-induced autophagy contributed to the increased survival of infected flies and the limitation of bacterial load. We further showed that OMS triggered AMP-activated protein kinase (AMPK) activation, which was required for OMS-mediated phagosome maturation and antimicrobial responses against Mtb. Moreover, treating BMDMs with OMS led to dose-dependent inhibition of macrophage inflammatory responses, which was also dependent on AMPK activation. Collectively, these data show that OMS is a promising candidate for new anti-mycobacterial therapeutics by activating antibacterial autophagy via AMPK-dependent signaling and suppressing excessive inflammation during Mtb infections.

  20. Regulation of AMP-activated protein kinase by natural and synthetic activators

    Directory of Open Access Journals (Sweden)

    David Grahame Hardie

    2016-01-01

    Full Text Available The AMP-activated protein kinase (AMPK is a sensor of cellular energy status that is almost universally expressed in eukaryotic cells. While it appears to have evolved in single-celled eukaryotes to regulate energy balance in a cell-autonomous manner, during the evolution of multicellular animals its role has become adapted so that it also regulates energy balance at the whole body level, by responding to hormones that act primarily on the hypothalamus. AMPK monitors energy balance at the cellular level by sensing the ratios of AMP/ATP and ADP/ATP, and recent structural analyses of the AMPK heterotrimer that have provided insight into the complex mechanisms for these effects will be discussed. Given the central importance of energy balance in diseases that are major causes of morbidity or death in humans, such as type 2 diabetes, cancer and inflammatory disorders, there has been a major drive to develop pharmacological activators of AMPK. Many such activators have been described, and the various mechanisms by which these activate AMPK will be discussed. A particularly large class of AMPK activators are natural products of plants derived from traditional herbal medicines. While the mechanism by which most of these activate AMPK has not yet been addressed, I will argue that many of them may be defensive compounds produced by plants to deter infection by pathogens or grazing by insects or herbivores, and that many of them will turn out to be inhibitors of mitochondrial function.

  1. Reduced activity of AMP-activated protein kinase protects against genetic models of motor neuron disease.

    Science.gov (United States)

    Lim, M A; Selak, M A; Xiang, Z; Krainc, D; Neve, R L; Kraemer, B C; Watts, J L; Kalb, R G

    2012-01-18

    A growing body of research indicates that amyotrophic lateral sclerosis (ALS) patients and mouse models of ALS exhibit metabolic dysfunction. A subpopulation of ALS patients possesses higher levels of resting energy expenditure and lower fat-free mass compared to healthy controls. Similarly, two mutant copper zinc superoxide dismutase 1 (mSOD1) mouse models of familial ALS possess a hypermetabolic phenotype. The pathophysiological relevance of the bioenergetic defects observed in ALS remains largely elusive. AMP-activated protein kinase (AMPK) is a key sensor of cellular energy status and thus might be activated in various models of ALS. Here, we report that AMPK activity is increased in spinal cord cultures expressing mSOD1, as well as in spinal cord lysates from mSOD1 mice. Reducing AMPK activity either pharmacologically or genetically prevents mSOD1-induced motor neuron death in vitro. To investigate the role of AMPK in vivo, we used Caenorhabditis elegans models of motor neuron disease. C. elegans engineered to express human mSOD1 (G85R) in neurons develops locomotor dysfunction and severe fecundity defects when compared to transgenic worms expressing human wild-type SOD1. Genetic reduction of aak-2, the ortholog of the AMPK α2 catalytic subunit in nematodes, improved locomotor behavior and fecundity in G85R animals. Similar observations were made with nematodes engineered to express mutant tat-activating regulatory (TAR) DNA-binding protein of 43 kDa molecular weight. Altogether, these data suggest that bioenergetic abnormalities are likely to be pathophysiologically relevant to motor neuron disease.

  2. Demodex-associated bacterial proteins induce neutrophil activation.

    LENUS (Irish Health Repository)

    2012-02-01

    Background: Patients with rosacea demonstrate a higher density of Demodex mites in their skin than controls. A bacterium isolated from a Demodex mite from a patient with papulopustular rosacea (PPR) was previously shown to provoke an immune response in patients with PPR or ocular rosacea thus suggesting a possible role for bacterial proteins in the etiology of this condition. Objectives: To examine the response of neutrophils to proteins derived from a bacterium isolated from a Demodex mite. Methods: Bacterial cells were lysed and proteins were partially purified by AKTA-FPLC. Isolated neutrophils were exposed to bacterial proteins and monitored for alterations in migration, degranulation and cytokine production. Results: Neutrophils exposed to proteins from Bacillus cells demonstrated increased levels of migration and elevated release of MMP-9, an enzyme known to degrade collagen and cathelicidin, an antimicrobial peptide. In addition neutrophils exposed to the bacterial proteins demonstrated elevated rates of Il-8 and TNF-alpha production. Conclusions: Proteins produced by a bacterium isolated from a Demodex mite have the ability to increase the migration, degranulation and cytokine production abilities of neutrophils. These results suggest that bacteria may play a role in the inflammatory erythema associated with rosacea.

  3. Double-stranded RNA-induced activation of activating protein-1 promoter is differentially regulated by the non-structural protein 1 of avian influenza A viruses.

    Science.gov (United States)

    Munir, Muhammad; Zohari, Siamak; Belák, Sándor; Berg, Mikael

    2012-02-01

    Non-structural protein 1 (NS1) of influenza A viruses is a multifunctional protein that antagonizes the host immune response by interfering with several host signaling pathways. Based on putative amino acid sequences, NS1 proteins are categorized into two gene pools, allele A and allele B. Here we identified that allele A NS1 proteins of H6N8 and H4N6 are able to inhibit double-stranded RNA (dsRNA)-induced activating protein-1 (AP-1) promoter in cultured cell lines (human A549 and mink lung cells). Allele B NS1 proteins from corresponding subtypes of influenza A viruses are weak in this inhibition, despite significant levels of expression of each NS1 protein in human A549 cells. Furthermore, the capability to inhibit AP-1 promoter was mapped in the effector domain, since RNA binding domain alone lost its ability to inhibit this promoter activation. Chimeric forms of NS1 protein, composed of either RNA binding domain of allele A or B and effector domain of allele A or B, showed comparable inhibition to that of their wild-type NS1 proteins, or to the effector domain of corresponding NS1 proteins. Both alleles A and B NS1 proteins of H6N8 and H4N6 were expressed to significant levels, and were localized predominantly in the nucleus of human A549 cells. These results underscore the importance of the effector domain in inhibiting AP-1 promoter activation, and the biological function of the effector domain in stabilizing the RNA binding domain. Further, we revealed the versatile nature of NS1 in inhibiting the AP-1 transcription factor, in a manner dependent on allele type. Comprehensive studies, focusing on the molecular mechanisms behind this differential inhibition, may facilitate exploration of the zoonotic and pathogenic potential of influenza A viruses.

  4. The yeast Pan2 protein is required for poly(A)-binding protein-stimulated poly(A)-nuclease activity.

    Science.gov (United States)

    Boeck, R; Tarun, S; Rieger, M; Deardorff, J A; Müller-Auer, S; Sachs, A B

    1996-01-05

    The removal of the mRNA poly(A) tail in the yeast Saccharomyces cerevisiae is stimulated by the poly(A)-binding protein (Pab1p). A large scale purification of the Pab1p-stimulated poly(A) ribonuclease (PAN) identifies a 76-kDa and two 135-Da polypeptides as candidate enzyme subunits. Antibodies against the Pan1p protein, which is the minor 135-kDa protein in the preparation, can immunodeplete Pan1p but not PAN activity. The protein sequence of the major 135-kDa protein, Pan2p, reveals a novel protein that was also found in the previously reported PAN purification (Sachs, A. B., and Deardorff, J. A. (1992) Cell 70, 961-973). Deletion of the non-essential PAN2 gene results in an increase of the average length of mRNA poly(A) tails in vivo, and a loss of Pab1p-stimulated PAN activity in crude extracts. These data confirm that Pan2p and not Pan1p is required for PAN activity, and they suggest that ribonucleases other than the Pab1p-stimulated PAN are capable of shortening poly(A) tails in vivo.

  5. Fibroblast activation protein alpha expression identifies activated fibroblasts after myocardial infarction.

    Science.gov (United States)

    Tillmanns, Jochen; Hoffmann, Daniel; Habbaba, Yasmin; Schmitto, Jan D; Sedding, Daniel; Fraccarollo, Daniela; Galuppo, Paolo; Bauersachs, Johann

    2015-10-01

    Fibroblast activation protein α (FAP) is a membrane-bound serine protease expressed by activated fibroblasts during wound healing in the skin. Expression of FAP after myocardial infarction (MI) and potential effects on cardiac wound healing are largely unknown. MI was induced in rats and FAP expression was analyzed at 3, 7 and 28 days post-MI by microarray, Western blot and immunohistochemistry. In human hearts after MI, a FAP(+) fibroblast population was identified, and characterized by immunohistochemistry for prolyl-4-hydroxylase β, α-smooth muscle actin, Thy-1 and vimentin. Signaling pathways leading to FAP expression were studied in human cardiac fibroblasts by Western blot and ELISA using TGFβ1, TGF-beta type I-receptor (TGFbR1)-inhibitor SB431542 or the MAPK-inhibitor U0126 as well as siRNA targeting SMAD2 and SMAD3. Finally, fibroblasts were assayed for FAP-dependent migration (modified Boyden-chamber), proliferation (BrdU-assay) and gelatinolytic activity by gelatin zymography. In rats, FAP expression was increased after MI especially in the peri-infarct area peaking at 7 days post-MI. Co-localization analysis identified the majority of FAP(+) cells as activated proto-myofibroblasts and myofibroblasts. Concordantly, FAP(+) fibroblasts were abundant in ischemic tissue of human hearts after MI, but not in healthy control hearts. In vitro, FAP was induced by TGFβ1 via the canonical SMAD2/SMAD3 pathway. Depletion of FAP in fibroblasts reduced migratory capacity, while proliferation was not affected. Gelatin zymography revealed gelatinase activity by fibroblast-derived FAP. In this study, we show for the first time the expression of FAP in activated fibroblasts after MI and its activation by TGFβ1. Effects of FAP on fibroblast migration and gelatinolytic activity indicate a potential role in cardiac wound healing and remodeling. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. The Increasing Impact of Activity-Based Protein Profiling in Plant Science.

    Science.gov (United States)

    Morimoto, Kyoko; van der Hoorn, Renier A L

    2016-03-01

    The active proteome dictates plant physiology. Yet, active proteins are difficult to predict based on transcript or protein levels, because protein activities are regulated post-translationally in their microenvironments. Over the past 10 years, activity-based protein profiling (ABPP) is increasingly used in plant science. ABPP monitors the activities of hundreds of plant proteins using tagged chemical probes that react with the active site of proteins in a mechanism-dependent manner. Since labeling is covalent and irreversible, labeled proteins can be detected and identified on protein gels and by mass spectrometry using tagged fluorophores and/or biotin. Here, we discuss general concepts, approaches and practical considerations of ABPP, before we summarize the discoveries made using 40 validated probes representing 14 chemotypes that can monitor the active state of >4,500 plant proteins. These discoveries and new opportunities indicate that this emerging functional proteomic technology is a powerful discovery tool that will have an increasing impact on plant science. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  7. Strategies for production of active eukaryotic proteins in bacterial expression system

    OpenAIRE

    Orawan Khow; Sunutcha Suntrarachun

    2012-01-01

    Bacteria have long been the favorite expression system for recombinant protein production. However, the flaw of the system is that insoluble and inactive proteins are co-produced due to codon bias, protein folding, phosphorylation, glycosylation, mRNA stability and promoter strength. Factors are cited and the methods to convert to soluble and active proteins are described, for example a tight control of Escherichia coli milieu, refolding from inclusion body and through fusion technology.

  8. Vitamin K dependent protein activity and incident ischemic cardiovascular disease: The multi ethnic study of atherosclerosis

    Science.gov (United States)

    OBJECTIVE: Vitamin K-dependent proteins (VKDPs), which require post-translational modification to achieve biological activity, seem to contribute to thrombus formation, vascular calcification, and vessel stiffness. Whether VKDP activity is prospectively associated with incident cardiovascular diseas...

  9. Mitogen activated protein kinase signaling in the kidney: Target for intervention?

    NARCIS (Netherlands)

    de Borst, M.H.; Wassef, L.; Kelly, D.J.; van Goor, H.; Navis, Ger Jan

    2006-01-01

    Mitogen activated protein kinases (MAPKs) are intracellular signal transduction molecules, which connect cell-surface receptor signals to intracellular processes. MAPKs regulate a range of cellular activities including cell proliferation, gene expression, apoptosis, cell differentiation and cytokine

  10. Effects of protein kinase C activators and staurosporine on protein kinase activity, cell survival, and proliferation in Tetrahymena thermophila

    DEFF Research Database (Denmark)

    Straarup, EM; Schousboe, P; Hansen, HQ

    1997-01-01

    with either PMA or OAG, or at 2,500 cells ml-1. At 500 cells ml-1 PMA induced the in vivo phosphorylation of at least six proteins. The myelin basic protein fragment 4-14 was phosphorylated in vitro in crude extracts of a culture of 250,000 cells ml-1. Both the in vivo and the in vitro phosphorylation were...

  11. 5'-Monophosphate-activated protein kinase (AMPK improves autophagic activity in diabetes and diabetic complications

    Directory of Open Access Journals (Sweden)

    Fan Yao

    2016-01-01

    Full Text Available Diabetes mellitus (DM, an endocrine disorder, will be one of the leading causes of death world-wide in about two decades. Cellular injuries and disorders of energy metabolism are two key factors in the pathogenesis of diabetes, which also become the important causes for the process of diabetic complications. AMPK is a key enzyme in maintaining metabolic homeostasis and has been implicated in the activation of autophagy in distinct tissues. An increasing number of researchers have confirmed that autophagy is a potential factor to affect or induce diabetes and its complications nowadays, which could remove cytotoxic proteins and dysfunctional organelles. This review will summarize the regulation of autophagy and AMPK in diabetes and its complications, and explore how AMPK stimulates autophagy in different diabetic syndromes. A deeper understanding of the regulation and activity of AMPK in autophagy would enhance its development as a promising therapeutic target for diabetes treatment.

  12. A novel antifungal protein with lysozyme-like activity from seeds of Clitoria ternatea.

    Science.gov (United States)

    K, Ajesh; K, Sreejith

    2014-06-01

    An antifungal protein with a molecular mass of 14.3 kDa was isolated from the seeds of butterfly pea (Clitoria ternatea) and designated as Ct protein. The antifungal protein was purified using different methods including ammonium sulphate precipitation, ion exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-50 column. Ct protein formed a single colourless rod-shaped crystal by hanging drop method after 7 days of sample loading. The protein showed lytic activity against Micrococcus luteus and broad-spectrum, fungicidal activity, particularly against the most clinically relevant yeasts, such as Cryptococcus neoformans, Cryptococcus albidus, Cryptococcus laurentii, Candida albicans and Candida parapsilosis. It also exerted an inhibitory activity on mycelial growth in several mould species including Curvularia sp., Alternaria sp., Cladosporium sp., Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Rhizopus sp., and Sclerotium sp. The present study adds to the literature on novel seed proteins with antifungal activity.

  13. Demodex-associated bacterial proteins induce neutrophil activation

    OpenAIRE

    O'Reilly, N.; Bergin, D.; Reeves, E.P.; McElvaney, N.G.; Kavanagh, K.

    2012-01-01

    Background: Patients with rosacea demonstrate a higher density of Demodex mites in their skin than do controls. A bacterium isolated from a Demodex mite from a patient with papulopustular rosacea (PPR) was previously shown to provoke an immune response in patients with PPR or ocular rosacea, thus suggesting a possible role for bacterial proteins in the aetiology of this condition. Objectives To examine the response of neutrophils to proteins derived from a bacterium isolated...

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

    Science.gov (United States)

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

    2018-02-01

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

  15. Thymic Stromal Lymphopoietin Promotes Fibrosis and Activates Mitogen-Activated Protein Kinases in MRC-5 Cells.

    Science.gov (United States)

    Li, Li; Tang, Su; Tang, Xiaodong

    2016-07-06

    BACKGROUND Acute lung injury (ALI) is a life-threatening hypoxemic respiratory disorder with high incidence and mortality. ALI usually manifests as widespread inflammation and lung fibrosis with the accumulation of pro-inflammatory and pro-fibrotic factors and collagen. Thymic stromal lymphopoietin (TSLP) has a significant role in regulation of inflammation but little is known about its roles in lung fibrosis or ALI. This study aimed to define the role and possible regulatory mechanism of TSLP in lung fibrosis. MATERIAL AND METHODS We cultured human lung fibroblast MRC-5 cells and overexpressed or inhibited TSLP by the vector or small interfering RNA transfection. Then, the pro-fibrotic factors skeletal muscle actin alpha (α-SMA) and collagen I, and the 4 mitogen-activated protein kinases (MAPKs) - MAPK7, p38, extracellular signal-regulated kinase 1 (ERK1), and c-Jun N-terminal kinase 1 (JNK1) - were detected by Western blot. RESULTS Results showed that TSLP promoted the production of α-SMA and collagen I (PMRC-5 cell fibrosis. It also activated the expression of MAPK7, p-p38, p-ERK1, and p-JNK1, but the total MAPK7, p-38, ERK1, and JNK1 protein levels were mostly unchanged, indicating the activated MAPK pathways that might contribute to the promotion of cell fibrosis. CONCLUSIONS This study shows the pro-fibrotic role of TSLP in MRC-5 cells, suggesting TSLP is a potential therapeutic target for treating lung fibrosis in ALI. It possibly functions via activating MAPKs. These findings add to our understanding of the mechanism of fibrosis.

  16. Factors affecting antioxidant activity of soybean meal and caseine protein hydrolysates

    International Nuclear Information System (INIS)

    Korczak, J.

    1998-01-01

    Antioxidative activity of protein hydrolysates was dependent on the raw material, condition of hydrolysis and lipid substrate used in model systems. Soybean meal hydrolysate was more active in lard and in linoleic acid emulsion than caseine hydrolysate, whereas caseine was more active in vegetable oils. Antioxidant activity of evaluated protein hydrolysates in all lipid systems, with or without oxidation catalysts, suggests them as natural food additives for lipid stabilization, thus for improvement of its nutritional value and sensory properties

  17. Probing intracellular motor protein activity using an inducible cargo trafficking assay

    NARCIS (Netherlands)

    L.C. Kapitein (Lukas); M.A. Schlager (Max); W.A. van der Zwan (Wouter); P. Wulf (Phebe); N. Keijzer (Nanda); C.C. Hoogenraad (Casper)

    2010-01-01

    textabstractAlthough purified cytoskeletal motor proteins have been studied extensively with the use of in vitro approaches, a generic approach to selectively probe actin and microtubule-based motor protein activity inside living cells is lacking. To examine specific motor activity inside living

  18. Soluble Milk Protein Supplementation with Moderate Physical Activity Improves Locomotion Function in Aging Rats.

    Directory of Open Access Journals (Sweden)

    Aude Lafoux

    Full Text Available Aging is associated with a loss of muscle mass and functional capacity. Present study was designed to compare the impact of specific dairy proteins on muscular function with or without a low-intensity physical activity program on a treadmill in an aged rat model. We investigated the effects of nutritional supplementation, five days a week over a 2-month period with a slow digestible protein, casein or fast digestible proteins, whey or soluble milk protein, on strength and locomotor parameters in sedentary or active aged Wistar RjHan rats (17-19 months of age. An extensive gait analysis was performed before and after protein supplementation. After two months of protein administration and activity program, muscle force was evaluated using a grip test, spontaneous activity using an open-field and muscular mass by specific muscle sampling. When aged rats were supplemented with proteins without exercise, only minor effects of different diets on muscle mass and locomotion were observed: higher muscle mass in the casein group and improvement of stride frequencies with soluble milk protein. By contrast, supplementation with soluble milk protein just after physical activity was more effective at improving overall skeletal muscle function in old rats compared to casein. For active old rats supplemented with soluble milk protein, an increase in locomotor activity in the open field and an enhancement of static and dynamic gait parameters compared to active groups supplemented with casein or whey were observed without any differences in muscle mass and forelimb strength. These results suggest that consumption of soluble milk protein as a bolus immediately after a low intensity physical activity may be a suitable nutritional intervention to prevent decline in locomotion in aged rats and strengthen the interest to analyze the longitudinal aspect of locomotion in aged rodents.

  19. Soluble Milk Protein Supplementation with Moderate Physical Activity Improves Locomotion Function in Aging Rats.

    Science.gov (United States)

    Lafoux, Aude; Baudry, Charlotte; Bonhomme, Cécile; Le Ruyet, Pascale; Huchet, Corinne

    2016-01-01

    Aging is associated with a loss of muscle mass and functional capacity. Present study was designed to compare the impact of specific dairy proteins on muscular function with or without a low-intensity physical activity program on a treadmill in an aged rat model. We investigated the effects of nutritional supplementation, five days a week over a 2-month period with a slow digestible protein, casein or fast digestible proteins, whey or soluble milk protein, on strength and locomotor parameters in sedentary or active aged Wistar RjHan rats (17-19 months of age). An extensive gait analysis was performed before and after protein supplementation. After two months of protein administration and activity program, muscle force was evaluated using a grip test, spontaneous activity using an open-field and muscular mass by specific muscle sampling. When aged rats were supplemented with proteins without exercise, only minor effects of different diets on muscle mass and locomotion were observed: higher muscle mass in the casein group and improvement of stride frequencies with soluble milk protein. By contrast, supplementation with soluble milk protein just after physical activity was more effective at improving overall skeletal muscle function in old rats compared to casein. For active old rats supplemented with soluble milk protein, an increase in locomotor activity in the open field and an enhancement of static and dynamic gait parameters compared to active groups supplemented with casein or whey were observed without any differences in muscle mass and forelimb strength. These results suggest that consumption of soluble milk protein as a bolus immediately after a low intensity physical activity may be a suitable nutritional intervention to prevent decline in locomotion in aged rats and strengthen the interest to analyze the longitudinal aspect of locomotion in aged rodents.

  20. A Nucleotide Phosphatase Activity in the Nucleotide Binding Domain of an Orphan Resistance Protein from Rice*

    Science.gov (United States)

    Fenyk, Stepan; de San Eustaquio Campillo, Alba; Pohl, Ehmke; Hussey, Patrick J.; Cann, Martin J.

    2012-01-01

    Plant resistance proteins (R-proteins) are key components of the plant immune system activated in response to a plethora of different pathogens. R-proteins are P-loop NTPase superfamily members, and current models describe their main function as ATPases in defense signaling pathways. Here we show that a subset of R-proteins have evolved a new function to combat pathogen infection. This subset of R-proteins possesses a nucleotide phosphatase activity in the nucleotide-binding domain. Related R-proteins that fall in the same phylogenetic clade all show the same nucleotide phosphatase activity indicating a conserved function within at least a subset of R-proteins. R-protein nucleotide phosphatases catalyze the production of nucleoside from nucleotide with the nucleotide monophosphate as the preferred substrate. Mutation of conserved catalytic residues substantially reduced activity consistent with the biochemistry of P-loop NTPases. Kinetic analysis, analytical gel filtration, and chemical cross-linking demonstrated that the nucleotide-binding domain was active as a multimer. Nuclear magnetic resonance and nucleotide analogues identified the terminal phosphate bond as the target of a reaction that utilized a metal-mediated nucleophilic attack by water on the phosphoester. In conclusion, we have identified a group of R-proteins with a unique function. This biochemical activity appears to have co-evolved with plants in signaling pathways designed to resist pathogen attack. PMID:22157756

  1. A nucleotide phosphatase activity in the nucleotide binding domain of an orphan resistance protein from rice.

    Science.gov (United States)

    Fenyk, Stepan; Campillo, Alba de San Eustaquio; Pohl, Ehmke; Hussey, Patrick J; Cann, Martin J

    2012-02-03

    Plant resistance proteins (R-proteins) are key components of the plant immune system activated in response to a plethora of different pathogens. R-proteins are P-loop NTPase superfamily members, and current models describe their main function as ATPases in defense signaling pathways. Here we show that a subset of R-proteins have evolved a new function to combat pathogen infection. This subset of R-proteins possesses a nucleotide phosphatase activity in the nucleotide-binding domain. Related R-proteins that fall in the same phylogenetic clade all show the same nucleotide phosphatase activity indicating a conserved function within at least a subset of R-proteins. R-protein nucleotide phosphatases catalyze the production of nucleoside from nucleotide with the nucleotide monophosphate as the preferred substrate. Mutation of conserved catalytic residues substantially reduced activity consistent with the biochemistry of P-loop NTPases. Kinetic analysis, analytical gel filtration, and chemical cross-linking demonstrated that the nucleotide-binding domain was active as a multimer. Nuclear magnetic resonance and nucleotide analogues identified the terminal phosphate bond as the target of a reaction that utilized a metal-mediated nucleophilic attack by water on the phosphoester. In conclusion, we have identified a group of R-proteins with a unique function. This biochemical activity appears to have co-evolved with plants in signaling pathways designed to resist pathogen attack.

  2. Heat Shock Proteins and Mitogen-activated Protein Kinases in Steatotic Livers Undergoing Ischemia-Reperfusion: Some Answers

    Science.gov (United States)

    Massip-Salcedo, Marta; Casillas-Ramirez, Araní; Franco-Gou, Rosah; Bartrons, Ramón; Ben Mosbah, Ismail; Serafin, Anna; Roselló-Catafau, Joan; Peralta, Carmen

    2006-01-01

    Ischemic preconditioning protects steatotic livers against ischemia-reperfusion (I/R) injury, but just how this is achieved is poorly understood. Here, I/R or preconditioning plus I/R was induced in steatotic and nonsteatotic livers followed by investigating the effect of pharmacological treatments that modulate heat shock proteins (HSPs) and mitogen-activated protein kinases (MAPKs). MAPKs, HSPs, protein kinase C, and transaminase levels were measured after reperfusion. We report that preconditioning increased HSP72 and heme-oxygenase-1 (HO-1) at 6 and 24 hours of reperfusion, respectively. Unlike nonsteatotic livers, steatotic livers benefited from HSP72 activators (geranylgeranylacetone) throughout reperfusion. This protection seemed attributable to HO-1 induction. In steatotic livers, preconditioning and geranylgeranylacetone treatment (which are responsible for HO-1 induction) increased protein kinase C activity. HO-1 activators (cobalt(III) protoporphyrin IX) protected both liver types. Preconditioning reduced p38 MAPK and c-Jun N-terminal kinase (JNK), resulting in HSP72 induction though HO-1 remained unmodified. Like HSP72, both p38 and JNK appeared not to be crucial in preconditioning, and inhibitors of p38 (SB203580) and JNK (SP600125) were less effective against hepatic injury than HO-1 activators. These results provide new data regarding the mechanisms of preconditioning and may pave the way to the development of new pharmacological strategies in liver surgery. PMID:16651615

  3. The protein C omega-loop substitution Asn2Ile is associated with reduced protein C anticoagulant activity.

    LENUS (Irish Health Repository)

    Preston, Roger J S

    2012-02-01

    We report a kindred with heritable protein C (PC) deficiency in which two siblings with severe thrombosis showed a composite type I and IIb PC deficiency phenotype, identified using commercial PC assays (proband: PC antigen 42 u\\/dl, amidolytic activity 40 u\\/dl, anticoagulant activity 9 u\\/dl). The independent PROC nucleotide variations c.669C>A (predictive of Ser181Arg) and c.131C>T (predictive of Asn2Ile) segregated with the type I and type IIb PC deficiency phenotypes respectively, but co-segregated in the siblings with severe thrombosis. Soluble thrombomodulin (sTM)-mediated inhibition of plasma thrombin generation from an individual with PC-Asn2Ile was lower (endogenous thrombin potential (ETP) 56 +\\/- 1% that of ETP determined without sTM) than control plasma (ETP 15 +\\/- 2%) indicating reduced PC anticoagulant activity. Recombinant APC-Asn2Ile exhibited normal amidolytic activity but impaired anticoagulant activity. Protein S (PS)-dependent anticoagulant activity of recombinant APC-Asn2Ile and binding of recombinant APC-Asn2Ile to endothelial protein C receptor (EPCR) were reduced compared to recombinant wild-type APC. Asn2 lies within the omega-loop of the PC\\/APC Gla domain and this region is critical for calcium-induced folding and subsequent interactions with anionic phospholipids, EPCR and PS. The disruption of these interactions in this naturally-occurring PC variant highlights their collective importance in mediating APC anticoagulant activity in vivo.

  4. The NAP motif of activity-dependent neuroprotective protein (ADNP) regulates dendritic spines through microtubule end binding proteins.

    Science.gov (United States)

    Oz, S; Kapitansky, O; Ivashco-Pachima, Y; Malishkevich, A; Giladi, E; Skalka, N; Rosin-Arbesfeld, R; Mittelman, L; Segev, O; Hirsch, J A; Gozes, I

    2014-10-01

    The NAP motif of activity-dependent neuroprotective protein (ADNP) enhanced memory scores in patients suffering from mild cognitive impairment and protected activities of daily living in schizophrenia patients, while fortifying microtubule (MT)-dependent axonal transport, in mice and flies. The question is how does NAP fortify MTs? Our sequence analysis identified the MT end-binding protein (EB1)-interacting motif SxIP (SIP, Ser-Ile-Pro) in ADNP/NAP and showed specific SxIP binding sites in all members of the EB protein family (EB1-3). Others found that EB1 enhancement of neurite outgrowth is attenuated by EB2, while EB3 interacts with postsynaptic density protein 95 (PSD-95) to modulate dendritic plasticity. Here, NAP increased PSD-95 expression in dendritic spines, which was inhibited by EB3 silencing. EB1 or EB3, but not EB2 silencing inhibited NAP-mediated cell protection, which reflected NAP binding specificity. NAPVSKIPQ (SxIP=SKIP), but not NAPVAAAAQ mimicked NAP activity. ADNP, essential for neuronal differentiation and brain formation in mouse, a member of the SWI/SNF chromatin remodeling complex and a major protein mutated in autism and deregulated in schizophrenia in men, showed similar EB interactions, which were enhanced by NAP treatment. The newly identified shared MT target of NAP/ADNP is directly implicated in synaptic plasticity, explaining the breadth and efficiency of neuroprotective/neurotrophic capacities.

  5. The Cytotoxicity of Elderberry Ribosome-Inactivating Proteins Is Not Solely Determined by Their Protein Translation Inhibition Activity.

    Directory of Open Access Journals (Sweden)

    Chenjing Shang

    Full Text Available Although the protein translation inhibition activity of ribosome inactivating proteins (RIPs is well documented, little is known about the contribution of the lectin chain to the biological activity of these proteins. In this study, we compared the in vitro and intracellular activity of several S. nigra (elderberry RIPs and non-RIP lectins. Our data demonstrate that RIPs from elderberry are much more toxic to HeLa cells than to primary fibroblasts. Differences in the cytotoxicity between the elderberry proteins correlated with differences in glycan specificity of their lectin domain, cellular uptake efficiency and intracellular destination. Despite the fact that the bulk of the RIPs accumulated in the lysosomes and partly in the Golgi apparatus, we could demonstrate effective inhibition of protein synthesis in cellula. As we also observed cytotoxicity for non-RIP lectins, it is clear that the lectin chain triggers additional pathways heralding cell death. Our data suggest that one of these pathways involves the induction of autophagy.

  6. Activation of AMP-activated protein kinase attenuates hepatocellular carcinoma cell adhesion stimulated by adipokine resistin

    International Nuclear Information System (INIS)

    Yang, Chen-Chieh; Chang, Shun-Fu; Chao, Jian-Kang; Lai, Yi-Liang; Chang, Wei-En; Hsu, Wen-Hsiu; Kuo, Wu-Hsien

    2014-01-01

    Resistin, adipocyte-secreting adipokine, may play critical role in modulating cancer pathogenesis. The aim of this study was to investigate the effects of resistin on HCC adhesion to the endothelium, and the mechanism underlying these resistin effects. Human SK-Hep1 cells were used to study the effect of resistin on intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expressions as well as NF-κB activation, and hence cell adhesion to human umbilical vein endothelial cells (HUVECs). 5-Aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR), an AMP-activated protein kinase (AMPK) activator, was used to determine the regulatory role of AMPK on HCC adhesion to the endothelium in regard to the resistin effects. Treatment with resistin increased the adhesion of SK-Hep1 cells to HUVECs and concomitantly induced NF-κB activation, as well as ICAM-1 and VCAM-1 expressions in SK-Hep1 cells. Using specific blocking antibodies and siRNAs, we found that resistin-induced SK-Hep1 cell adhesion to HUVECs was through NF-κB-regulated ICAM-1 and VCAM-1 expressions. Moreover, treatment with AICAR demonstrated that AMPK activation in SK-Hep1 cells significantly attenuates the resistin effect on SK-Hep1 cell adhesion to HUVECs. These results clarify the role of resistin in inducing HCC adhesion to the endothelium and demonstrate the inhibitory effect of AMPK activation under the resistin stimulation. Our findings provide a notion that resistin play an important role to promote HCC metastasis and implicate AMPK may be a therapeutic target to against HCC metastasis

  7. Characterization of the interactions between the active site of a protein tyrosine kinase and a divalent metal activator

    Directory of Open Access Journals (Sweden)

    Ayrapetov Marina K

    2005-11-01

    Full Text Available Abstract Background Protein tyrosine kinases are important enzymes for cell signalling and key targets for anticancer drug discovery. The catalytic mechanisms of protein tyrosine kinase-catalysed phosphorylation are not fully understood. Protein tyrosine kinase Csk requires two Mg2+ cations for activity: one (M1 binds to ATP, and the other (M2 acts as an essential activator. Results Experiments in this communication characterize the interaction between M2 and Csk. Csk activity is sensitive to pH in the range of 6 to 7. Kinetic characterization indicates that the sensitivity is not due to altered substrate binding, but caused by the sensitivity of M2 binding to pH. Several residues in the active site with potential of binding M2 are mutated and the effect on metal activation studied. An active mutant of Asn319 is generated, and this mutation does not alter the metal binding characteristics. Mutations of Glu236 or Asp332 abolish the kinase activity, precluding a positive or negative conclusion on their role in M2 coordination. Finally, the ability of divalent metal cations to activate Csk correlates to a combination of ionic radius and the coordination number. Conclusion These studies demonstrate that M2 binding to Csk is sensitive to pH, which is mainly responsible for Csk activity change in the acidic arm of the pH response curve. They also demonstrate critical differences in the metal activator coordination sphere in protein tyrosine kinase Csk and a protein Ser/Thr kinase, the cAMP-dependent protein kinase. They shed light on the physical interactions between a protein tyrosine kinase and a divalent metal activator.

  8. Screening of activators and inhibitors of nuclear protein synthesis using labeled compounds

    International Nuclear Information System (INIS)

    Saitmuratova, O.H.

    2004-01-01

    Full text: With the purpose of definition of physiological activity of some known and yet not investigated natural and synthetic compounds (only 40 compounds) their action on protein synthesis ability (PSA) of nucleus of not sharing cells of a brain of rabbits with use of the marked amino acid - C 14 -lysine has been investigated. As a result of our researches activators of non ribosomal synthesis of protein are revealed: N-(β-chlorethyl)-decahydroquinoline, licorine, lupinine, anabasine hydrochloride, peptides: enkephalin, epitalanine, ACTH 4-7 , proteinkinase C, nitrocel, benzolaminopurine - synthetic cytokinin and inhibitors: cocaine, strychnine, aminazine, venom of a cobra snake, indolyl acetic acid, lectin like proteins, extensin like proteins, polyprenol, nitroglycerine. These received results can be used for the decision of the following problems: - Regulation of biosynthesis of the certain kinds of proteins; - Definition of a functional role of studying proteins; - he classification of studying compounds on activity for research of the certain processes in a cellular level

  9. Proteolytic activity of prostate-specific antigen (PSA towards protein substrates and effect of peptides stimulating PSA activity.

    Directory of Open Access Journals (Sweden)

    Johanna M Mattsson

    Full Text Available Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3 exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA.

  10. Proteolytic activity of prostate-specific antigen (PSA) towards protein substrates and effect of peptides stimulating PSA activity.

    Science.gov (United States)

    Mattsson, Johanna M; Ravela, Suvi; Hekim, Can; Jonsson, Magnus; Malm, Johan; Närvänen, Ale; Stenman, Ulf-Håkan; Koistinen, Hannu

    2014-01-01

    Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3) exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA.

  11. Proteolytic Activity of Prostate-Specific Antigen (PSA) towards Protein Substrates and Effect of Peptides Stimulating PSA Activity

    Science.gov (United States)

    Mattsson, Johanna M.; Ravela, Suvi; Hekim, Can; Jonsson, Magnus; Malm, Johan; Närvänen, Ale; Stenman, Ulf-Håkan; Koistinen, Hannu

    2014-01-01

    Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3) exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA. PMID:25237904

  12. Protein-Nanoparticle Interactions: Improving Immobilized Lytic Enzyme Activity and Surface Energy Effects

    Science.gov (United States)

    Downs, Emily Elizabeth

    Protein-nanostructure conjugates, particularly particles, are a subject of significant interest due to changes in their fundamental behavior compared to bulk surfaces. As the size scale of nano-structured materials and proteins are on the same order of magnitude, nanomaterial properties can heavily influence how proteins adsorb and conform to the surface. Previous work has demonstrated the ability of nanoscale surfaces to modulate protein activity, conformation, and retention by modifying the particle surface curvature, morphology, and surface charge. This work has improved our understanding of the protein material interactions, but a complete understanding is still lacking. The goal of this thesis is to investigate two missing areas of understanding using two distinct systems. The first system utilizes a particle with controlled surface energy to observe the impact of surface energy on protein-particle interactions, while the second system uses a modified Listeria-specific protein to determine how protein structure and flexibility affects protein adsorption and activity on particles. Spherical, amorphous, and uniformly doped Zn-silica particles with tailored surface energies were synthesized to understand the impact of surface energy on protein adsorption behavior. Particle surface energy increased with a decrease in particle size and greater dopant concentrations. Protein adsorption and structural loss increased with both particle size and particle surface energy. Higher surface energies promoted protein-particle association and increased protein unfolding. Particle curvature and protein steric hindrance effects limited adsorption and structural loss on smaller particles. Protein surface charge heterogeneity was also found to be linked to both protein adsorption and unfolding behavior on larger particles. Greater surface charge heterogeneity led to higher adsorption concentrations and multilayer formation. These multilayers transitioned from protein

  13. Neofunctionalization of zona pellucida proteins enhances freeze-prevention in the eggs of Antarctic notothenioids

    Science.gov (United States)

    Cao, Lixue; Huang, Qiao; Wu, Zhichao; Cao, Dong-dong; Ma, Zhanling; Xu, Qianghua; Hu, Peng; Fu, Yanxia; Shen, Yu; Chan, Jiulin; Zhou, Cong-zhao; Zhai, Wanying; Chen, Liangbiao

    2016-01-01

    The mechanisms by which the eggs of the Antarctic notothenioid fishes avoid freezing are not fully understood. Zona pellucida proteins (ZPs) are constituents of the chorion which forms a protective matrix surrounding the egg. Here we report occurrence of freezing temperature-related gene expansion and acquisition of unusual ice melting-promoting (IMP) activity in a family of Antarctic notothenioid ZPs (AnnotoZPs). Members of AnnotoZPs are shown to bind with ice and non-colligatively depress the melting point of a solution in a range of 0.26 to 0.65 °C at a moderate concentration. Eggs of zebrafishes expressing an AnnotoZP transgene show improved melting point depression and enhanced survival in freezing conditions. Mutational analyses in a representative AnnotoZP indicate the ZP domain and patches of acidic residues are essential structures for the IMP activity. AnnotoZPs, therefore, represent a group of macromolecules that prevent freezing by a unique ZP–ice interaction mechanism distinct from the known antifreeze proteins. PMID:27698404

  14. Protein - Sparing Activity of Lipid and Carbohydrate in the Giant ...

    African Journals Online (AJOL)

    ADCP/ REP80/1 l. Mangalik, A (1986). Dietary energy requirements of t channel catfish. Ph. D dissertation Auburn. University. Auburn, AL l26pp. Pranther, EE; Lovell, RT (1973). Response of intensively fed channel catfish to diets containing various protein~energy ratios. Proc. Annu. Conf. S.E. Assos. Game Fish. Comm.

  15. One step purification of biological active human interleukin-2 protein ...

    African Journals Online (AJOL)

    Administrator

    2011-10-31

    Oct 31, 2011 ... Pharmacological importance of recombinant human interleukin-2 protein has increased the demand to establish effective ... Extracellular expression of mrhIL-2 in the culture supernatant was ~210 mg/L. Cell free culture .... bottomed micro plate (tissue culture grade) starting with 10 ng/ml in a volume of 100 ...

  16. Antimicrobial activity of Brassica nectar lipid transfer protein

    Science.gov (United States)

    Antimicrobial peptides (AMPs) provide an ancient, innate immunity conserved in all multicellular organisms. In plants, there are several large families of AMPs defined by sequence similarity. The nonspecific lipid transfer protein (LTP) family is defined by a conserved signature of eight cysteines a...

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

    African Journals Online (AJOL)

    AJL

    2012-05-15

    May 15, 2012 ... was bought from the China Center for Type Culture Collection of. Wuhan University (Wuhan, China). ... cultures were exposed to 100 µl of the crude protein fractions. (dilution rates were 1:1, 1:10 or 1:100) on .... The values represent the average of three replications. Asterisks denote significant differences ...

  18. Genome-wide regulation of TATA-binding protein activity

    NARCIS (Netherlands)

    van Werven, F.J.

    2009-01-01

    Transcription, the synthesis of RNA from a DNA template, is a well-controlled process. TATA binding protein (TBP) recruitment to promoters is essential for transcription by all three RNA polymerases, and often is the rate-limiting step of transcription initiation. TBP is incorporated into different

  19. Cholinergic and anticholinesterase activities of total protein extract ...

    African Journals Online (AJOL)

    Traditional herbal medicines such as Morinda morindoïdes are used for treatment of intestinal disorders including constipation in Ivory Coast. The aim of present study was to investigate the effect of total protein of Morinda morindoïdes extract (PT-Mm) on rabbit duodenum contractility and the involved possible ...

  20. Protein kinase and phosphatase activities of thylakoid membranes

    International Nuclear Information System (INIS)

    Michel, H.; Shaw, E.K.; Bennett, J.

    1987-01-01

    Dephosphorylation of the 25 and 27 kDa light-harvesting Chl a/b proteins (LHCII) of the thylakoid membranes is catalyzed by a phosphatase which differs from previously reported thylakoid-bound phosphatases in having an alkaline pH optimum (9.0) and a requirement for Mg 2+ ions. Dephosphorylation of the 8.3 kDa psb H gene product requires a Mg 2+ ion concentration more than 200 fold higher than that for dephosphorylation of LHC II. The 8.3 kDa and 27 kDa proteins appear to be phosphorylated by two distinct kinases, which differ in substrate specificity and sensitivity to inhibitors. The plastoquinone antagonist 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone (DBMIB) inhibits phosphorylation of the 27 kDa LHC II much more readily than phosphorylation of the 8.3 kDa protein. A similar pattern of inhibition is seen for two synthetic oligopeptides (MRKSATTKKAVC and ATQTLESSSRC) which are analogs of the phosphorylation sites of the two proteins. Possible modes of action of DBMIB are discussed. 45 refs., 7 figs., 3 tabs

  1. One step purification of biological active human interleukin-2 protein ...

    African Journals Online (AJOL)

    The mrhIL-2 was expressed extracellularly under methanol inducible AOX1 promoter of P. pastoris. Extracellular expression of mrhIL-2 in the culture supernatant was ~210 mg/L. Cell free culture supernatant containing mrhIL-2 protein was concentrated and buffer exchanged by diafiltration by tangential flow filtration system.

  2. Dipeptidyl peptidase IV inhibitory activity of protein hydrolyzates ...

    African Journals Online (AJOL)

    Background: Type 2 diabetes is a chronic metabolic disorder. Recently, dipeptidyl peptidase IV (DPP-IV) inhibitors that protect incretin hormones from being cleaved by DPP-IV have been used as drugs to control glycemia. This study examined the potential hypoglycemic effect of amaranth grain storage protein hydrolyzates ...

  3. Hydrodynamic collective effects of active protein machines in solution and lipid bilayers.

    Science.gov (United States)

    Mikhailov, Alexander S; Kapral, Raymond

    2015-07-14

    The cytoplasm and biomembranes in biological cells contain large numbers of proteins that cyclically change their shapes. They are molecular machines that can function as molecular motors or carry out various other tasks in the cell. Many enzymes also undergo conformational changes within their turnover cycles. We analyze the advection effects that nonthermal fluctuating hydrodynamic flows induced by active proteins have on other passive molecules in solution or membranes. We show that the diffusion constants of passive particles are enhanced substantially. Furthermore, when gradients of active proteins are present, a chemotaxis-like drift of passive particles takes place. In lipid bilayers, the effects are strongly nonlocal, so that active inclusions in the entire membrane contribute to local diffusion enhancement and the drift. All active proteins in a biological cell or in a membrane contribute to such effects and all passive particles, and the proteins themselves, will be subject to them.

  4. Biological activities of the antiviral protein BE27 from sugar beet (Beta vulgaris L.).

    Science.gov (United States)

    Iglesias, Rosario; Citores, Lucía; Di Maro, Antimo; Ferreras, José M

    2015-02-01

    The ribosome inactivating protein BE27 displays several biological activities in vitro that could result in a broad action against several types of pathogens. Beetin 27 (BE27), a ribosome-inactivating protein (RIP) from sugar beet (Beta vulgaris L.) leaves, is an antiviral protein induced by virus and signaling compounds such as hydrogen peroxide and salicylic acid. Its role as a defense protein has been attributed to its RNA polynucleotide:adenosine glycosidase activity. Here we tested other putative activities of BE27 that could have a defensive role against pathogens finding that BE27 displays rRNA N-glycosidase activity against yeast and Agrobacterium tumefaciens ribosomes, DNA polynucleotide:adenosine glycosidase activity against herring sperm DNA, and magnesium-dependent endonuclease activity against the supercoiled plasmid PUC19 (nicking activity). The nicking activity could be a consequence of an unusual conformation of the BE27 active site, similar to that of PD-L1, a RIP from Phytolacca dioica L. leaves. Additionally, BE27 possesses superoxide dismutase activity, thus being able to produce the signal compound hydrogen peroxide. BE27 is also toxic to COLO 320 cells, inducing apoptosis in these cells by either activating the caspase pathways and/or inhibiting protein synthesis. The combined effect of these biological activities could result in a broad action against several types of pathogens such as virus, bacteria, fungi or insects.

  5. Damage-induced DNA replication stalling relies on MAPK-activated protein kinase 2 activity

    DEFF Research Database (Denmark)

    Köpper, Frederik; Bierwirth, Cathrin; Schön, Margarete

    2013-01-01

    DNA damage can obstruct replication forks, resulting in replicative stress. By siRNA screening, we identified kinases involved in the accumulation of phosphohistone 2AX (γH2AX) upon UV irradiation-induced replication stress. Surprisingly, the strongest reduction of phosphohistone 2AX followed...... knockdown of the MAP kinase-activated protein kinase 2 (MK2), a kinase currently implicated in p38 stress signaling and G2 arrest. Depletion or inhibition of MK2 also protected cells from DNA damage-induced cell death, and mice deficient for MK2 displayed decreased apoptosis in the skin upon UV irradiation...... replication impaired by gemcitabine or by Chk1 inhibition. This rescue strictly depended on translesion DNA polymerases. In conclusion, instead of being an unavoidable consequence of DNA damage, alterations of replication speed and origin firing depend on MK2-mediated signaling....

  6. Structural basis for activation of G-protein-coupled receptors

    DEFF Research Database (Denmark)

    Gether, Ulrik; Asmar, Fazila; Meinild, Anne Kristine

    2002-01-01

    into conformational changes accompanying GPCR activation and the underlying molecular mechanism governing transition of the receptor between its active and inactive states. Using the beta2-adrenergic receptor as a model system we have obtained evidence for an evolutionary conserved activation mechanism where...... changes and receptor activation. At the current stage we are exploring the possibility of reaching this goal by direct in situ labeling of the beta2-adrenergic receptor in Xenopus laevis oocytes with conformationally sensitive fluorescent probes and parallel detection of receptor activation by co...

  7. Stimulation of protein phosphatase activity by insulin and growth factors in 3T3 cells

    International Nuclear Information System (INIS)

    Chan, C.P.; McNall, S.J.; Krebs, E.G.; Fischer, E.H.

    1988-01-01

    Incubation of Swiss mouse 3T3-D1 cells with physiological concentrations of insulin resulted in a rapid and transient activation of protein phosphatase activity as measure by using [ 32 P]phosphorylase α as substrate. Activation reached a maximum level (140% of control value) within 5 min of addition and returned to control levels within 20 min. The effect of insulin was dose-dependent with half-maximal activation occurring at ∼5 nM insulin. This activity could be completely inhibited by addition of the heat-stable protein inhibitor 2, which suggests the presence of an activated type-1 phosphatase. Similar effects on phosphatase activity were seen when epidermal growth factor and platelet-derived growth factor were tested. These results suggest that some of the intracellular effects caused by insulin and growth factors are mediated through the activation of a protein phosphatase

  8. The Ginkgo biloba Extract EGb 761 Modulates Proteasome Activity and Polyglutamine Protein Aggregation

    Directory of Open Access Journals (Sweden)

    Marcel Stark

    2014-01-01

    Full Text Available The standardized Ginkgo biloba extract EGb 761 has well-described antioxidative activities and effects on different cytoprotective signaling pathways. Consequently, a potential use of EGb 761 in neurodegenerative diseases has been proposed. A common characteristic feature of a variety of such disorders is the pathologic formation of protein aggregates, suggesting a crucial role for protein homeostasis. In this study, we show that EGb 761 increased the catalytic activity of the proteasome and enhanced protein degradation in cultured cells. We further investigated this effect in a cellular model of Huntington’s disease (HD by employing cells expressing pathologic variants of a polyglutamine protein (polyQ protein. We show that EGb 761 affected these cells by (i increasing proteasome activity and (ii inducing a more efficient degradation of aggregation-prone proteins. These results demonstrate a novel activity of EGb 761 on protein aggregates by enhancing proteasomal protein degradation, suggesting a therapeutic use in neurodegenerative disorders with a disturbed protein homeostasis.

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

    Directory of Open Access Journals (Sweden)

    Kalpesh B Ishnava

    2014-10-01

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

  10. Light-activated DNA binding in a designed allosteric protein

    Energy Technology Data Exchange (ETDEWEB)

    Strickland, Devin; Moffat, Keith; Sosnick, Tobin R. (UC)

    2008-09-03

    An understanding of how allostery, the conformational coupling of distant functional sites, arises in highly evolvable systems is of considerable interest in areas ranging from cell biology to protein design and signaling networks. We reasoned that the rigidity and defined geometry of an {alpha}-helical domain linker would make it effective as a conduit for allosteric signals. To test this idea, we rationally designed 12 fusions between the naturally photoactive LOV2 domain from Avena sativa phototropin 1 and the Escherichia coli trp repressor. When illuminated, one of the fusions selectively binds operator DNA and protects it from nuclease digestion. The ready success of our rational design strategy suggests that the helical 'allosteric lever arm' is a general scheme for coupling the function of two proteins.

  11. Protein retention and liver aminotransferase activities in Atlantic salmon fed diets containing different energy sources

    Science.gov (United States)

    Fynn-Aikins, K.; Hughes, S.G.; Vandenberg, G.W.

    1995-01-01

    Atlantic salmon (Salmo salar) fingerlings (14.4 g) were fed diets containing either glucose, dextrin, raw corn starch and lipid, or a high protein U.S. Fish and Wildlife Service open-formula diet (ASD2-30) for 12 weeks. Significant differences in weight gain and feed: gain ratio were not observed among salmon fed the diets containing glucose, dextrin or ASD2-30. Diets containing dextrin and glucose supported greater protein retention and reduction in alanine aminotransferase activity than the other diets. Activity of aspartate aminotransferase was not affected by the dietary treatment. Protein retention correlated highly with alanine aminotransferase activity.

  12. Peptidomic analysis reveals proteolytic activity of kefir microorganisms on bovine milk proteins.

    Science.gov (United States)

    Dallas, David C; Citerne, Florine; Tian, Tian; Silva, Vitor L M; Kalanetra, Karen M; Frese, Steven A; Robinson, Randall C; Mills, David A; Barile, Daniela

    2016-04-15

    The microorganisms that make up kefir grains are well known for lactose fermentation, but the extent to which they hydrolyze and consume milk proteins remains poorly understood. Peptidomics technologies were used to examine the proteolytic activity of kefir grains on bovine milk proteins. Gel electrophoresis revealed substantial digestion of milk proteins by kefir grains, with mass spectrometric analysis showing the release of 609 protein fragments and alteration of the abundance of >1500 peptides that derived from 27 milk proteins. Kefir contained 25 peptides identified from the literature as having biological activity, including those with antihypertensive, antimicrobial, immunomodulatory, opioid and anti-oxidative functions. 16S rRNA and shotgun metagenomic sequencing identified the principle taxa in the culture as Lactobacillus species. The model kefir sample contained thousands of protein fragments released in part by kefir microorganisms and in part by native milk proteases. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Jojoba seed meal proteins associated with proteolytic and protease inhibitory activities.

    Science.gov (United States)

    Shrestha, Madan K; Peri, Irena; Smirnoff, Patricia; Birk, Yehudith; Golan-Goldhirsh, Avi

    2002-09-25

    The jojoba, Simmondsia chinensis, is a characteristic desert plant native to the Sonoran desert. The jojoba meal after oil extraction is rich in protein. The major jojoba proteins were albumins (79%) and globulins (21%), which have similar amino acid compositions and also showed a labile thrombin-inhibitory activity. SDS-PAGE showed two major proteins at 50 kDa and 25 kDa both in the albumins and in the globulins. The 25 kDa protein has trypsin- and chymotrypsin-inhibitory activities. In vitro digestibility of the globulins and albumins resembled that of casein and soybean protein concentrates and was increased after heat treatment. The increased digestibility achieved by boiling may be attributed to inactivation of the protease inhibitors and denaturation of proteins.

  14. MEK-1 phosphorylation by MEK kinase, Raf, and mitogen-activated protein kinase: analysis of phosphopeptides and regulation of activity.

    OpenAIRE

    Gardner, A M; Vaillancourt, R R; Lange-Carter, C A; Johnson, G L

    1994-01-01

    MEK-1 is a dual threonine and tyrosine recognition kinase that phosphorylates and activates mitogen-activated protein kinase (MAPK). MEK-1 is in turn activated by phosphorylation. Raf and MAPK/extracellular signal-regulated kinase kinase (MEKK) independently phosphorylate and activate MEK-1. Recombinant MEK-1 is also capable of autoactivation. Purified recombinant wild type MEK-1 and a mutant kinase inactive MEK-1 were used as substrates for MEKK, Raf, and autophosphorylation. MEK-1 phosphory...

  15. Design and application of natural product derived probes for activity based protein profiling

    OpenAIRE

    Battenberg, Oliver Alexander

    2015-01-01

    The identification of new antibacterial protein targets by activity based protein profiling (ABPP) is an important approach to face the increasing emergence of resistant bacteria. The scope of this work focuses on three new strategies for the labeling of antibacterial protein-targets with natural product derived ABPP-probes: A.) Evaluation of the intrinsic photo-reactivity of α-pyrones and pyrimidones for use as photo-crosslinkers. B.) Synthesis of a benzophenone-tag that combines photo-cross...

  16. Early expression of nucleolar SURF-6 protein in mouse spleen lymphocytes activated for proliferation in vitro.

    Science.gov (United States)

    Moraleva, A A; Malysheva, M V; Magoulas, Ch; Polzikov, M A; Zatsepina, O V

    2009-05-01

    Using specific antibodies we studied the content of nucleolar SURF-6 protein, which participates in rRNA processing, in mouser spleen lymphocytes activated for proliferation with concanavalin A and compared it with the content of nucleolar nucleophosmin/B23 protein and DNA replication factor PCNA, well-known markers of proliferating cells. Using immunocytochemistry and immunoblotting methods we demonstrate that the concentration of all these proteins increases simultaneously with increasing the proportion of proliferating cells. Unlike nucleophosmin/B23, SURF-6 protein was not revealed in quiescent lymphocyte nucleoli, while the increase of its level in activated lymphocytes preceded elevation of PCNA level. These observations suggest that nucleolar protein SURF-6 can act as a marker of early T lymphocyte activation for proliferation and that it could participate in cell cycle regulation in mammals.

  17. Cetyltrimethylammonium bromide discontinuous gel electrophoresis: Mr-based separation of proteins with retention of enzymatic activity.

    Science.gov (United States)

    Akins, R E; Levin, P M; Tuan, R S

    1992-04-01

    A discontinuous polyacrylamide and agarose gel electrophoresis system is presented here which allows the fine separation of proteins based on molecular weight with the concomitant retention of native enzymatic activity. This system, referred to as the CAT gel, uses the cationic detergent cetyltrimethylammonium bromide (CTAB) and includes a stacking gel based on the zwitterion arginine and the buffer N-tris(hydroxymethyl)-methylglycine. The CAT gel system allows specific enzyme histochemical detection and localization of proteins after gel electrophoresis. We present evidence that the CAT system stacked and separated a broad range of proteins into discrete bands which migrate as a linear function of log Mr. We have also assessed the effect of CTAB solubilization on the activity of several proteins and showed that some proteins separated by CAT electrophoresis maintain high levels of native enzymatic activity and may be detected histochemically in situ.

  18. The HIV-1 Nef protein and phagocyte NADPH oxidase activation

    DEFF Research Database (Denmark)

    Vilhardt, Frederik; Plastre, Olivier; Sawada, Makoto

    2002-01-01

    of Rac by Clostridium difficile toxin B abolished the Nef effect. (ii) The fraction of activated Rac1 was increased in Nef-transduced cells, and (iii) the dominant positive Rac1(V12) mutant mimicked the effect of Nef. These results are to our knowledge the first analysis of the effect of Rac activation...

  19. Spinacia oleracea proteins with antiviral activity against tobacco ...

    African Journals Online (AJOL)

    The results show that the crude extract and the fraction of 0 to 40% ammonium sulfate precipitation could protect tobacco from TMV infection. An active fraction was further purified with cation-exchange chromatography (SP-sepharose HP) and activity screening. The inhibitory rate for TMV in vitro was up to 94.35% with 50 ...

  20. Plasma Renin Activity in Children with Protein Energy Malnutrition ...

    African Journals Online (AJOL)

    Plasma renin activity was measured by bio-assay in 100 children with kwashiorkor and in 20 healthy children, and also by radio-immunoassay in another 26 children with kwashiorkor and in another 20 healthy children. Both methods showed that (compared with healthy children) renin activity was significantly increased in ...

  1. An active form of calcium and calmodulin dependant protein kinase ...

    African Journals Online (AJOL)

    The removal of the auto-inhibitory domain that negatively regulates the kinase activity in M. truncatula results in a constitutively-active form, inducing symbiotic responses in the absence of bacterial signals. In this study, we verified the functionality of a DMI3 variant and its ability to induce spontaneous nodules in M.

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

    African Journals Online (AJOL)

    AJL

    2012-05-15

    May 15, 2012 ... (2004). The chitosan, extracted from M. domestica was found to have effect on fungus and bacteria (Ai et al., 2008, 2012) while the extract from the larvae of the housefly exhibited antibacterial activity and in vitro anti-tumor activity (Hou et al., 2007a). In addition, Hf-1, a novel antibacterial peptide, was also.

  3. Aqueous geochemistry in icy world interiors: Equilibrium fluid, rock, and gas compositions, and fate of antifreezes and radionuclides

    Science.gov (United States)

    Neveu, Marc; Desch, Steven J.; Castillo-Rogez, Julie C.

    2017-09-01

    The geophysical evolution of many icy moons and dwarf planets seems to have provided opportunities for interaction between liquid water and rock (silicate and organic solids). Here, we explore two ways by which water-rock interaction can feed back on geophysical evolution: the production or consumption of antifreeze compounds, which affect the persistence and abundance of cold liquid; and the potential leaching into the fluid of lithophile radionuclides, affecting the distribution of a long-term heat source. We compile, validate, and use a numerical model, implemented with the PHREEQC code, of the interaction of chondritic rock with pure water and with C, N, S-bearing cometary fluid, thought to be the materials initially accreted by icy worlds, and describe the resulting equilibrium fluid and rock assemblages at temperatures, pressures, and water-to-rock ratios of 0-200 ° C, 1-1000 bar, and 0.1-10 by mass, respectively. Our findings suggest that water-rock interaction can strongly alter the nature and amount of antifreezes, resulting in solutions rich in reduced nitrogen and carbon, and sometimes dissolved H2, with additional sodium, calcium, chlorine, and/or oxidized carbon. Such fluids can remain partially liquid down to 176 K if NH3 is present. The prominence of Cl in solution seems to hinge on its primordial supply in ices, which is unconstrained by the meteoritical record. Equilibrium assemblages, rich in serpentine and saponite clays, retain thorium and uranium radionuclides unless U-Cl or U-HCO3 complexing, which was not modeled, significantly enhances U solubility. However, the radionuclide 40 K can be leached at high water:rock ratio and/or low temperature at which K is exchanged with ammonium in minerals. We recommend the inclusion of these effects in future models of the geophysical evolution of ocean-bearing icy worlds. Our simulation products match observations of chloride salts on Europa and Enceladus; CI chondrites mineralogies; the observation of

  4. Cloning, overexpression and purification of functionally active Saccharomyces cerevisiae Hop1 protein from Escherichia coli.

    Science.gov (United States)

    Khan, Krishnendu; Madhavan, T P Vipin; Muniyappa, K

    2010-07-01

    One of the major limitations to the application of high-resolution biophysical techniques such as X-crystallography and spectroscopic analyses to structure-function studies of Saccharomyces cerevisiae Hop1 protein has been the non-availability of sufficient quantities of functionally active pure protein. This has, indeed, been the case of many proteins, including yeast synaptonemal complex proteins. In this study, we have performed expression screening in Escherichia coli host strains, capable of high-level expression of soluble S. cerevisiae Hop1 protein. A new protocol has been developed for expression and purification of S. cerevisiae Hop1 protein, based on the presence of hexa-histidine tag and double-stranded DNA-Cellulose chromatography. Recombinant S. cerevisiae Hop1 protein was >98% pure and exhibited DNA-binding activity with high-affinity to the Holliday junction. The availability of the recombinant HOP1 expression vector and active Hop1 protein would facilitate structure-function investigations as well as the generation of appropriate truncated and site-directed mutant proteins, respectively. Copyright 2010 Elsevier Inc. All rights reserved.

  5. Histone acetyltransferase inhibitors antagonize AMP-activated protein kinase in postmortem glycolysis

    Directory of Open Access Journals (Sweden)

    Qiong Li

    2017-06-01

    Full Text Available Objective The purpose of this study was to investigate the influence of AMP-activated protein kinase (AMPK activation on protein acetylation and glycolysis in postmortem muscle to better understand the mechanism by which AMPK regulates postmortem glycolysis and meat quality. Methods A total of 32 mice were randomly assigned to four groups and intraperitoneally injected with 5-Aminoimidazole-4-carboxamide1-β-D-ribofuranoside (AICAR, a specific activator of AMPK, AICAR and histone acetyltransferase inhibitor II, or AICAR, Trichostatin A (TSA, an inhibitor of histone deacetylase I and II and Nicotinamide (NAM, an inhibitor of the Sirt family deacetylases. After mice were euthanized, the Longissimus dorsi muscle was collected at 0 h, 45 min, and 24 h postmortem. AMPK activity, protein acetylation and glycolysis in postmortem muscle were measured. Results Activation of AMPK by AICAR significantly increased glycolysis in postmortem muscle. At the same time, it increased the total acetylated proteins in muscle 45 min postmortem. Inhibition of protein acetylation by histone acetyltransferase inhibitors reduced AMPK activation induced increase in the total acetylated proteins and glycolytic rate in muscle early postmortem, while histone deacetylase inhibitors further promoted protein acetylation and glycolysis. Several bands of proteins were detected to be differentially acetylated in muscle with different glycolytic rates. Conclusion Protein acetylation plays an important regulatory role in postmortem glycolysis. As AMPK mediates the effects of pre-slaughter stress on postmortem glycolysis, protein acetylation is likely a mechanism by which antemortem stress influenced postmortem metabolism and meat quality though the exact mechanism is to be elucidated.

  6. The Adaptor Protein and Arf GTPase-activating Protein Cat-1/Git-1 Is Required for Cellular Transformation

    Science.gov (United States)

    Yoo, Sungsoo M.; Antonyak, Marc A.; Cerione, Richard A.

    2012-01-01

    Cat-1/Git-1 is a multifunctional protein that acts as a GTPase-activating protein (GAP) for Arf GTPases, as well as serves as a scaffold for a number of different signaling proteins. Cat-1 is best known for its role in regulating cell shape and promoting cell migration. However, whether Cat-1 might also contribute to cellular transformation is currently unknown. Here we show that ∼95% of cervical tumor samples examined overexpress Cat-1, suggesting that the up-regulation of Cat-1 expression is a frequent occurrence in this type of cancer. We demonstrate further that knocking down Cat-1 from NIH3T3 fibroblasts expressing an activated form of Cdc42 (Cdc42 F28L), or from the human cervical carcinoma (HeLa) cell line, inhibits the ability of these cells to form colonies in soft agar, an in vitro measure of tumorgenicity. The requirement for Cat-1 when assaying the anchorage-independent growth of transformed fibroblasts and HeLa cells is dependent on its ability to bind paxillin, while being negatively impacted by its Arf-GAP activity. Moreover, the co-expression of Cat-1 and an activated form of Arf6 in fibroblasts was sufficient to induce their transformation. These findings highlight novel roles for Cat-1 and its interactions with the Arf GTPases and paxillin in oncogenic transformation. PMID:22807447

  7. Solubilization, Activation, and Insecticidal Activity of Bacillus thuringiensis Serovar thompsoni HD542 Crystal Proteins

    NARCIS (Netherlands)

    Naimov, S.; Boncheva, R.; Karlova, R.B.; Dukiandjiev, S.; Minkov, I.; Maagd, de R.A.

    2008-01-01

    Cry15Aa protein, produced by Bacillus thuringiensis serovar thompsoni HD542 in a crystal together with a 40 kDa accompanying protein is one of a small group of non-typical, less well-studied members of the Cry family of insecticidal proteins, and may provide an alternative for the more commonly used

  8. Mini-G proteins: Novel tools for studying GPCRs in their active conformation.

    Science.gov (United States)

    Nehmé, Rony; Carpenter, Byron; Singhal, Ankita; Strege, Annette; Edwards, Patricia C; White, Courtney F; Du, Haijuan; Grisshammer, Reinhard; Tate, Christopher G

    2017-01-01

    Mini-G proteins are the engineered GTPase domains of Gα subunits. They couple to GPCRs and recapitulate the increase in agonist affinity observed upon coupling of a native heterotrimeric G protein. Given the small size and stability of mini-G proteins, and their ease of expression and purification, they are ideal for biophysical studies of GPCRs in their fully active state. The first mini-G protein developed was mini-Gs. Here we extend the family of mini-G proteins to include mini-Golf, mini-Gi1, mini-Go1 and the chimeras mini-Gs/q and mini-Gs/i. The mini-G proteins were shown to couple to relevant GPCRs and to form stable complexes with purified receptors that could be purified by size exclusion chromatography. Agonist-bound GPCRs coupled to a mini-G protein showed higher thermal stability compared to the agonist-bound receptor alone. Fusion of GFP at the N-terminus of mini-G proteins allowed receptor coupling to be monitored by fluorescence-detection size exclusion chromatography (FSEC) and, in a separate assay, the affinity of mini-G protein binding to detergent-solubilised receptors was determined. This work provides the foundation for the development of any mini-G protein and, ultimately, for the structure determination of GPCRs in a fully active state.

  9. Roles of coactosin-like protein (CLP) and 5-lipoxygenase-activating protein (FLAP) in cellular leukotriene biosynthesis.

    Science.gov (United States)

    Basavarajappa, Devaraj; Wan, Min; Lukic, Ana; Steinhilber, Dieter; Samuelsson, Bengt; Rådmark, Olof

    2014-08-05

    5-Lipoxygenase (5LO) is a key enzyme in leukotriene (LT) biosynthesis. Two accessory proteins, coactosin-like protein (CLP) and 5-lipoxygenase-activating protein (FLAP), can support 5LO activity. To study the roles of CLP and FLAP, we knocked down these proteins in the human monocytic cell line Mono Mac 6 (MM6). Expression of CLP increased MM6 cellular 5LO activity for all stimuli tested. CLP is not absolutely crucial, however; some 5LO activity remained in all incubations of CLP knockdown cells. FLAP knockdown had minor effects in the presence of exogenous arachidonic acid, but led to prominent reductions in 5LO product formation from endogenous substrate. Similar effects were observed after CLP and FLAP knockdown in human primary macrophages as well. In addition, FLAP knockdown reduced conversion of leukotriene A4 to leukotriene C4 (LTC4), suggesting a role for the activity of LTC4 synthase. After stimulation of MM6 cells by phorbol myristate acetate and ionophore A23187, a perinuclear ring pattern was observed for 5LO. This redistribution from cytosolic to perinuclear was clearly compromised in both CLP- and FLAP-deficient cells. In addition, association of CLP with the nucleus was almost absent after 5LO knockdown, and was clearly reduced in FLAP knockdown cells. Coimmunoprecipitation experiments indicated that 5LO-CLP complex formation in MM6 cells was increased by stimulation with ionophore, and that this complex was formed to the same extent in FLAP knockdown cells. A possible interpretation of our findings is that on cell stimulation, formation of the 5LO-CLP complex augments the translocation from cytosol to nucleus, whereas FLAP stabilizes association of this complex with the perinuclear membrane.

  10. Recombinant human activated protein C: current insights into its mechanism of action

    NARCIS (Netherlands)

    Levi, Marcel; van der Poll, Tom

    2007-01-01

    Impairment of the protein C pathway plays a central role in the pathogenesis of sepsis. Administration of recombinant human activated protein C (rhAPC) may correct the dysregulated anticoagulant mechanism and prevent propagation of thrombin generation and formation of microvascular thrombosis.

  11. Effect of diet of Varying Protein concentrations on the Activity of ...

    African Journals Online (AJOL)

    Alterations in protein diet have been reported to result in alterations in calcium homeostasis in the body. Ca2+Mg2+ATPase is an ubiquitous enzyme important in calcium homeostasis in the body. The effect of varying protein diet on the activities of Ca2+ pump across cell membranes is however yet to be fully elucidated.

  12. The antiviral activity of the milk protein lactoferrin against the human immunodeficiency virus type 1

    NARCIS (Netherlands)

    Berkhout, Ben; Floris, René; Recio, Isidra; Visser, Servaas

    2004-01-01

    Milk forms a rich source of biologically interesting components and the protein fraction is known to facilitate many different biological functions. In this manuscript, we review the antiviral properties of the milk protein lactoferrin (LF). In particular, we will describe its antiviral activity

  13. Single-molecule analysis of ligand efficacy in beta(2)AR-G-protein activation

    DEFF Research Database (Denmark)

    Gregorio, G. Glenn; Masureel, Matthieu; Hilger, Daniel

    2017-01-01

    G-protein-coupled receptor (GPCR)-mediated signal transduction is central to human physiology and disease intervention, yet the molecular mechanisms responsible for ligand-dependent signalling responses remain poorly understood. In class A GPCRs, receptor activation and G-protein coupling entail ...

  14. [Isolation and identification of proteins with anti-tumor and fibrinolysogen kinase activities from Eisenia foetida].

    Science.gov (United States)

    Zhao, Rui; Ji, Jian-Guo; Tong, Yuan-Peng; Chen, Qian; Pu, Hai; Ru, Bing-Gen

    2002-09-01

    Proteins from Eisenia foetida possess many biological activities. A group of proteins precipitated by ethanol were isolated and purified by Sephadex G-75 and HiPrep 16/60 DEAE columns, then identified by one- or two- dimensional electrophoresis and mass spectrometry. 2D gel experiments displayed that the pI of proteins from Eisenia foetida were mainly from 3.0 to 4.0. Anti-tumor and kinase activities were determined by in vitro experiments. The enthanol fraction D2(8) showed both of the activities. These ethanol-precipitated proteins were identified further by native polyacrylamide electrophoresis, the protein spots were cut off from gels and digested by trypsin, the peptide mass fingerprints (PMFs) were determined by mass spectrometry. PMF, molecular weight, amino acid composition and N-terminus of 6 proteins were characterized, and band 9 was identified as D2(8). The results suggested that there exist proteins in Eisenia foetida possessed both anti-tumor and fibrinolysogen kinase activities. These methods can be used for identification of the natural bioactive proteins.

  15. Structural and functional analysis of negatively charged milk proteins with anti-HIV activity

    NARCIS (Netherlands)

    Berkhout, B.; Derksen, G. C.; Back, N. K.; Klaver, B.; de Kruif, C. G.; Visser, S.

    1997-01-01

    Several polyanionic reagents such as dextran sulfates, heparin sulfates, and negatively charged proteins have been reported to exhibit anti-HIV activity in vitro. Particularly potent inhibition has been reported for the milk protein beta-lactoglobulin (betaLG) on modification by 3-hydroxyphthalic

  16. Isolation of a 60 kDa protein with in vitro anticancer activity against ...

    African Journals Online (AJOL)

    A 60 kDa protein was purified from secreted purple fluid of A. dactylomela, a sea hare from Persian Gulf. The protein purification procedure consisted basically of ammonium sulfate precipitation, ion exchange chromatography using DEAE– Sepharose and ultra-filtration method. In vitro antiproliferative and cytotoxic activity ...

  17. Role of 5'AMP-activated protein kinase in skeletal muscle

    DEFF Research Database (Denmark)

    Treebak, Jonas Thue; Wojtaszewski, Jørgen F. P.

    2008-01-01

    5'AMP-activated protein kinase (AMPK) is recognized as an important intracellular energy sensor, shutting down energy-consuming processes and turning on energy-generating processes. Discovery of target proteins of AMPK has dramatically increased in the past 10 years. Historically, AMPK was first...

  18. Subtype activation and interaction of protein kinase C and mitogen-activated protein kinase controlling receptor expression in cerebral arteries and microvessels after subarachnoid hemorrhage

    DEFF Research Database (Denmark)

    Ansar, S.; Edvinsson, L.

    2008-01-01

    BACKGROUND AND PURPOSE: The pathogenesis of cerebral ischemia associated with subarachnoid hemorrhage (SAH) still remains elusive. The aim of this study was to examine the involvement of mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) subtypes in the pathophysiology of cerebral...... ischemia after SAH in cerebral arteries and microvessels and to examine temporal activation of the kinases. We hypothesize that treatment with a MAPK or PKC inhibitor will prevent the SAH-induced kinase activation in brain vessels. METHODS: SAH was induced by injecting 250 microL blood...... into the prechiasmatic cistern in the rat. The activation of different MAPK and PKC isotypes in large circle of Willis cerebral arteries and intracerebral microvessels was examined at 0, 1, 3, 6, 12, 24, and 48 hours after SAH and after intrathecal treatment with PKC or MAPK inhibitor by use of Western blot. RESULTS...

  19. Measles virus C protein impairs production of defective copyback double-stranded viral RNA and activation of protein kinase R.

    Science.gov (United States)

    Pfaller, Christian K; Radeke, Monte J; Cattaneo, Roberto; Samuel, Charles E

    2014-01-01

    Measles virus (MV) lacking expression of C protein (C(KO)) is a potent activator of the double-stranded RNA (dsRNA)-dependent protein kinase (PKR), whereas the isogenic parental virus expressing C protein is not. Here, we demonstrate that significant amounts of dsRNA accumulate during C(KO) mutant infection but not following parental virus infection. dsRNA accumulated during late stages of infection and localized with virus replication sites containing N and P proteins. PKR autophosphorylation and stress granule formation correlated with the timing of dsRNA appearance. Phospho-PKR localized to dsRNA-containing structures as revealed by immunofluorescence. Production of dsRNA was sensitive to cycloheximide but resistant to actinomycin D, suggesting that dsRNA is a viral product. Quantitative PCR (qPCR) analyses revealed reduced viral RNA synthesis and a steepened transcription gradient in C(KO) virus-infected cells compared to those in parental virus-infected cells. The observed alterations were further reflected in lower viral protein expression levels and reduced C(KO) virus infectious yield. RNA deep sequencing confirmed the viral RNA expression profile differences seen by qPCR between C(KO) mutant and parental viruses. After one subsequent passage of the C(KO) virus, defective interfering RNA (DI-RNA) with a duplex structure was obtained that was not seen with the parental virus. We conclude that in the absence of C protein, the amount of PKR activator RNA, including DI-RNA, is increased, thereby triggering innate immune responses leading to impaired MV growth.

  20. Characterization of Carbohydrate Active Enzymes Involved in Arabinogalactan Protein Metabolism

    DEFF Research Database (Denmark)

    Knoch, Eva

    and tissues, their functions and synthesis are still poorly understood. The aim of the research presented in the thesis was to characterize carbohydrate active enzymes involved in AGP biosynthesis and modification to gain insights into the biosynthesis of the glycoproteins in plants. Candidate....... The enzymatic activity of a hydrolase from GH family 17 was investigated, without successful determination of the activity. Members of hydrolase family 43 appeared to be localized in the Golgi-apparatus, which is also the compartment for glycan biosynthesis. The localization of these glycoside hydrolases...

  1. [Effects of Bt corn straw insecticidal proteins on enzyme activities of Eisenia fetida].

    Science.gov (United States)

    Shu, Ying-hua; Ma, Hong-hui; Du, Yan; Wang, Jian-wu

    2011-08-01

    Bacillus thuringiensis (Bt) proteins released from Bt corn can enter soil ecosystem via returning straw into field, root exudation, and pollen fluttering-down. In this study, the straws of Bt corn and its near-isogenic non-Bt line were added into soil with an application rate of 5% and 7.5% to breed Eisenia fetida, and the total protein content and the activities of acetylcholine esterase (AchE), glutathione peroxidase (GSH-PX), catalase (CAT), and superoxide dismutase (SOD) in E. fetida were determined after 7 and 14 days. Under the same application rate of the straws, the total protein content and GSH-PX activity of E. fetida decreased while the AchE, CAT, and SOD activities increased on the 14th day, compared with those on the 7th day. The Bt corn straw increased the SOD activity and decreased the AchE and GSH-PX activities, but had less effects on the total protein content and CAT activity, compared with non-Bt corn straw. All the results suggested that Bt corn straw had no inhibitory effect on E. fetida total protein but could inhibit the AchE and GSH-PX activities, and could not induce CAT activity but induce SOD activity within a short time.

  2. Unc-51 controls active zone density and protein composition by downregulating ERK signaling.

    Science.gov (United States)

    Wairkar, Yogesh P; Toda, Hirofumi; Mochizuki, Hiroaki; Furukubo-Tokunaga, Katsuo; Tomoda, Toshifumi; Diantonio, Aaron

    2009-01-14

    Efficient synaptic transmission requires the apposition of neurotransmitter release sites opposite clusters of postsynaptic neurotransmitter receptors. Transmitter is released at active zones, which are composed of a large complex of proteins necessary for synaptic development and function. Many active zone proteins have been identified, but little is known of the mechanisms that ensure that each active zone receives the proper complement of proteins. Here we use a genetic analysis in Drosophila to demonstrate that the serine threonine kinase Unc-51 acts in the presynaptic motoneuron to regulate the localization of the active zone protein Bruchpilot opposite to glutamate receptors at each synapse. In the absence of Unc-51, many glutamate receptor clusters are unapposed to Bruchpilot, and ultrastructural analysis demonstrates that fewer active zones contain dense body T-bars. In addition to the presence of these aberrant synapses, there is also a decrease in the density of all synapses. This decrease in synaptic density and abnormal active zone composition is associated with impaired evoked transmitter release. Mechanistically, Unc-51 inhibits the activity of the MAP kinase ERK to promote synaptic development. In the unc-51 mutant, increased ERK activity leads to the decrease in synaptic density and the absence of Bruchpilot from many synapses. Hence, activated ERK negatively regulates synapse formation, resulting in either the absence of active zones or the formation of active zones without their proper complement of proteins. The Unc-51-dependent inhibition of ERK activity provides a potential mechanism for synapse-specific control of active zone protein composition and release probability.

  3. Presenilin-2 regulates the degradation of RBP-Jk protein through p38 mitogen-activated protein kinase.

    Science.gov (United States)

    Kim, Su-Man; Kim, Mi-Yeon; Ann, Eun-Jung; Mo, Jung-Soon; Yoon, Ji-Hye; Park, Hee-Sae

    2012-03-01

    Transcriptional regulation performs a central role in Notch1 signaling by recombining binding protein Suppressor of Hairless (RBP-Jk)--a signaling pathway that is widely involved in determination of cell fate. Our earlier work demonstrated the possible regulation of the Notch1-RBP-Jk pathway through protein degradation of RBP-Jk; however, the potential regulator for the degradation of RBP-Jk remains to be determined. Here, we report that the expression of endogenous and exogenous RBP-Jk was increased significantly in cells treated with proteasome- and lysosome-specific inhibitors. The effects of these inhibitors on RBP-Jk occurred in a dose- and time-dependent manner. The level of RBP-Jk protein was higher in presenilin-2 (PS2)-knockout cells than in presenilin-1 (PS1)-knockout cells. Furthermore, the level of RBP-Jk was decreased by expression of PS2 in PS1 and PS2 double-knockout cells. We also found that PS1-knockout cells treated with a specific inhibitor of p38 mitogen-activated protein kinase ∂ (MAPK) had significantly increased levels of RBP-Jk. p38 MAPK phosphorylates RBP-Jk at Thr339 by physical binding, which subsequently induces the degradation and ubiquitylation of the RBP-Jk protein. Collectively, our results indicate that PS2 modulates the degradation of RBP-Jk through phosphorylation by p38 MAPK.

  4. Heterogeneous nuclear ribonucleoprotein B1 protein impairs DNA repair mediated through the inhibition of DNA-dependent protein kinase activity

    International Nuclear Information System (INIS)

    Iwanaga, Kentaro; Sueoka, Naoko; Sato, Akemi; Hayashi, Shinichiro; Sueoka, Eisaburo

    2005-01-01

    Heterogeneous nuclear ribonucleoprotein B1, an RNA binding protein, is overexpressed from the early stage of lung cancers; it is evident even in bronchial dysplasia, a premalignant lesion. We evaluated the proteins bound with hnRNP B1 and found that hnRNP B1 interacted with DNA-dependent protein kinase (DNA-PK) complex, and recombinant hnRNP B1 protein dose-dependently inhibited DNA-PK activity in vitro. To test the effect of hnRNP B1 on DNA repair, we performed comet assay after irradiation, using normal human bronchial epithelial (HBE) cells treated with siRNA for hnRNP A2/B1: reduction of hnRNP B1 treated with siRNA for hnRNP A2/B1 induced faster DNA repair in normal HBE cells. Considering these results, we assume that overexpression of hnRNP B1 occurring in the early stage of carcinogenesis inhibits DNA-PK activity, resulting in subsequent accumulation of erroneous rejoining of DNA double-strand breaks, causing tumor progression

  5. Kinesin-Binding Protein Controls Microtubule Dynamics and Cargo Trafficking by Regulating Kinesin Motor Activity

    NARCIS (Netherlands)

    Kevenaar, Josta T|info:eu-repo/dai/nl/338771042; Bianchi, Sarah; van Spronsen, Myrrhe|info:eu-repo/dai/nl/337616655; Olieric, Natacha; Lipka, Joanna|info:eu-repo/dai/nl/369403142; Frias, Cátia P; Mikhaylova, Marina; Harterink, Martin|info:eu-repo/dai/nl/304075655; Keijzer, Nanda; Wulf, Phebe S; Hilbert, Manuel; Kapitein, Lukas C|info:eu-repo/dai/nl/298806630; de Graaff, Esther|info:eu-repo/dai/nl/148374646; Akhmanova, Anna|info:eu-repo/dai/nl/156410591; Steinmetz, Michel O; Hoogenraad, Casper C|info:eu-repo/dai/nl/227263502

    2016-01-01

    Kinesin motor proteins play a fundamental role for normal neuronal development by controlling intracellular cargo transport and microtubule (MT) cytoskeleton organization. Regulating kinesin activity is important to ensure their proper functioning, and their misregulation often leads to severe human

  6. Chronic regulation of colonic epithelial secretory function by activation of G protein-coupled receptors.

    LENUS (Irish Health Repository)

    Toumi, F

    2011-02-01

    Enteric neurotransmitters that act at G protein-coupled receptors (GPCRs) are well known to acutely promote epithelial Cl(-) and fluid secretion. Here we examined if acute GPCR activation might have more long-term consequences for epithelial secretory function.

  7. Resistance to activated protein C is a risk factor for fibrostenosis in Crohn’s disease

    OpenAIRE

    Novacek, Gottfried; Miehsler, Wolfgang; Palkovits, Julia; Reinisch, Walter; Waldhör, Thomas; Kapiotis, Stylianos; Gangl, Alfred; Vogelsang, Harald

    2006-01-01

    AIM: To evaluate the effect of resistance to activated protein C (aPCR), the most common known inherited thrombophilic disorder, on the risk of intestinal operation of fibrostenosis in patients with Crohn’s disease (CD).

  8. Syntenin-1 and ezrin proteins link activated leukocyte cell adhesion molecule to the actin cytoskeleton

    NARCIS (Netherlands)

    Tudor, Cicerone; te Riet, J.; Eich, C.; Harkes, R.; Smisdom, N.; Bouhuijzen Wenger, J.; Ameloot, M.; Holt, M.; Kanger, Johannes S.; Figdor, Carl; Cambi, A.; Subramaniam, Vinod

    2014-01-01

    Activated leukocyte cell adhesion molecule (ALCAM) is a type I transmembrane protein member of the immunoglobulin superfamily of cell adhesion molecules. Involved in important pathophysiological processes such as the immune response, cancer metastasis, and neuronal development, ALCAM undergoes both

  9. Hypocholesterolaemic and antioxidant activities of chickpea (Cicer arietinum L.) protein hydrolysates.

    Science.gov (United States)

    Yust, María del Mar; Millán-Linares, María del Carmen; Alcaide-Hidalgo, Juan María; Millán, Francisco; Pedroche, Justo

    2012-07-01

    Some dietary proteins possess biological properties which make them potential ingredients of functional or health-promoting foods. Many of these properties are attributed to bioactive peptides that can be released by controlled hydrolysis using exogenous proteases. The aim of this work was to test the improvement of hypocholesterolaemic and antioxidant activities of chickpea protein isolate by means of hydrolysis with alcalase and flavourzyme. All hydrolysates tested exhibited better hypocholesterolaemic activity when compared with chickpea protein isolate. The highest cholesterol micellar solubility inhibition (50%) was found after 60 min of treatment with alcalase followed by 30 min of hydrolysis with flavourzyme. To test antioxidant activity of chickpea proteins three methods were used: β-carotene bleaching method, reducing power and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging effect since antioxidant activity of protein hydrolysates may not be attributed to a single mechanism. Chickpea hydrolysates showed better antioxidant activity in all assays, especially reducing power and DPPH scavenging effect than chickpea protein isolate. The results of this study showed the good potential of chickpea protein hydrolysates as bioactive ingredients. The highest bioactive properties could be obtained by selecting the type of proteases and the hydrolysis time. Copyright © 2012 Society of Chemical Industry.

  10. Lactobacillus Proteins Are Associated with the Bactericidal Activity against E. coli of Female Genital Tract Secretions

    Science.gov (United States)

    Kalyoussef, Sabah; Nieves, Edward; Dinerman, Ellen; Carpenter, Colleen; Shankar, Viswanathan; Oh, Jamie; Burd, Berta; Angeletti, Ruth H.; Buckheit, Karen W.; Fredricks, David N.; Madan, Rebecca P.; Keller, Marla J.; Herold, Betsy C.

    2012-01-01

    Background Female genital tract secretions are bactericidal for Escherichia (E.) coli ex vivo. However, the intersubject variability and molecules that contribute to this activity have not been defined. Methods The bactericidal activity and concentration of immune mediators in cervicovaginal lavage (CVL) collected from 99 healthy women were determined. Results CVL reduced the number of E. coli colonies by 68% [−26, 100] (median [range]). CVL were active against laboratory and clinical isolates of E. coli, but were inactive against Lactobacillus species. Bactericidal activity correlated with the concentration of protein recovered (p90% inhibitory activity (active) and two withbactericidal for E. coli. Conclusion Both host and commensal microbiota proteins contribute to mucosal defense. Identification of these proteins will facilitate the development of strategies to maintain a healthy vaginal microbiome and prevent colonization with pathogenic bacteria such as E. coli that increase the risk for urinary tract infections, preterm labor and perinatal infection. PMID:23185346

  11. Zoanthid mucus as new source of useful biologically active proteins.

    Science.gov (United States)

    Guarnieri, Míriam Camargo; de Albuquerque Modesto, Jeanne Claíne; Pérez, Carlos Daniel; Ottaiano, Tatiana Fontes; Ferreira, Rodrigo da Silva; Batista, Fabrício Pereira; de Brito, Marlon Vilela; Campos, Ikaro Henrique Mendes Pinto; Oliva, Maria Luiza Vilela

    2018-03-01

    Palythoa caribaeorum is a very common colonial zoanthid in the coastal reefs of Brazil. It is known for its massive production of mucus, which is traditionally used in folk medicine by fishermen in northeastern Brazil. This study identified biologically active compounds in P. caribaerum mucus. Crude mucus was collected during low tides by the manual scraping of colonies; samples were maintained in an ice bath, homogenized, and centrifuged at 16,000 g for 1 h at 4 °C; the supernatant (mucus) was kept at -80 °C until use. The enzymatic (proteolytic and phospholipase A 2 ), inhibitory (metallo, cysteine and serine proteases), and hemagglutinating (human erythrocyte) activities were determined. The results showed high levels of cysteine and metallo proteases, intermediate levels of phosholipase A 2 , low levels of trypsin, and no elastase and chymotrypsin like activities. The mucus showed potent inhibitory activity on snake venom metalloproteases and cysteine proteinase papain. In addition, it showed agglutinating activity towards O + , B + , and A + erythrocyte types. The hemostatic results showed that the mucus prolongs the aPTT and PT, and strongly inhibited platelet aggregation induced by arachidonic acid, collagen, epinephrine, ADP, and thrombin. The antimicrobial activity was tested on 15 strains of bacteria and fungi through the radial diffusion assay in agar, and no activity was observed. Compounds in P. caribaeorum mucus were analyzed for the first time in this study, and our results show potential pharmacological activities in these compounds, which are relevant for use in physiopathological investigations. However, the demonstration of these activities indicates caution in the use of crude mucus in folk medicine. Furthermore, the present or absent activities identified in this mucus suggest that the studied P. caribaeorum colonies were in thermal stress conditions at the time of sample collection; these conditions may precede the bleaching

  12. Optically and biologically active mussel protein-coated double-walled carbon nanotubes.

    Science.gov (United States)

    Jung, Yong Chae; Muramatsu, Hiroyuki; Fujisawa, Kazunori; Kim, Jin Hee; Hayashi, Takuya; Kim, Yoong Ahm; Endo, Morinobu; Terrones, Mauricio; Dresselhaus, Mildred S

    2011-12-02

    A method of dispersing strongly bundled double-walled carbon nanotubes (DWNTs) via a homogeneous coating of mussel protein in an aqueous solution is presented. Optical activity, mechanical strength, as well as electrical conductivity coming from the nanotubes and the versatile biological activity from the mussel protein make mussel-coated DWNTs promising as a multifunctional scaffold and for anti-fouling materials. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Discovery of a Stress-Activated Protein Kinase Inhibitor for Lymphatic Filariasis.

    Science.gov (United States)

    Tummalapalli, Sreedhar R; Bhat, Rohit; Chojnowski, Agnieszka; Prorok, Monika; Kreiss, Tamara; Goldberg, Ronald; Canan, Stacie; Hawryluk, Natalie; Mortensen, Deborah; Khetani, Vikram; Zeldis, Jerome; Siekierka, John J; Rotella, David P

    2018-03-08

    Lymphatic filariasis infects over 120 million people worldwide and can lead to significant disfigurement and disease. Resistance is emerging with current treatments, and these therapies have dose limiting adverse events; consequently new targets are needed. One approach to achieve this goal is inhibition of parasitic protein kinases involved in circumventing host defense mechanisms. This report describes structure-activity relationships leading to the identification of a potent, orally bioavailable stress activated protein kinase inhibitor that may be used to investigate this hypothesis.

  14. Mining frequent patterns for AMP-activated protein kinase regulation on skeletal muscle

    OpenAIRE

    Chen, Qingfeng; Chen, Yi-Ping Phoebe

    2006-01-01

    Abstract Background AMP-activated protein kinase (AMPK) has emerged as a significant signaling intermediary that regulates metabolisms in response to energy demand and supply. An investigation into the degree of activation and deactivation of AMPK subunits under exercise can provide valuable data for understanding AMPK. In particular, the effect of AMPK on muscle cellular energy status makes this protein a promising pharmacological target for disease treatment. As more AMPK regulation data ar...

  15. Activated protein synthesis and suppressed protein breakdown signaling in skeletal muscle of critically ill patients

    DEFF Research Database (Denmark)

    Jespersen, Jakob G; Nedergaard, Anders; Reitelseder, Søren

    2011-01-01

    involved in muscle mass regulation, we investigated the phosphorylation and expression of key factors in these protein synthesis and breakdown signaling pathways in thigh skeletal muscle of critically ill intensive care unit (ICU) patients compared with healthy controls.......Skeletal muscle mass is controlled by myostatin and Akt-dependent signaling on mammalian target of rapamycin (mTOR), glycogen synthase kinase 3ß (GSK3ß) and forkhead box O (FoxO) pathways, but it is unknown how these pathways are regulated in critically ill human muscle. To describe factors...

  16. Activation and translocation of p38 mitogen-activated protein kinase after stimulation of monocytes with contact sensitizers.

    Science.gov (United States)

    Brand, Pia; Plochmann, Sibylle; Valk, Elke; Zahn, Sabine; Saloga, Joachim; Knop, Jürgen; Becker, Detlef

    2002-07-01

    Recently we described the induction of tyrosine phosphorylation by contact sensitizers as an early molecular event during the activation of antigen- presenting cells. In this study, the role of the p38 mitogen-activated protein kinase for the activation of human monocytes after exposure to four structurally unrelated contact sensitizers was analyzed in comparison with the irritant benzalkonium chloride and an inductor of oxidative stress (H2O2) using immunofluorescence, Western blotting, and enzyme-linked immunosorbent assay techniques. Bio chemical analysis revealed a translocation of p38 from the cytoplasm to the detergent-resistant cell fraction only upon stimulation with contact sensitizers. The activity of p38 was studied by quantification of its phosphorylated active form with a specific antibody and by kinase assay. Although all stimulants used in this study led to the activation of p38, a translocation to the detergent-resistant fraction as well phosphorylation of the mitogen-activated protein kinase dependent transcription factor Elk-1 was induced only by contact sensitizers. Evidence for a functional relevance of mitogen-activated protein kinase activation was provided by measurement of the hapten-induced production of the proinflammatory cytokine interleukin-1beta. Its release was inhibited by blocking p38-mediated signaling using the imidazole compounds SB203580 and SB202190. These data show that contact sensitizers are strong activators of the p38 mitogen-activated protein kinase. Although activation of this stress-associated pathway has been reported for many other stimuli, a unique translocation of p38 from the cytoplasm to the detergent-resistant fraction seems to be a specific event during hapten-induced activation of antigen-presenting cells.

  17. Activation of protein kinase C inhibits synthesis and release of decidual prolactin

    International Nuclear Information System (INIS)

    Harman, I.; Costello, A.; Ganong, B.; Bell, R.M.; Handwerger, S.

    1986-01-01

    Activation of calcium-activated, phospholipid-dependent protein kinase C by diacylglycerol and phorbol esters has been shown to mediate release of hormones in many systems. To determine whether protein kinase C activation is also involved in the regulation of prolactin release from human decidual, the authors have examined the effects of various acylglycerols and phorbol esters on the synthesis and release of prolactin from cultured human decidual cells. sn-1,2-Dioctanolyglycerol (diC 8 ), which is known to stimulate protein kinase C in other systems, inhibited prolactin release in a dose-dependent manner with maximal inhibition of 53.1% at 100 μM. Diolein (100 μM), which also stimulates protein kinase C activity in some systems, inhibited prolactin release by 21.3%. Phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-didecanoate, and 4β-phorbol 12,13-dibutyrate, which activate protein kinase C in other systems, also inhibited the release of prolactin, which the protein kinase C inactivate 4α-phorbol-12,13-didecanoate was without effect. The inhibition of prolactin release was secondary to a decrease in prolactin synthesis. Although diC 8 and PMA inhibited the synthesis and release of prolactin, these agents had no effect on the synthesis or release of trichloroacetic acid-precipitable [ 35 S]methionine-labeled decidual proteins and did not cause the release of the cytosolic enzymes lactic dehydrogenase and alkaline phosphatase. DiC 8 and PMA stimulates the specific activity of protein kinase C in decidual tissue by 14.6 and 14.0-fold, respectively. The inhibition of the synthesis and release of prolactin by diC 8 and phorbol esters strongly implicates protein kinase C in the regulation of the production and release of prolactin from the decidua

  18. Active protein aggregates induced by terminally attached self-assembling peptide ELK16 in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Zhou Bihong

    2011-02-01

    Full Text Available Abstract Background In recent years, it has been gradually realized that bacterial inclusion bodies (IBs could be biologically active. In particular, several proteins including green fluorescent protein, β-galactosidase, β-lactamase, alkaline phosphatase, D-amino acid oxidase, polyphosphate kinase 3, maltodextrin phosphorylase, and sialic acid aldolase have been successfully produced as active IBs when fused to an appropriate partner such as the foot-and-mouth disease virus capsid protein VP1, or the human β-amyloid peptide Aβ42(F19D. As active IBs may have many attractive advantages in enzyme production and industrial applications, it is of considerable interest to explore them further. Results In this paper, we report that an ionic self-assembling peptide ELK16 (LELELKLK2 was able to effectively induce the formation of cytoplasmic inclusion bodies in Escherichia coli (E. coli when attached to the carboxyl termini of four model proteins including lipase A, amadoriase II, β-xylosidase, and green fluorescent protein. These aggregates had a general appearance similar to the usually reported cytoplasmic inclusion bodies (IBs under transmission electron microscopy or fluorescence confocal microscopy. Except for lipase A-ELK16 fusion, the three other fusion protein aggregates retained comparable specific activities with the native counterparts. Conformational analyses by Fourier transform infrared spectroscopy revealed the existence of newly formed antiparallel beta-sheet structures in these ELK16 peptide-induced inclusion bodies, which is consistent with the reported assembly of the ELK16 peptide. Conclusions This has been the first report where a terminally attached self-assembling β peptide ELK16 can promote the formation of active inclusion bodies or active protein aggregates in E. coli. It has the potential to render E. coli and other recombinant hosts more efficient as microbial cell factories for protein production. Our observation might

  19. A variant of green fluorescent protein exclusively deposited to active intracellular inclusion bodies.

    Science.gov (United States)

    Raghunathan, Govindan; Munussami, Ganapathiraman; Moon, Hyojin; Paik, Hyun-jong; An, Seong Soo A; Kim, Yong-Sung; Kang, Sebyung; Lee, Sun-Gu

    2014-05-16

    Inclusion bodies (IBs) were generally considered to be inactive protein deposits and did not hold any attractive values in biotechnological applications. Recently, some IBs of recombinant proteins were confirmed to show their functional properties such as enzyme activities, fluorescence, etc. Such biologically active IBs are not commonly formed, but they have great potentials in the fields of biocatalysis, material science and nanotechnology. In this study, we characterized the IBs of DL4, a deletion variant of green fluorescent protein which forms active intracellular aggregates. The DL4 proteins expressed in Escherichia coli were exclusively deposited to IBs, and the IBs were estimated to be mostly composed of active proteins. The spectral properties and quantum yield of the DL4 variant in the active IBs were almost same with those of its native protein. Refolding and stability studies revealed that the deletion mutation in DL4 didn't affect the folding efficiency of the protein, but destabilized its structure. Analyses specific for amyloid-like structures informed that the inner architecture of DL4 IBs might be amorphous rather than well-organized. The diameter of fluorescent DL4 IBs could be decreased up to 100-200 nm by reducing the expression time of the protein in vivo. To our knowledge, DL4 is the first GFP variant that folds correctly but aggregates exclusively in vivo without any self-aggregating/assembling tags. The fluorescent DL4 IBs have potentials to be used as fluorescent biomaterials. This study also suggests that biologically active IBs can be achieved through engineering a target protein itself.

  20. Real-time visualization of heterotrimeric G protein Gq activation in living cells

    Directory of Open Access Journals (Sweden)

    Manders Erik MM

    2011-05-01

    Full Text Available Abstract Background Gq is a heterotrimeric G protein that plays an important role in numerous physiological processes. To delineate the molecular mechanisms and kinetics of signalling through this protein, its activation should be measurable in single living cells. Recently, fluorescence resonance energy transfer (FRET sensors have been developed for this purpose. Results In this paper, we describe the development of an improved FRET-based Gq activity sensor that consists of a yellow fluorescent protein (YFP-tagged Gγ2 subunit and a Gαq subunit with an inserted monomeric Turquoise (mTurquoise, the best cyan fluorescent protein variant currently available. This sensor enabled us to determine, for the first time, the kon (2/s of Gq activation. In addition, we found that the guanine nucleotide exchange factor p63RhoGEF has a profound effect on the number of Gq proteins that become active upon stimulation of endogenous histamine H1 receptors. The sensor was also used to measure ligand-independent activation of the histamine H1 receptor (H1R upon addition of a hypotonic stimulus. Conclusions Our observations reveal that the application of a truncated mTurquoise as donor and a YFP-tagged Gγ2 as acceptor in FRET-based Gq activity sensors substantially improves their dynamic range. This optimization enables the real-time single cell quantification of Gq signalling dynamics, the influence of accessory proteins and allows future drug screening applications by virtue of its sensitivity.

  1. Noise exposure immediately activates cochlear mitogen-activated protein kinase signaling

    Directory of Open Access Journals (Sweden)

    Kumar N Alagramam

    2014-01-01

    Full Text Available Noise-induced hearing loss (NIHL is a major public health issue worldwide. Uncovering the early molecular events associated with NIHL would reveal mechanisms leading to the hearing loss. Our aim is to investigate the immediate molecular responses after different levels of noise exposure and identify the common and distinct pathways that mediate NIHL. Previous work showed mice exposed to 116 decibels sound pressure level (dB SPL broadband noise for 1 h had greater threshold shifts than the mice exposed to 110 dB SPL broadband noise, hence we used these two noise levels in this study. Groups of 4-8-week-old CBA/CaJ mice were exposed to no noise (control or to broadband noise for 1 h, followed by transcriptome analysis of total cochlear RNA isolated immediately after noise exposure. Previously identified and novel genes were found in all data sets. Following exposure to noise at 116 dB SPL, the earliest responses included up-regulation of 243 genes and down-regulation of 61 genes, while a similar exposure at 110 dB SPL up-regulated 155 genes and down-regulated 221 genes. Bioinformatics analysis indicated that mitogen-activated protein kinase (MAPK signaling was the major pathway in both levels of noise exposure. Nevertheless, both qualitative and quantitative differences were noticed in some MAPK signaling genes, after exposure to different noise levels. Cacna1b , Cacna1g , and Pla2g6 , related to calcium signaling were down-regulated after 110 dB SPL exposure, while the fold increase in the expression of Fos was relatively lower than what was observed after 116 dB SPL exposure. These subtle variations provide insight on the factors that may contribute to the differences in NIHL despite the activation of a common pathway.

  2. Noise exposure immediately activates cochlear mitogen-activated protein kinase signaling.

    Science.gov (United States)

    Alagramam, Kumar N; Stepanyan, Ruben; Jamesdaniel, Samson; Chen, Daniel H-C; Davis, Rickie R

    2014-01-01

    Noise-induced hearing loss (NIHL) is a major public health issue worldwide. Uncovering the early molecular events associated with NIHL would reveal mechanisms leading to the hearing loss. Our aim is to investigate the immediate molecular responses after different levels of noise exposure and identify the common and distinct pathways that mediate NIHL. Previous work showed mice exposed to 116 decibels sound pressure level (dB SPL) broadband noise for 1 h had greater threshold shifts than the mice exposed to 110 dB SPL broadband noise, hence we used these two noise levels in this study. Groups of 4-8-week-old CBA/CaJ mice were exposed to no noise (control) or to broadband noise for 1 h, followed by transcriptome analysis of total cochlear RNA isolated immediately after noise exposure. Previously identified and novel genes were found in all data sets. Following exposure to noise at 116 dB SPL, the earliest responses included up-regulation of 243 genes and down-regulation of 61 genes, while a similar exposure at 110 dB SPL up-regulated 155 genes and down-regulated 221 genes. Bioinformatics analysis indicated that mitogen-activated protein kinase (MAPK) signaling was the major pathway in both levels of noise exposure. Nevertheless, both qualitative and quantitative differences were noticed in some MAPK signaling genes, after exposure to different noise levels. Cacna1b , Cacna1g , and Pla2g6 , related to calcium signaling were down-regulated after 110 dB SPL exposure, while the fold increase in the expression of Fos was relatively lower than what was observed after 116 dB SPL exposure. These subtle variations provide insight on the factors that may contribute to the differences in NIHL despite the activation of a common pathway.

  3. Mitogen-activated protein kinase and abscisic acid signal transduction

    NARCIS (Netherlands)

    Heimovaara-Dijkstra, S.; Testerink, C.; Wang, M.

    1998-01-01

    The phytohormone abscisic acid (ABA) is a classical plant hormone, responsible for regulation of abscission, diverse aspects of plant and seed development, stress responses and germination. It was found that ABA signal transduction in plants can involve the activity of type 2C-phosphatases (PP2C),

  4. -Glucosidase inhibitory activity of thermostability lectin protein from ...

    African Journals Online (AJOL)

    Personal

    2012-05-24

    May 24, 2012 ... into monosaccharides in the gut are used in the treatment of insulin-independent diabetes mellitus type. 2. Archidendron jiringa .... means to control this type of diabetes by diminishing the absorption rate of glucose ..... isolated to date is from Momordica charantia, which has a maximal activity at 55°C ...

  5. Relationship of amino acid composition and molecular weight of antifreeze glycopeptides to non-colligative freezing point depression.

    Science.gov (United States)

    Schrag, J D; O'Grady, S M; DeVries, A L

    1982-08-06

    Many polar fishes synthesize a group of eight glycopeptides that exhibit a non-colligative lowering of the freezing point of water. These glycopeptides range in molecular weight between 2600 and 33 700. The largest glycopeptides [1-5] lower the freezing point more than the small ones on a weight basis and contain only two amino acids, alanine and threonine, with the disaccharide galactose-N-acetyl-galactosamine attached to threonine. The small glycopeptides, 6, 7, and 8, also lower the freezing point and contain proline, which periodically substitutes for alanine. Glycopeptides with similar antifreeze properties isolated from the saffron cod and the Atlantic tomcod contain an additional amino acid, arginine, which substitutes for threonine in glycopeptide 6. In this study we address the question of whether differences in amino acid composition or molecular weight between large and small glycopeptides are responsible for the reduced freezing point depressing capability of the low molecular weight glycopeptides. The results indicate that the degree of amino acid substitutions that occur in glycopeptides 6-8 do not have a significant effect on the unusual freezing point lowering and that the observed decrease in freezing point depression with smaller glycopeptides can be accounted for on the basis of molecular weight.

  6. Isolation of two biologically active cell surface proteins from Brucella abortus by chromatofocusing

    International Nuclear Information System (INIS)

    Tabatabai, L.B.; Deyoe, B.L.

    1983-01-01

    Brucella abortus contains a group of immunogenic cell surface proteins which have potential value as a vaccine or as a diagnostic reagent for the prevention and diagnosis of bovine brucellosis. Under nondenaturing conditions, these proteins range in molecular weight from 10,000-124,000, as determined by high performance liquid chromatography (HPLC) on TSK 3000sw. By analytical isoelectrofocusing, 6 major protein bands could be distinguished with pI's ranging from 4.0 to 6.0 and 3 additional major proteins with pI's of 7.5, 9.5, and 10. By chromatofocusing on Polybuffer Exchanger 94 with a pH gradient from 6-4, two of the six proteins from pI 4-6 were separated, a pI 4.9 and a pI 4.7 protein; a third fraction contained the high pI proteins. The former two proteins were homogeneous by analytical isoelectrofocusing, and a molecular weight of 54,000 daltons was found for both protein species by HPLC on TSK 3000sw. The pI 4-6 and not the pI 9.5 and 10 proteins, could be radiolabeled when intact cells were radioiodinated with diazotized ( 125 I)-iodosulfanilic acid. Biological activity of the proteins as assessed in lemmings indicated that immunization with the pI 4.7 and 4.9 proteins afforded better protection against experimental brucellosis than immunization with the high pI proteins. These results support our view that a single surface protein may be sufficient for the prevention of experimental brucellosis

  7. Isolation of two biologically active cell surface proteins from Brucella abortus by chromatofocusing

    Energy Technology Data Exchange (ETDEWEB)

    Tabatabai, L.B.; Deyoe, B.L.

    1983-01-01

    Brucella abortus contains a group of immunogenic cell surface proteins which have potential value as a vaccine or as a diagnostic reagent for the prevention and diagnosis of bovine brucellosis. Under nondenaturing conditions, these proteins range in molecular weight from 10,000-124,000, as determined by high performance liquid chromatography (HPLC) on TSK 3000sw. By analytical isoelectrofocusing, 6 major protein bands could be distinguished with pI's ranging from 4.0 to 6.0 and 3 additional major proteins with pI's of 7.5, 9.5, and 10. By chromatofocusing on Polybuffer Exchanger 94 with a pH gradient from 6-4, two of the six proteins from pI 4-6 were separated, a pI 4.9 and a pI 4.7 protein; a third fraction contained the high pI proteins. The former two proteins were homogeneous by analytical isoelectrofocusing, and a molecular weight of 54,000 daltons was found for both protein species by HPLC on TSK 3000sw. The pI 4-6 and not the pI 9.5 and 10 proteins, could be radiolabeled when intact cells were radioiodinated with diazotized (/sup 125/I)-iodosulfanilic acid. Biological activity of the proteins as assessed in lemmings indicated that immunization with the pI 4.7 and 4.9 proteins afforded better protection against experimental brucellosis than immunization with the high pI proteins. These results support our view that a single surface protein may be sufficient for the prevention of experimental brucellosis.

  8. The Role of Protein-Ligand Contacts in Allosteric Regulation of the Escherichia coli Catabolite Activator Protein*

    Science.gov (United States)

    Townsend, Philip D.; Rodgers, Thomas L.; Glover, Laura C.; Korhonen, Heidi J.; Richards, Shane A.; Colwell, Lucy J.; Pohl, Ehmke; Wilson, Mark R.; Hodgson, David R. W.; McLeish, Tom C. B.; Cann, Martin J.

    2015-01-01

    Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distant site. Both experimental and theoretical evidence demonstrate that allostery can be communicated through altered slow relaxation protein dynamics without conformational change. The catabolite activator protein (CAP) of Escherichia coli is an exemplar for the analysis of such entropically driven allostery. Negative allostery in CAP occurs between identical cAMP binding sites. Changes to the cAMP-binding pocket can therefore impact the allosteric properties of CAP. Here we demonstrate, through a combination of coarse-grained modeling, isothermal calorimetry, and structural analysis, that decreasing the affinity of CAP for cAMP enhances negative cooperativity through an entropic penalty for ligand binding. The use of variant cAMP ligands indicates the data are not explained by structural heterogeneity between protein mutants. We observe computationally that altered interaction strength between CAP and cAMP variously modifies the change in allosteric cooperativity due to second site CAP mutations. As the degree of correlated motion between the cAMP-contacting site and a second site on CAP increases, there is a tendency for computed double mutations at these sites to drive CAP toward noncooperativity. Naturally occurring pairs of covarying residues in CAP do not display this tendency, suggesting a selection pressure to fine tune allostery on changes to the CAP ligand-binding pocket without a drive to a noncooperative state. In general, we hypothesize an evolutionary selection pressure to retain slow relaxation dynamics-induced allostery in proteins in which evolution of the ligand-binding site is occurring. PMID:26187469

  9. The Role of Protein-Ligand Contacts in Allosteric Regulation of the Escherichia coli Catabolite Activator Protein.

    Science.gov (United States)

    Townsend, Philip D; Rodgers, Thomas L; Glover, Laura C; Korhonen, Heidi J; Richards, Shane A; Colwell, Lucy J; Pohl, Ehmke; Wilson, Mark R; Hodgson, David R W; McLeish, Tom C B; Cann, Martin J

    2015-09-04

    Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distant site. Both experimental and theoretical evidence demonstrate that allostery can be communicated through altered slow relaxation protein dynamics without conformational change. The catabolite activator protein (CAP) of Escherichia coli is an exemplar for the analysis of such entropically driven allostery. Negative allostery in CAP occurs between identical cAMP binding sites. Changes to the cAMP-binding pocket can therefore impact the allosteric properties of CAP. Here we demonstrate, through a combination of coarse-grained modeling, isothermal calorimetry, and structural analysis, that decreasing the affinity of CAP for cAMP enhances negative cooperativity through an entropic penalty for ligand binding. The use of variant cAMP ligands indicates the data are not explained by structural heterogeneity between protein mutants. We observe computationally that altered interaction strength between CAP and cAMP variously modifies the change in allosteric cooperativity due to second site CAP mutations. As the degree of correlated motion between the cAMP-contacting site and a second site on CAP increases, there is a tendency for computed double mutations at these sites to drive CAP toward noncooperativity. Naturally occurring pairs of covarying residues in CAP do not display this tendency, suggesting a selection pressure to fine tune allostery on changes to the CAP ligand-binding pocket without a drive to a noncooperative state. In general, we hypothesize an evolutionary selection pressure to retain slow relaxation dynamics-induced allostery in proteins in which evolution of the ligand-binding site is occurring. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Abnormal activation of glial cells in the brains of prion protein-deficient mice ectopically expressing prion protein-like protein, PrPLP/Dpl.

    Science.gov (United States)

    Atarashi, R; Sakaguchi, S; Shigematsu, K; Arima, K; Okimura, N; Yamaguchi, N; Li, A; Kopacek, J; Katamine, S

    2001-12-01

    Some lines of mice homozygous for a disrupted prion protein gene (Prnp), including Ngsk Prnp(0/0) mice, exhibit Purkinje cell degeneration as a consequence of the ectopic overexpression of the downstream gene for prion protein-like protein (PrPLP/Dpl) in the brain, but others, such as Zrch I Prnp(0/0) mice, show neither the neurodegeneration nor the expression of PrPLP/Dpl. In the present study, we found that Ngsk Prnp(0/0), but not Zrch I Prnp(0/0) mice, developed gliosis involving both astrocytes and microglia in the brain. The brains from wild-type (Prnp(+/+)), Ngsk Prnp(0/0), Zrch I Prnp(0/0), and reconstituted Ngsk Prnp(0/0) mice carrying a mouse PrP transgene, designated Tg(P) Ngsk Prnp(0/0) mice, were subjected into Northern blotting and in situ hybridization using probes of glial fibrillary acidic protein (GFAP) and lysozyme M (LM) specific for astrocytes and microglia, respectively. Immunohistochemistry was also performed on the brain sections using anti-GFAP and anti-F4/80 antibodies. Northern blotting demonstrated upregulated expression of the genes for GFAP and LM in the brains of Ngsk Prnp(0/0), but not in Zrch I Prnp(0/0) mice. A transgene for normal mouse PrP(C) successfully rescued Ngsk Prnp(0/0) mice from the glial activation. In situ hybridization and immunohistochemistry revealed activated astrocytes and microglia mainly in the white matter of both the forebrains and cerebella. In contrast, there was no evidence of neuronal injury except for the Purkinje cell degeneration. Moreover, the glial cell activation was notable well before the onset of the Purkinje cell degeneration. These findings strongly suggest that ectopic PrPLP/Dpl in the absence of PrP(C) is actively involved in the glial-cell activation in the brain.

  11. Variation in the Subcellular Localization and Protein Folding Activity among Arabidopsis thaliana Homologs of Protein Disulfide Isomerase

    Directory of Open Access Journals (Sweden)

    Christen Y. L. Yuen

    2013-10-01

    Full Text Available Protein disulfide isomerases (PDIs catalyze the formation, breakage, and rearrangement of disulfide bonds to properly fold nascent polypeptides within the endoplasmic reticulum (ER. Classical animal and yeast PDIs possess two catalytic thioredoxin-like domains (a, a′ and two non-catalytic domains (b, b′, in the order a-b-b′-a′. The model plant, Arabidopsis thaliana, encodes 12 PDI-like proteins, six of which possess the classical PDI domain arrangement (AtPDI1 through AtPDI6. Three additional AtPDIs (AtPDI9, AtPDI10, AtPDI11 possess two thioredoxin domains, but without intervening b-b′ domains. C-terminal green fluorescent protein (GFP fusions to each of the nine dual-thioredoxin PDI homologs localized predominantly to the ER lumen when transiently expressed in protoplasts. Additionally, expression of AtPDI9:GFP-KDEL and AtPDI10: GFP-KDDL was associated with the formation of ER bodies. AtPDI9, AtPDI10, and AtPDI11 mediated the oxidative folding of alkaline phosphatase when heterologously expressed in the Escherichia coli protein folding mutant, dsbA−. However, only three classical AtPDIs (AtPDI2, AtPDI5, AtPDI6 functionally complemented dsbA−. Interestingly, chemical inducers of the ER unfolded protein response were previously shown to upregulate most of the AtPDIs that complemented dsbA−. The results indicate that Arabidopsis PDIs differ in their localization and protein folding activities to fulfill distinct molecular functions in the ER.

  12. AMP-activated protein kinase (AMPK mediates nutrient regulation of thioredoxin-interacting protein (TXNIP in pancreatic beta-cells.

    Directory of Open Access Journals (Sweden)

    Maayan Shaked

    Full Text Available Thioredoxin-interacting protein (TXNIP regulates critical biological processes including inflammation, stress and apoptosis. TXNIP is upregulated by glucose and is a critical mediator of hyperglycemia-induced beta-cell apoptosis in diabetes. In contrast, the saturated long-chain fatty acid palmitate, although toxic to the beta-cell, inhibits TXNIP expression. The mechanisms involved in the opposing effects of glucose and fatty acids on TXNIP expression are unknown. We found that both palmitate and oleate inhibited TXNIP in a rat beta-cell line and islets. Palmitate inhibition of TXNIP was independent of fatty acid beta-oxidation or esterification. AMP-activated protein kinase (AMPK has an important role in cellular energy sensing and control of metabolic homeostasis; therefore we investigated its involvement in nutrient regulation of TXNIP. As expected, glucose inhibited whereas palmitate stimulated AMPK. Pharmacologic activators of AMPK mimicked fatty acids by inhibiting TXNIP. AMPK knockdown increased TXNIP expression in presence of high glucose with and without palmitate, indicating that nutrient (glucose and fatty acids effects on TXNIP are mediated in part via modulation of AMPK activity. TXNIP is transcriptionally regulated by carbohydrate response element-binding protein (ChREBP. Palmitate inhibited glucose-stimulated ChREBP nuclear entry and recruitment to the Txnip promoter, thereby inhibiting Txnip transcription. We conclude that AMPK is an important regulator of Txnip transcription via modulation of ChREBP activity. The divergent effects of glucose and fatty acids on TXNIP expression result in part from their opposing effects on AMPK activity. In light of the important role of TXNIP in beta-cell apoptosis, its inhibition by fatty acids can be regarded as an adaptive/protective response to glucolipotoxicity. The finding that AMPK mediates nutrient regulation of TXNIP may have important implications for the pathophysiology and treatment

  13. Active zone proteins are transported via distinct mechanisms regulated by Par-1 kinase.

    Directory of Open Access Journals (Sweden)

    Kara R Barber

    2017-02-01

    Full Text Available Disruption of synapses underlies a plethora of neurodevelopmental and neurodegenerative disease. Presynaptic specialization called the active zone plays a critical role in the communication with postsynaptic neuron. While the role of many proteins at the active zones in synaptic communication is relatively well studied, very little is known about how these proteins are transported to the synapses. For example, are there distinct mechanisms for the transport of active zone components or are they all transported in the same transport vesicle? Is active zone protein transport regulated? In this report we show that overexpression of Par-1/MARK kinase, a protein whose misregulation has been implicated in Autism spectrum disorders (ASDs and neurodegenerative disorders, lead to a specific block in the transport of an active zone protein component- Bruchpilot at Drosophila neuromuscular junctions. Consistent with a block in axonal transport, we find a decrease in number of active zones and reduced neurotransmission in flies overexpressing Par-1 kinase. Interestingly, we find that Par-1 acts independently of Tau-one of the most well studied substrates of Par-1, revealing a presynaptic function for Par-1 that is independent of Tau. Thus, our study strongly suggests that there are distinct mechanisms that transport components of active zones and that they are tightly regulated.

  14. Cell type-specific neuroprotective activity of untranslocated prion protein.

    Directory of Open Access Journals (Sweden)

    Elena Restelli

    2010-10-01

    Full Text Available A key pathogenic role in prion diseases was proposed for a cytosolic form of the prion protein (PrP. However, it is not clear how cytosolic PrP localization influences neuronal viability, with either cytotoxic or anti-apoptotic effects reported in different studies. The cellular mechanism by which PrP is delivered to the cytosol of neurons is also debated, and either retrograde transport from the endoplasmic reticulum or inefficient translocation during biosynthesis has been proposed. We investigated cytosolic PrP biogenesis and effect on cell viability in primary neuronal cultures from different mouse brain regions.Mild proteasome inhibition induced accumulation of an untranslocated form of cytosolic PrP in cortical and hippocampal cells, but not in cerebellar granules. A cyclopeptolide that interferes with the correct insertion of the PrP signal sequence into the translocon increased the amount of untranslocated PrP in cortical and hippocampal cells, and induced its synthesis in cerebellar neurons. Untranslocated PrP boosted the resistance of cortical and hippocampal neurons to apoptotic insults but had no effect on cerebellar cells.These results indicate cell type-dependent differences in the efficiency of PrP translocation, and argue that cytosolic PrP targeting might serve a physiological neuroprotective function.

  15. Molecular Characteristics and Biological Functions of Surface-Active and Surfactant Proteins.

    Science.gov (United States)

    Sunde, Margaret; Pham, Chi L L; Kwan, Ann H

    2017-06-20

    Many critical biological processes take place at hydrophobic:hydrophilic interfaces, and a wide range of organisms produce surface-active proteins and peptides that reduce surface and interfacial tension and mediate growth and development at these boundaries. Microorganisms produce both small lipid-associated peptides and amphipathic proteins that allow growth across water:air boundaries, attachment to surfaces, predation, and improved bioavailability of hydrophobic substrates. Higher-order organisms produce surface-active proteins with a wide variety of functions, including the provision of protective foam environments for vulnerable reproductive stages, evaporative cooling, and gas exchange across airway membranes. In general, the biological functions supported by these diverse polypeptides require them to have an amphipathic nature, and this is achieved by a diverse range of molecular structures, with some proteins undergoing significant conformational change or intermolecular association to generate the structures that are surface active.

  16. The Biological Activity of alpha-Mangostin, a Larvicidal Botanic Mosquito Sterol Carrier Protein-2 Inhibitor

    Science.gov (United States)

    2010-01-01

    consider- ingG.mangostana is a tropical evergreen that ismainly found in India , Myanmar, Sri Lanka, and Thailand (Jung et al. 2006). Almost a decade...unclassified c. THIS PAGE unclassified Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18 ferase ( GST ) activities (Bullangpoti et al...Thermo ScientiÞc, Rockford, IL). The esterase activity was expressed as mol/mg protein/min substrate hydrolyzed. GST Activity. GST activity samples

  17. Effect of mitochondrial complex I inhibition on Fe-S cluster protein activity

    Energy Technology Data Exchange (ETDEWEB)

    Mena, Natalia P. [Department of Biology, Faculty of Sciences, Universidad de Chile, Las Palmeras 3425, Santiago (Chile); Millennium Institute of Cell Dynamics and Biotechnology, Santiago (Chile); Bulteau, Anne Laure [UPMC Univ Paris 06, UMRS 975 - UMR 7725, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); Inserm, U 975, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); CNRS, UMR 7225, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, Paris 75013 (France); Salazar, Julio [Millennium Institute of Cell Dynamics and Biotechnology, Santiago (Chile); Hirsch, Etienne C. [UPMC Univ Paris 06, UMRS 975 - UMR 7725, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); Inserm, U 975, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); CNRS, UMR 7225, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, Paris 75013 (France); Nunez, Marco T., E-mail: mnunez@uchile.cl [Department of Biology, Faculty of Sciences, Universidad de Chile, Las Palmeras 3425, Santiago (Chile); Millennium Institute of Cell Dynamics and Biotechnology, Santiago (Chile)

    2011-06-03

    Highlights: {yields} Mitochondrial complex I inhibition resulted in decreased activity of Fe-S containing enzymes mitochondrial aconitase and cytoplasmic aconitase and xanthine oxidase. {yields} Complex I inhibition resulted in the loss of Fe-S clusters in cytoplasmic aconitase and of glutamine phosphoribosyl pyrophosphate amidotransferase. {yields} Consistent with loss of cytoplasmic aconitase activity, an increase in iron regulatory protein 1 activity was found. {yields} Complex I inhibition resulted in an increase in the labile cytoplasmic iron pool. -- Abstract: Iron-sulfur (Fe-S) clusters are small inorganic cofactors formed by tetrahedral coordination of iron atoms with sulfur groups. Present in numerous proteins, these clusters are involved in key biological processes such as electron transfer, metabolic and regulatory processes, DNA synthesis and repair and protein structure stabilization. Fe-S clusters are synthesized mainly in the mitochondrion, where they are directly incorporated into mitochondrial Fe-S cluster-containing proteins or exported for cytoplasmic and nuclear cluster-protein assembly. In this study, we tested the hypothesis that inhibition of mitochondrial complex I by rotenone decreases Fe-S cluster synthesis and cluster content and activity of Fe-S cluster-containing enzymes. Inhibition of complex I resulted in decreased activity of three Fe-S cluster-containing enzymes: mitochondrial and cytosolic aconitases and xanthine oxidase. In addition, the Fe-S cluster content of glutamine phosphoribosyl pyrophosphate amidotransferase and mitochondrial aconitase was dramatically decreased. The reduction in cytosolic aconitase activity was associated with an increase in iron regulatory protein (IRP) mRNA binding activity and with an increase in the cytoplasmic labile iron pool. Since IRP activity post-transcriptionally regulates the expression of iron import proteins, Fe-S cluster inhibition may result in a false iron deficiency signal. Given that

  18. Increased prothrombin activation in protein S-deficient plasma under flow conditions on endothelial cell matrix: an independent anticoagulant function of protein S in plasma

    NARCIS (Netherlands)

    van't Veer, C.; Hackeng, T. M.; Biesbroeck, D.; Sixma, J. J.; Bouma, B. N.

    1995-01-01

    Protein S is a vitamin K-dependent nonenzymatic coagulation factor involved in the regulation of activated protein C (aPC). In this study, we report an aPC-independent anticoagulant function of protein S in plasma under flow conditions. Plasma, anticoagulated with low-molecular-weight heparin

  19. Protein turnover, amino acid requirements and recommendations for athletes and active populations

    International Nuclear Information System (INIS)

    Poortmans, J.R.; Carpentier, A.; Pereira-Lancha, L.O.; Lancha, A. Jr.

    2012-01-01

    Skeletal muscle is the major deposit of protein molecules. As for any cell or tissue, total muscle protein reflects a dynamic turnover between net protein synthesis and degradation. Noninvasive and invasive techniques have been applied to determine amino acid catabolism and muscle protein building at rest, during exercise and during the recovery period after a single experiment or training sessions. Stable isotopic tracers ( 13 C-lysine, 15 N-glycine, 2 H 5 -phenylalanine) and arteriovenous differences have been used in studies of skeletal muscle and collagen tissues under resting and exercise conditions. There are different fractional synthesis rates in skeletal muscle and tendon tissues, but there is no major difference between collagen and myofibrillar protein synthesis. Strenuous exercise provokes increased proteolysis and decreased protein synthesis, the opposite occurring during the recovery period. Individuals who exercise respond differently when resistance and endurance types of contractions are compared. Endurance exercise induces a greater oxidative capacity (enzymes) compared to resistance exercise, which induces fiber hypertrophy (myofibrils). Nitrogen balance (difference between protein intake and protein degradation) for athletes is usually balanced when the intake of protein reaches 1.2 g·kg −1 ·day −1 compared to 0.8 g·kg −1 ·day −1 in resting individuals. Muscular activities promote a cascade of signals leading to the stimulation of eukaryotic initiation of myofibrillar protein synthesis. As suggested in several publications, a bolus of 15-20 g protein (from skimmed milk or whey proteins) and carbohydrate (± 30 g maltodextrine) drinks is needed immediately after stopping exercise to stimulate muscle protein and tendon collagen turnover within 1 h

  20. Protein turnover, amino acid requirements and recommendations for athletes and active populations

    Energy Technology Data Exchange (ETDEWEB)

    Poortmans, J.R.; Carpentier, A. [Laboratory for Biometry and Sport Nutrition, Faculty of Motor Sciences, Free University of Brussels, Brussels (Belgium); Pereira-Lancha, L.O. [Departamento de Nutrição, Instituto Vita, São Paulo, SP (Brazil); Lancha, A. Jr. [Laboratório de Nutrição Aplicada à Atividade Motora, Escola de Educação Física e Esporte, Universidade de São Paulo, São Paulo, SP (Brazil)

    2012-06-08

    Skeletal muscle is the major deposit of protein molecules. As for any cell or tissue, total muscle protein reflects a dynamic turnover between net protein synthesis and degradation. Noninvasive and invasive techniques have been applied to determine amino acid catabolism and muscle protein building at rest, during exercise and during the recovery period after a single experiment or training sessions. Stable isotopic tracers ({sup 13}C-lysine, {sup 15}N-glycine, {sup 2}H{sub 5}-phenylalanine) and arteriovenous differences have been used in studies of skeletal muscle and collagen tissues under resting and exercise conditions. There are different fractional synthesis rates in skeletal muscle and tendon tissues, but there is no major difference between collagen and myofibrillar protein synthesis. Strenuous exercise provokes increased proteolysis and decreased protein synthesis, the opposite occurring during the recovery period. Individuals who exercise respond differently when resistance and endurance types of contractions are compared. Endurance exercise induces a greater oxidative capacity (enzymes) compared to resistance exercise, which induces fiber hypertrophy (myofibrils). Nitrogen balance (difference between protein intake and protein degradation) for athletes is usually balanced when the intake of protein reaches 1.2 g·kg{sup −1}·day{sup −1} compared to 0.8 g·kg{sup −1}·day{sup −1} in resting individuals. Muscular activities promote a cascade of signals leading to the stimulation of eukaryotic initiation of myofibrillar protein synthesis. As suggested in several publications, a bolus of 15-20 g protein (from skimmed milk or whey proteins) and carbohydrate (± 30 g maltodextrine) drinks is needed immediately after stopping exercise to stimulate muscle protein and tendon collagen turnover within 1 h.

  1. Protein turnover, amino acid requirements and recommendations for athletes and active populations.

    Science.gov (United States)

    Poortmans, J R; Carpentier, A; Pereira-Lancha, L O; Lancha, A

    2012-10-01

    Skeletal muscle is the major deposit of protein molecules. As for any cell or tissue, total muscle protein reflects a dynamic turnover between net protein synthesis and degradation. Noninvasive and invasive techniques have been applied to determine amino acid catabolism and muscle protein building at rest, during exercise and during the recovery period after a single experiment or training sessions. Stable isotopic tracers ((13)C-lysine, (15)N-glycine, ²H5-phenylalanine) and arteriovenous differences have been used in studies of skeletal muscle and collagen tissues under resting and exercise conditions. There are different fractional synthesis rates in skeletal muscle and tendon tissues, but there is no major difference between collagen and myofibrillar protein synthesis. Strenuous exercise provokes increased proteolysis and decreased protein synthesis, the opposite occurring during the recovery period. Individuals who exercise respond differently when resistance and endurance types of contractions are compared. Endurance exercise induces a greater oxidative capacity (enzymes) compared to resistance exercise, which induces fiber hypertrophy (myofibrils). Nitrogen balance (difference between protein intake and protein degradation) for athletes is usually balanced when the intake of protein reaches 1.2 g · kg(-1) · day(-1) compared to 0.8 g · kg(-1) · day(-1) in resting individuals. Muscular activities promote a cascade of signals leading to the stimulation of eukaryotic initiation of myofibrillar protein synthesis. As suggested in several publications, a bolus of 15-20 g protein (from skimmed milk or whey proteins) and carbohydrate (± 30 g maltodextrine) drinks is needed immediately after stopping exercise to stimulate muscle protein and tendon collagen turnover within 1 h.

  2. Development and enzyme activity of protein bodies in proteinoplasts of tobacco root cells.

    Science.gov (United States)

    Vigil, E L; Ruddat, M

    1985-01-01

    The development of protein bodies in proteinoplasts of tobacco (Nicotiana tabacum L. var. Wis. 38) roots was investigated with TEM, HVEM, and enzyme cytochemistry. These plastids contain a three-dimensional network of fenestrated tubules which originate from invaginations of the inner membrane of the plastid envelope. Elaboration of the network occurs in parallel with cell differentiation: slender tubules common to plastids in meristematic cells undergo dilation as protein accumulates during cell differentiation; proteinoplasts of vacuolate and root cap cells usually contain a large protein body. The contents of the peripheral tubules, originating from the inner membrane, are less electron dense than the tubules making up the central network. Localized dilations within the tubular network result in the formation of dense spheroidal structures, protein bodies, apparently as a result of continued protein accumulation via tubules connecting to the central network. Protein might be imported from segments of rough ER attached to or apposed to the outer membrane of the proteinoplast envelope. The presence of catalase (E.C. 1.11.1.6), peroxidase (E.C. 1.11.1.7), and cytochrome oxidase (E.C. 1.9.3.1) was demonstrated by cytochemistry with diaminobenzidine (DAB) as substrate. Oxidized DAB was found in protein bodies after incubation in each of the specific reaction media. While aminotriazole and sodium azide inhibited oxidation of DAB by catalase and peroxidase, respectively, only potassium cyanide completely inhibited oxidation of DAB in protein bodies. We conclude that protein bodies of proteinoplasts in tobacco roots are not sites for storage of protein, rather protein bodies contain heme protein(s) with strong oxidase activity that may convey a specific function to proteinoplasts.

  3. Protein turnover, amino acid requirements and recommendations for athletes and active populations

    Directory of Open Access Journals (Sweden)

    J.R. Poortmans

    2012-10-01

    Full Text Available Skeletal muscle is the major deposit of protein molecules. As for any cell or tissue, total muscle protein reflects a dynamic turnover between net protein synthesis and degradation. Noninvasive and invasive techniques have been applied to determine amino acid catabolism and muscle protein building at rest, during exercise and during the recovery period after a single experiment or training sessions. Stable isotopic tracers (13C-lysine, 15N-glycine, ²H5-phenylalanine and arteriovenous differences have been used in studies of skeletal muscle and collagen tissues under resting and exercise conditions. There are different fractional synthesis rates in skeletal muscle and tendon tissues, but there is no major difference between collagen and myofibrillar protein synthesis. Strenuous exercise provokes increased proteolysis and decreased protein synthesis, the opposite occurring during the recovery period. Individuals who exercise respond differently when resistance and endurance types of contractions are compared. Endurance exercise induces a greater oxidative capacity (enzymes compared to resistance exercise, which induces fiber hypertrophy (myofibrils. Nitrogen balance (difference between protein intake and protein degradation for athletes is usually balanced when the intake of protein reaches 1.2 g·kg-1·day-1 compared to 0.8 g·kg-1·day-1 in resting individuals. Muscular activities promote a cascade of signals leading to the stimulation of eukaryotic initiation of myofibrillar protein synthesis. As suggested in several publications, a bolus of 15-20 g protein (from skimmed milk or whey proteins and carbohydrate (± 30 g maltodextrine drinks is needed immediately after stopping exercise to stimulate muscle protein and tendon collagen turnover within 1 h.

  4. Engineering and evolution of molecular chaperones and protein disaggregases with enhanced activity

    Directory of Open Access Journals (Sweden)

    Korrie eMack

    2016-03-01

    Full Text Available Cells have evolved a sophisticated proteostasis network to ensure that proteins acquire and retain their native structure and function. Critical components of this network include molecular chaperones and protein disaggregases, which function to prevent and reverse deleterious protein misfolding. Nevertheless, proteostasis networks have limits, which when exceeded can have fatal consequences as in various neurodegenerative disorders, including Parkinson’s disease and amyotrophic lateral sclerosis. A promising strategy is to engineer proteostasis networks to counter challenges presented by specific diseases or specific proteins. Here, we review efforts to enhance the activity of individual molecular chaperones or protein disaggregases via engineering and directed evolution. Remarkably, enhanced global activity or altered substrate specificity of various molecular chaperones, including GroEL, Hsp70, ClpX, and Spy, can be achieved by minor changes in primary sequence and often a single missense mutation. Likewise, small changes in the primary sequence of Hsp104 yield potentiated protein disaggregases that reverse the aggregation and buffer toxicity of various neurodegenerative disease proteins, including α-synuclein, TDP-43, and FUS. Collectively, these advances have revealed key mechanistic and functional insights into chaperone and disaggregase biology. They also suggest that enhanced chaperones and disaggregases could have important applications in treating human disease as well as in the purification of valuable proteins in the pharmaceutical sector.

  5. Computational screening for active compounds targeting protein sequences: methodology and experimental validation.

    Science.gov (United States)

    Wang, Fei; Liu, Dongxiang; Wang, Heyao; Luo, Cheng; Zheng, Mingyue; Liu, Hong; Zhu, Weiliang; Luo, Xiaomin; Zhang, Jian; Jiang, Hualiang

    2011-11-28

    The three-dimensional (3D) structures of most protein targets have not been determined so far, with many of them not even having a known ligand, a truly general method to predict ligand-protein interactions in the absence of three-dimensional information would be of great potential value in drug discovery. Using the support vector machine (SVM) approach, we constructed a model for predicting ligand-protein interaction based only on the primary sequence of proteins and the structural features of small molecules. The model, trained by using 15,000 ligand-protein interactions between 626 proteins and over 10,000 active compounds, was successfully used in discovering nine novel active compounds for four pharmacologically important targets (i.e., GPR40, SIRT1, p38, and GSK-3β). To our knowledge, this is the first example of a successful sequence-based virtual screening campaign, demonstrating that our approach has the potential to discover, with a single model, active ligands for any protein.

  6. Identification and Antithrombotic Activity of Peptides from Blue Mussel (Mytilus edulis Protein

    Directory of Open Access Journals (Sweden)

    Meiling Qiao

    2018-01-01

    Full Text Available The blue mussel (Mytilus edulis reportedly contains many bioactive components of nutritional value. Water-, salt- and acid-soluble M. edulis protein fractions were obtained and the proteins were trypsinized. The resultant peptides were analyzed by ultra-performance liquid chromatography quadrupole time of flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS. 387 unique peptides were identified that matched 81 precursor proteins. Molecular mass distributions of the proteins and peptides were analyzed by sodium dodecyl sulfate-polyacryl amide gel electrophoresis (SDS-PAGE. The differences between the three protein samples were studied by Venn diagram of peptide and protein compositions. Toxicity, allergic and antithrombotic activity of peptides was predicted using database website and molecular docking respectively. The antithrombotic activity of enzymatic hydrolysate from water-, salt- and acid-soluble M. edulis protein were 40.17%, 85.74%, 82.00% at 5 mg/mL, respectively. Active mechanism of antithrombotic peptide (ELEDSLDSER was also research about amino acid binding sites and interaction, simultaneously.

  7. Antioxidant activity of pea protein hydrolysates produced by batch fermentation with lactic acid bacteria

    Directory of Open Access Journals (Sweden)

    Stanisavljević Nemanja S.

    2015-01-01

    Full Text Available Nine Lactobacillus strains known for surface proteinase activity were chosen from our collection and tested for their ability to grow in pea seed protein-based medium, and to hydrolyze purified pea proteins in order to produce peptides with antioxidant (AO activity. Two strains, Lactobacillus rhamnosus BGT10 and Lactobacillus zeae LMG17315, exhibited strong proteolytic activity against pea proteins. The AO activity of the pea hydrolysate fraction, MW <10 kDa, obtained by the fermentation of purified pea proteins with Lactobacillus rhamnosus BGT10, was tested by standard spectrophotometric assays (DPPH, ABTS, Fe3+-reducing capacity and the recently developed direct current (DC polarographic assay. The low molecular weight fraction of the obtained hydrolysate was separated using ion exchange chromatography, while the AO activity of eluted fractions was determined by means of a sensitive DC polarographic assay without previous concentration of samples. Results revealed that the fraction present in low abundance that contained basic peptides possessed the highest antioxidant activity. Based on the obtained results, it can be concluded that Lactobacillus rhamnosus BGT10 should be further investigated as a candidate strain for large-scale production of bioactive peptides from legume proteins. [Projekat Ministartsva nauke Republike Srbije, br. 173005 i br. 173026

  8. Activated protein C attenuates acute ischaemia reperfusion injury in skeletal muscle.

    LENUS (Irish Health Repository)

    Dillon, J P

    2012-02-03

    Activated protein C (APC) is an endogenous anti-coagulant with anti-inflammatory properties. The purpose of the present study was to evaluate the effects of activated protein C in the setting of skeletal muscle ischaemia reperfusion injury (IRI). IRI was induced in rats by applying rubber bands above the levels of the greater trochanters bilaterally for a period of 2h followed by 12h reperfusion. Treatment groups received either equal volumes of normal saline or activated protein C prior to tourniquet release. Following 12h reperfusion, muscle function was assessed electrophysiologically by electrical field stimulation. The animals were then sacrificed and skeletal muscle harvested for evaluation. Activated protein C significantly attenuated skeletal muscle reperfusion injury as shown by reduced myeloperoxidase content, wet to dry ratio and electrical properties of skeletal muscle. Further in vitro work was carried out on neutrophils isolated from healthy volunteers to determine the direct effect of APC on neutrophil function. The effects of APC on TNF-alpha stimulated neutrophils were examined by measuring CD18 expression as well as reactive oxygen species generation. The in vitro work demonstrated a reduction in CD18 expression and reactive oxygen species generation. We conclude that activated protein C may have a protective role in the setting of skeletal muscle ischaemia reperfusion injury and that this is in part mediated by a direct inhibitory effect on neutrophil activation.

  9. Structural organization and expression of the gene for the mouse GM2 activator protein.

    Science.gov (United States)

    Bertoni, C; Appolloni, M G; Stirling, J L; Li, S C; Li, Y T; Orlacchio, A; Beccari, T

    1997-02-01

    The GM2 activator protein is an essential component for the degradation of GM2 ganglioside by hexosaminidase A in vivo. Mutations in the human gene coding for the GM2 activator protein cause the AB variant of GM2-gangliosidosis, a condition that is clinically indistinguishable from Tay-Sachs disease. To understand better factors affecting the expression of the GM2 activator protein gene (Gm2a) in mouse tissues, we have determined its exon-intron organization and analyzed its promoter region.Gm2a is about 14 kb, has four exons, and the 5' flanking region contains a CAAT box, Spl binding sites, AP-1, AP-2 sites, and a pair of IRE sites. A 1.2-kb fragment upstream from the initiation codon was shown to have promoter activity in NIH 3T3 cells. Similarities between the elements present in Gm2a and Hexa promoters might in part explain their similar expression patterns in mouse tissues. The different levels of GM2 activator protein mRNA in liver, kidney, brain, and testis are not owing to the use of different transcription start sites, because a single start site was found 50 bp upstream from the initiation codon in each these tissues. Northern blot analysis demonstrated variation in the GM2 activator protein mRNA expression during mouse development. Gm2a was mapped to Chromosome (Chr) 11, where it co-segregated with Csfgm.

  10. LIM domains regulate protein kinase C activity: a novel molecular function.

    Science.gov (United States)

    Maturana, Andrés D; Nakagawa, Noritaka; Yoshimoto, Nobuo; Tatematsu, Kenji; Hoshijima, Masahiko; Tanizawa, Katsuyuki; Kuroda, Shun'ichi

    2011-05-01

    Enigma homolog protein 1 (ENH1) acts as a scaffold that selectively associates protein kinases and transcription factors with cytoskeletal elements. ENH1 comprises an N-terminal PDZ domain and three C-terminal LIM domains. Through the LIM domains ENH1 interacts with the N-terminal region of protein kinase C βI (PKCβI). Here, we show that when ENH1 is co-expressed, PKCβI is translocated from the cytoplasm to the plasma membrane in the absence of any other stimulation. Moreover expression of ENH1 markedly increases PKCβI activity in the absence of PKC activators. A similar activation of PKCβI was observed with co-expression of Cypher1 or Enigma, but not other LIM proteins. The region including the three LIM domains of ENH1 (residues 415-591) appears to be sufficient for this PKCβI activation. Finally, interaction with ENH1 also increases the activity of PKCα and PKCγ, whereas it reduces PKCζ activity. These findings provide strong evidence that ENH1 activates conventional PKCs by directly binding through its LIM domains. Thus, LIM domains have a novel molecular function: the regulation of PKC activities in a PKC isoform-specific manner. Copyright © 2011 Elsevier Inc. All rights reserved.

  11. Modulation of Src Activity by Low Molecular Weight Protein Tyrosine Phosphatase During Osteoblast Differentiation

    NARCIS (Netherlands)

    Zambuzzi, Willian F.; Granjeiro, Jose M.; Parikh, Kaushal; Yuvaraj, Saravanan; Peppelenbosch, Maikel P.; Ferreira, Carmen V.

    2008-01-01

    Background: Src kinase plays a critical role in bone metabolism, particularly in osteoclasts. However, the ability of Src kinase to modulate the activity of other bone cells is less well understood. In this work, we examined the expression and activity of Src and low molecular weight protein

  12. Differential Requirement for Pten Lipid and Protein Phosphatase Activity during Zebrafish Embryonic Development.

    Science.gov (United States)

    Stumpf, Miriam; den Hertog, Jeroen

    2016-01-01

    The lipid- and protein phosphatase PTEN is one of the most frequently mutated tumor suppressor genes in human cancers and many mutations found in tumor samples directly affect PTEN phosphatase activity. In order to understand the functional consequences of these mutations in vivo, the aim of our study was to dissect the role of Pten phosphatase activities during zebrafish embryonic development. As in other model organisms, zebrafish mutants lacking functional Pten are embryonically lethal. Zebrafish have two pten genes and pten double homozygous zebrafish embryos develop a severe pleiotropic phenotype around 4 days post fertilization, which can be largely rescued by re-introduction of pten mRNA at the one-cell stage. We used this assay to characterize the rescue-capacity of Pten and variants with mutations that disrupt lipid, protein or both phosphatase activities. The pleiotropic phenotype at 4dpf could only be rescued by wild type Pten, indicating that both phosphatase activities are required for normal zebrafish embryonic development. An earlier aspect of the phenotype, hyperbranching of intersegmental vessels, however, was rescued by Pten that retained lipid phosphatase activity, independent of protein phosphatase activity. Lipid phosphatase activity was also required for moderating pAkt levels at 4 dpf. We propose that the role of Pten during angiogenesis mainly consists of suppressing PI3K signaling via its lipid phosphatase activity, whereas the complex process of embryonic development requires lipid and protein phosphatase of Pten.

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

    Directory of Open Access Journals (Sweden)

    Honghao Li

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

  14. Detergent disruption of bacterial inner membranes and recovery of protein translocation activity

    International Nuclear Information System (INIS)

    Cunningham, K.; Wickner, W.T.

    1989-01-01

    Isolation of the integral membrane components of protein translocation requires methods for fractionation and functional reconstitution. The authors treated inner-membrane vesicles of Escherichia coli with mixtures of octyl β-D-glucoside, phospholipids, and an integral membrane carrier protein under conditions that extract most of the membrane proteins into micellar solution. Upon dialysis, proteoliposomes were reconstituted that supported translocation of radiochemically pure [ 35 S]pro-OmpA (the precursor of outer membrane protein A). Translocation into these proteoliposomes required ATP hydrolysis and membrane proteins, indicating that the reaction is that of the inner membrane. The suspension of membranes in detergent was separated into supernatant and pellet fractions by ultracentrifugation. After reconstitution, translocation activity was observed in both fractions, but processing by leader peptidase of translocated pro-OmpA to OmpA was not detectable in the reconstituted pellet fraction. Processing activity was restored by addition of pure leader peptidase as long as this enzyme was added before detergent removal, indicating that the translocation activity is not associated with detergent-resistant membrane vesicles. These results show that protein translocation activity can be recovered from detergent-disrupted membrane vesicles, providing a first step towards the goal of isolating the solubilized components

  15. The protein phosphatase activity of PTEN is essential for regulating neural stem cell differentiation.

    Science.gov (United States)

    Lyu, Jingwen; Yu, Xiuya; He, Lingjie; Cheng, Tianlin; Zhou, Jingjing; Cheng, Cheng; Chen, Zhifang; Cheng, Guoqiang; Qiu, Zilong; Zhou, Wenhao

    2015-04-18

    The tumor suppressor gene Phosphatase and tensin homolog (PTEN) is highly expressed in neural progenitor cells (NPCs) and plays an important role in development of the central nervous system. As a dual-specificity phosphatase, the loss of PTEN phosphatase activity has been linked to various diseases. Here we report that the protein phosphatase activity of Pten is critical for regulating differentiation of neural progenitor cells. First we found that deletion of Pten promotes neuronal differentiation. To determine whether the protein or lipid phosphatase activity is required for regulating neuronal differentiation, we generated phosphatase domain-specific Pten mutations. Interestingly, only expression of protein phosphatase-deficient mutant Y138L could mimic the effect of knocking down Pten, suggesting the protein phosphatase of Pten is critical for regulating NPC differentiation. Importantly, we showed that the wild-type and lipid phosphatase mutant (G129E) forms of Pten are able to rescue neuronal differentiation in Pten knockout NPCs, but mutants containing protein phosphatase mutant cannot. We further found that Pten-dependent dephosphorylation of CREB is critical for neuronal differentiation. Our data indicate that the protein phosphatase activity of PTEN is critical for regulating differentiation of NSCs during cortical development.

  16. Structural basis for chemokine recognition and activation of a viral G protein-coupled receptor

    Energy Technology Data Exchange (ETDEWEB)

    Burg, John S.; Ingram, Jessica R.; Venkatakrishnan, A.J.; Jude, Kevin M.; Dukkipati, Abhiram; Feinberg, Evan N.; Angelini, Alessandro; Waghray, Deepa; Dror, Ron O.; Ploegh, Hidde L.; Garcia, K. Christopher (Stanford); (Stanford-MED); (Whitehead); (MIT)

    2015-03-05

    Chemokines are small proteins that function as immune modulators through activation of chemokine G protein-coupled receptors (GPCRs). Several viruses also encode chemokines and chemokine receptors to subvert the host immune response. How protein ligands activate GPCRs remains unknown. We report the crystal structure at 2.9 angstrom resolution of the human cytomegalovirus GPCR US28 in complex with the chemokine domain of human CX3CL1 (fractalkine). The globular body of CX3CL1 is perched on top of the US28 extracellular vestibule, whereas its amino terminus projects into the central core of US28. The transmembrane helices of US28 adopt an active-state-like conformation. Atomic-level simulations suggest that the agonist-independent activity of US28 may be due to an amino acid network evolved in the viral GPCR to destabilize the receptor’s inactive state.

  17. Characterization of a saccharide-binding protein from Talisia esculenta seeds with trypsin inhibitory activity.

    Science.gov (United States)

    Freire, Maria das Graças M; Vasconcelos, Ilka Maria; Oliveira, Marcos Vinïcius; Filho, Gonçalo Apolinário de Souza; Macedo, Maria Lïgia R

    2009-01-01

    Some proteins exhibit characteristics that suggest they have a primary, if not an exclusive role in nutrient reserve storage. The best studied examples are the storage proteins that accumulate specifically in developing seeds. Some of these protein demonstrate biological activities that could contribute to resistance to pest, pathogens or abiotic stresses. In this study we present the biochemical characterization and cloning of the major protein from seeds of T. esculenta (Talisin), a member of the Sapindaceae family. The N-terminal sequence of the protein isolated was used to produce a degenerated primer. This primer allowed the amplification of the Talisin cDNA by RTPCR from mRNA of the T. esculenta seeds protein. The sequence analysis of the cloned cDNA, demonstrated a 756 bp sequence encoding a peptide of 198 amino acids. The deduced peptide presented high similarity to a typical VSP, the 22-kDa protein in lychee (73 %) and 50.0 % identity to Theobroma bicolor reserve protein. Identities of 52.0 % and 44.0 % to trypsin inhibitors from Treobroma mammosum and Populus tremula respectively. In conclusion, we may suggest that Talisin could be a seed storage protein with affinity properties, i.e. interacts with carbohydrates and trypsin enzyme.

  18. Acetic acid activates the AMP-activated protein kinase signaling pathway to regulate lipid metabolism in bovine hepatocytes.

    Directory of Open Access Journals (Sweden)

    Xinwei Li

    Full Text Available The effect of acetic acid on hepatic lipid metabolism in ruminants differs significantly from that in monogastric animals. Therefore, the aim of this study was to investigate the regulation mechanism of acetic acid on the hepatic lipid metabolism in dairy cows. The AMP-activated protein kinase (AMPK signaling pathway plays a key role in regulating hepatic lipid metabolism. In vitro, bovine hepatocytes were cultured and treated with different concentrations of sodium acetate (neutralized acetic acid and BML-275 (an AMPKα inhibitor. Acetic acid consumed a large amount of ATP, resulting in an increase in AMPKα phosphorylation. The increase in AMPKα phosphorylation increased the expression and transcriptional activity of peroxisome proliferator-activated receptor α, which upregulated the expression of lipid oxidation genes, thereby increasing lipid oxidation in bovine hepatocytes. Furthermore, elevated AMPKα phosphorylation reduced the expression and transcriptional activity of the sterol regulatory element-binding protein 1c and the carbohydrate responsive element-binding protein, which reduced the expression of lipogenic genes, thereby decreasing lipid biosynthesis in bovine hepatocytes. In addition, activated AMPKα inhibited the activity of acetyl-CoA carboxylase. Consequently, the triglyceride content in the acetate-treated hepatocytes was significantly decreased. These results indicate that acetic acid activates the AMPKα signaling pathway to increase lipid oxidation and decrease lipid synthesis in bovine hepatocytes, thereby reducing liver fat accumulation in dairy cows.

  19. Activation of protein kinase A and exchange protein directly activated by cAMP promotes adipocyte differentiation of human mesenchymal stem cells

    DEFF Research Database (Denmark)

    Jia, Bingbing; Madsen, Lise; Petersen, Rasmus Koefoed

    2012-01-01

    ) and exchange protein directly activated by cAMP (Epac) in adipocyte conversion of human mesenchymal stem cells derived from adipose tissue (hMADS). We show that cAMP signaling involving the simultaneous activation of both PKA- and Epac-dependent signaling is critical for this process even in the presence......Human mesenchymal stem cells are primary multipotent cells capable of differentiating into several cell types including adipocytes when cultured under defined in vitro conditions. In the present study we investigated the role of cAMP signaling and its downstream effectors, protein kinase A (PKA...... results emphasize the need for cAMP signaling in concert with treatment with a PPARγ or PPARδ agonist to secure efficient adipocyte differentiation of human hMADS mesenchymal stem cells....

  20. Hepatitis C virus core protein potentiates proangiogenic activity of hepatocellular carcinoma cells.

    Science.gov (United States)

    Shao, Yu-Yun; Hsieh, Min-Shu; Wang, Han-Yu; Li, Yong-Shi; Lin, Hang; Hsu, Hung-Wei; Huang, Chung-Yi; Hsu, Chih-Hung; Cheng, Ann-Lii

    2017-10-17

    Increased angiogenic activity has been demonstrated in hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC), but the mechanism was unclear. To study the role of HCV core protein, we used tube formation and Matrigel plug assays to assess the proangiogenic activity of an HCC cell line, HuH7, and 2 of its stable clones-HuH7-core-high and HuH7-core-low, with high and low HCV core protein expression, respectively. In both assays, HuH7-core-high and HuH7-core-low cells dose-dependently induced stronger angiogenesis than control cells. HuH7 cells with HCV core protein expression showed increased mRNA and protein expression of vascular endothelial growth factor (VEGF). VEGF inhibition by bevacizumab reduced the proangiogenic activity of HuH7-core-high cells. The promotor region of VEGF contains the binding site of activator protein-1 (AP-1). Compared with controls, HuH7-core-high cells had an increased AP-1 activity and nuclear localization of phospho-c-jun. AP-1 inhibition using either RNA knockdown or AP-1 inhibitors reduced the VEGF mRNA expression and the proangiogenic activity of HuH7-core-high cells. Among 131 tissue samples from HCC patients, HCV-related HCC revealed stronger VEGF expression than did hepatitis B virus-related HCC. In conclusion, increased VEGF expression through AP-1 activation is a crucial mechanism underlying the proangiogenic activity of the HCV core protein in HCC cells.

  1. A histone-like protein of mycobacteria possesses ferritin superfamily protein-like activity and protects against DNA damage by Fenton reaction.

    Directory of Open Access Journals (Sweden)

    Masaki Takatsuka

    Full Text Available Iron is an essential metal for living organisms but its level must be strictly controlled in cells, because ferrous ion induces toxicity by generating highly active reactive oxygen, hydroxyl radicals, through the Fenton reaction. In addition, ferric ion shows low solubility under physiological conditions. To overcome these obstacles living organisms possess Ferritin superfamily proteins that are distributed in all three domains of life: bacteria, archaea, and eukaryotes. These proteins minimize hydroxyl radical formation by ferroxidase activity that converts Fe(2+ into Fe(3+ and sequesters iron by storing it as a mineral inside a protein cage. In this study, we discovered that mycobacterial DNA-binding protein 1 (MDP1, a histone-like protein, has similar activity to ferritin superfamily proteins. MDP1 prevented the Fenton reaction and protects DNA by the ferroxidase activity. The K(m values of the ferroxidase activity by MDP1 of Mycobacterium bovis bacillus Calmette-Guérin (BCG-3007c, Mycobacterium tuberculosis (Rv2986c, and Mycobacterium leprae (ML1683; ML-LBP were 0.292, 0.252, and 0.129 mM, respectively. Furthermore, one MDP1 molecule directly captured 81.4±19.1 iron atoms, suggesting the role of this protein in iron storage. This study describes for the first time a ferroxidase-iron storage protein outside of the ferritin superfamily proteins and the protective role of this bacterial protein from DNA damage.

  2. Viroporin Activity of the Foot-and-Mouth Disease Virus Non-Structural 2B Protein.

    Directory of Open Access Journals (Sweden)

    Da Ao

    Full Text Available Viroporins are a family of low-molecular-weight hydrophobic transmembrane proteins that are encoded by various animal viruses. Viroporins form transmembrane pores in host cells via oligomerization, thereby destroying cellular homeostasis and inducing cytopathy for virus replication and virion release. Among the Picornaviridae family of viruses, the 2B protein encoded by enteroviruses is well understood, whereas the viroporin activity of the 2B protein encoded by the foot-and-mouth disease virus (FMDV has not yet been described. An analysis of the FMDV 2B protein domains by computer-aided programs conducted in this study revealed that this protein may contain two transmembrane regions. Further biochemical, biophysical and functional studies revealed that the protein possesses a number of features typical of a viroporin when it is overexpressed in bacterial and mammalian cells as well as in FMDV-infected cells. The protein was found to be mainly localized in the endoplasmic reticulum (ER, with both the N- and C-terminal domains stretched into the cytosol. It exhibited cytotoxicity in Escherichia coli, which attenuated 2B protein expression. The release of virions from cells infected with FMDV was inhibited by amantadine, a viroporin inhibitor. The 2B protein monomers interacted with each other to form both intracellular and extracellular oligomers. The Ca(2+ concentration in the cells increased, and the integrity of the cytoplasmic membrane was disrupted in cells that expressed the 2B protein. Moreover, the 2B protein induced intense autophagy in host cells. All of the results of this study demonstrate that the FMDV 2B protein has properties that are also found in other viroporins and may be involved in the infection mechanism of FMDV.

  3. Interaction between Salt-inducible Kinase 2 and Protein Phosphatase 2A Regulates the Activity of Calcium/Calmodulin-dependent Protein Kinase I and Protein Phosphatase Methylesterase-1*

    Science.gov (United States)

    Lee, Chia-Wei; Yang, Fu-Chia; Chang, Hsin-Yun; Chou, Hanyi; Tan, Bertrand Chin-Ming; Lee, Sheng-Chung

    2014-01-01

    Salt-inducible kinase 2 (SIK2) is the only AMP-activated kinase (AMPK) family member known to interact with protein phosphatase 2 (PP2A). However, the functional aspects of this complex are largely unknown. Here we report that the SIK2·PP2A complex preserves both kinase and phosphatase activities. In this capacity, SIK2 attenuates the association of the PP2A repressor, the protein phosphatase methylesterase-1 (PME-1), thus preserving the methylation status of the PP2A catalytic subunit. Furthermore, the SIK2·PP2A holoenzyme complex dephosphorylates and inactivates Ca2+/calmodulin-dependent protein kinase I (CaMKI), an upstream kinase for phosphorylating PME-1/Ser15. The functionally antagonistic SIK2·PP2A and CaMKI and PME-1 networks thus constitute a negative feedback loop that modulates the phosphatase activity of PP2A. Depletion of SIK2 led to disruption of the SIK2·PP2A complex, activation of CaMKI, and downstream effects, including phosphorylation of HDAC5/Ser259, sequestration of HDAC5 in the cytoplasm, and activation of myocyte-specific enhancer factor 2C (MEF2C)-mediated gene expression. These results suggest that the SIK2·PP2A complex functions in the regulation of MEF2C-dependent transcription. Furthermore, this study suggests that the tightly linked regulatory loop comprised of the SIK2·PP2A and CaMKI and PME-1 networks may function in fine-tuning cell proliferation and stress response. PMID:24841198

  4. Chronic activation of AMP-activated protein kinase increases monocarboxylate transporter 2 and 4 expression in skeletal muscle.

    Science.gov (United States)

    England, E M; Shi, H; Matarneh, S K; Oliver, E M; Helm, E T; Scheffler, T L; Puolanne, E; Gerrard, D E

    2017-08-01

    Acute activation of AMP-activated protein kinase (AMPK) increases monocarboxylate transporter (MCT) expression in skeletal muscle. However, the impact of chronic activation of AMPK on MCT expression in skeletal muscle is unknown. To investigate, MCT1, MCT2, and MCT4 mRNA expression and protein abundance were measured in the longissimus lumborum (glycolytic), masseter (oxidative), and heart from wild-type (control) and AMPK γ3 pigs. The AMPK γ3 gain in function mutation results in AMPK being constitutively active in glycolytic skeletal muscle and increases energy producing pathways. The MCT1 and MCT2 mRNA expression in muscle was lower ( muscle, but MCT2 was greater ( muscles with an oxidative muscle phenotype. Monocarboxylate transporter 2 was also detected in muscle mitochondria and may explain the differences between muscles. The MCT4 mRNA expression was intermediate among all tissues tested and greater ( muscle.

  5. R26R-GR: a Cre-activable dual fluorescent protein reporter mouse.

    Directory of Open Access Journals (Sweden)

    You-Tzung Chen

    Full Text Available Green fluorescent protein (GFP and its derivatives are the most widely used molecular reporters for live cell imagining. The development of organelle-specific fusion fluorescent proteins improves the labeling resolution to a higher level. Here we generate a R26 dual fluorescent protein reporter mouse, activated by Cre-mediated DNA recombination, labeling target cells with a chromatin-specific enhanced green fluorescence protein (EGFP and a plasma membrane-anchored monomeric cherry fluorescent protein (mCherry. This dual labeling allows the visualization of mitotic events, cell shapes and intracellular vesicle behaviors. We expect this reporter mouse to have a wide application in developmental biology studies, transplantation experiments as well as cancer/stem cell lineage tracing.

  6. Interleukin-1 activates a novel protein kinase cascade that results in the phosphorylation of Hsp27.

    Science.gov (United States)

    Freshney, N W; Rawlinson, L; Guesdon, F; Jones, E; Cowley, S; Hsuan, J; Saklatvala, J

    1994-09-23

    An IL-1-stimulated protein kinase cascade resulting in phosphorylation of the small heat shock protein hsp27 has been identified in KB cells. It is distinct from the p42 MAP kinase cascade. An upstream activator kinase phosphorylated a 40 kDa kinase (p40) upon threonine and tyrosine residues, which in turn phosphorylated a 50 kDa kinase (p50) upon threonine (and some serine) residues. p50 phosphorylated hsp27 upon serine. p40 and p50 were purified to near homogeneity. All three components were inactivated by protein phosphatase 2A, and p40 was inactivated by protein tyrosine phosphatase 1B. The substrate specificity of p40 differed from that of p42 and p54 MAP kinases. The upstream activator was not a MAP kinase kinase. p50 resembled MAPKAPK-2 and may be identical.

  7. Sch proteins are localized on endoplasmic reticulum membranes and are redistributed after tyrosine kinase receptor activation

    DEFF Research Database (Denmark)

    Lotti, L V; Lanfrancone, L; Migliaccio, E

    1996-01-01

    The intracellular localization of Shc proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear...... and endocytic structures, such as coated pits and endosomes, and with the peripheral cytosol. Receptor activation in cells expressing phosphorylation-defective mutants of Shc and erbB-2 kinase showed that receptor autophosphorylation, but not Shc phosphorylation, is required for redistribution of Shc proteins....... The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein....

  8. Insecticidal activity of proteinous venom from tentacle of jellyfish Rhopilema esculentum Kishinouye.

    Science.gov (United States)

    Yu, Huahua; Liu, Xiguang; Dong, Xiangli; Li, Cuiping; Xing, Ronge; Liu, Song; Li, Pengcheng

    2005-11-15

    Insecticidal activity of proteinous venom from tentacle of jellyfish Rhopilema esculentum Kishinouye was determined against three pest species, Stephanitis pyri Fabriciusa, Aphis medicaginis Koch, and Myzus persicae Sulzer. R. esculentum full proteinous venom had different insecticidal activity against S. pyri Fabriciusa, A. medicaginis Koch, and M. persicae Sulzer. The 48 h LC50 values were 123.1, 581.6, and 716.3 microg/mL, respectively. Of the three pests, R. esculentum full proteinous venom had the most potent toxicity against S. pyri Fabriciusa, and the corrected mortality recorded at 48 h was 97.86%. So, S. pyri Fabriciusa could be a potential target pest of R. esculentum full proteinous venom.

  9. Transcriptional activation capacity of the novel PLAG family of zinc finger proteins.

    Science.gov (United States)

    Kas, K; Voz, M L; Hensen, K; Meyen, E; Van de Ven, W J

    1998-09-04

    We have isolated and characterized two novel cDNAs encoding C2H2 zinc finger proteins showing high sequence homology to PLAG1, a protein ectopically activated by promoter swapping or promoter substitution in pleomorphic adenomas with chromosomal abnormalities at chromosome 8q12. PLAG1 and the two new PLAG1 family members (PLAGL1 and PLAGL2) constitute a novel subfamily of zinc finger proteins that recognize DNA and/or RNA. To examine the potential of the three human proteins to modulate transcription, we constructed several PLAG/GAL4 DNA binding domain fusion proteins and measured their ability to activate transcription of a reporter gene construct in different mammalian cell lines and in yeast. Although the carboxyl-terminal part of PLAGL1 shows strong overall transcriptional activity in mesenchymal (COS-1) and epithelial cells (293), both PLAG1 and PLAGL2 transactivate in mesenchymal cells only if depleted from a repressing region. This effect is less profound in epithelial cells. These data suggest that the activation in pleomorphic adenomas of PLAG1 most likely results in uncontrolled activation of downstream target genes.

  10. Protein kinase C is activated in glomeruli from streptozotocin diabetic rats. Possible mediation by glucose

    International Nuclear Information System (INIS)

    Craven, P.A.; DeRubertis, F.R.

    1989-01-01

    Glomerular inositol content and the turnover of polyphosphoinositides was reduced by 58% in 1-2 wk streptozotocin diabetic rats. Addition of inositol to the incubation medium increased polyphosphoinositide turnover in glomeruli from diabetic rats to control values. Despite the reduction in inositol content and polyphosphoinositide turnover, protein kinase C was activated in glomeruli from diabetic rats, as assessed by an increase in the percentage of enzyme activity associated with the particulate cell fraction. Total protein kinase C activity was not different between glomeruli from control and diabetic rats. Treatment of diabetic rats with insulin to achieve near euglycemia prevented the increase in particulate protein kinase C. Moreover, incubation of glomeruli from control rats with glucose (100-1,000 mg/dl) resulted in a progressive increase in labeled diacylglycerol production and in the percentage of protein kinase C activity which was associated with the particulate fraction. These results support a role for hyperglycemia per se in the enhanced state of activation of protein kinase C seen in glomeruli from diabetic rats. Glucose did not appear to increase diacylglycerol by stimulating inositol phospholipid hydrolysis in glomeruli. Other pathways for diacylglycerol production, including de novo synthesis and phospholipase C mediated hydrolysis of phosphatidylcholine or phosphatidyl-inositol-glycan are not excluded

  11. Proteins labelling with 125I and experimental determination of their specific activity

    International Nuclear Information System (INIS)

    Caro, R.A.; Ciscato, V.A.; Giacomini, S.M.V. de; Quiroga, S.; Radicella, R.

    1975-11-01

    A standardization of the labelling technique of proteins with 125 I and the control of the obtained products, principally their specific activities was performed, in order to utilize them correctly in radioimmunoassays. The quantities of chloramine-T and sodium metabisulphite were lowered, with regard to the original method, to 3.6 and 9.6 μg respectively. Under these conditions, optimal yields and radioiodinated proteins with good immunological activities were obtained. It was found that the specific activity calculated, as usual, from the yield obtained by electrophoresis, is higher than the real value. For these reasons the yields and the corresponding specific activities were determined from ascending chromatographies performed with 70 per cent methanol as solvent, during two hours in darkness. The radioimmunoassay displacement curves obtained with proteins labelled which the proposed method and the specific activities of which were calculated from their radiochromatographic patterns, were reproducible and gave a percentage of bound radioiodinated protein in the absence of cold protein of 50 +- 4. (author) [es

  12. Conserved TRAM Domain Functions as an Archaeal Cold Shock Protein via RNA Chaperone Activity

    Directory of Open Access Journals (Sweden)

    Bo Zhang

    2017-08-01

    Full Text Available Cold shock proteins (Csps enable organisms to acclimate to and survive in cold environments and the bacterial CspA family exerts the cold protection via its RNA chaperone activity. However, most Archaea do not contain orthologs to the bacterial csp. TRAM, a conserved domain among RNA modification proteins ubiquitously distributed in organisms, occurs as an individual protein in most archaeal phyla and has a structural similarity to Csp proteins, yet its biological functions remain unknown. Through physiological and biochemical studies on four TRAM proteins from a cold adaptive archaeon Methanolobus psychrophilus R15, this work demonstrated that TRAM is an archaeal Csp and exhibits RNA chaperone activity. Three TRAM encoding genes (Mpsy_0643, Mpsy_3043, and Mpsy_3066 exhibited remarkable cold-shock induced transcription and were preferentially translated at lower temperature (18°C, while the fourth (Mpsy_2002 was constitutively expressed. They were all able to complement the cspABGE mutant of Escherichia coli BX04 that does not grow in cold temperatures and showed transcriptional antitermination. TRAM3066 (gene product of Mpsy_3066 and TRAM2002 (gene product of Mpsy_2002 displayed sequence-non-specific RNA but not DNA binding activity, and TRAM3066 assisted RNases in degradation of structured RNA, thus validating the RNA chaperone activity of TRAMs. Given the chaperone activity, TRAM is predicted to function beyond a Csp.

  13. Isolation and characterization of biologically active venom protein from sea snake Enhydrina schistosa.

    Science.gov (United States)

    Damotharan, Palani; Veeruraj, Anguchamy; Arumugam, Muthuvel; Balasubramanian, Thangavel

    2015-03-01

    The present study is designed to investigate the isolation and characterization of biological and biochemical active venom protein from sea snake, Enhydrina schistosa. The highest purification peaks in ion-exchange chromatography on DEAE-cellulose column were obtained for fraction numbers 39-49 when eluted with 0.35-0.45 M NaCl. Eighty per cent purity was obtained in the final stage of purification, and a single protein band of about 44 kDa was visualized in SDS-polyacrylamide gel under reducing condition. Purified venom protein expressed as haemolytic, cytotoxicity and proteolytic activities with lethal concentration (LC50 ) at 2.0 μg/mL. Venom protein exhibits enzymatic activity and hydrolyzed casein and gelatin. Gelatinolytic activity was optimal at pH 5-9. In conclusion, the present results suggested that the sea snake venom might be feasible sources for biologically active substances. Thus, this low molecular weight component of the venom protein could be used in potentially serve biological and pharmaceutical aspects. © 2014 Wiley Periodicals, Inc.

  14. Discovery of Cryoprotective Activity in Human Genome-Derived Intrinsically Disordered Proteins

    Directory of Open Access Journals (Sweden)

    Naoki Matsuo

    2018-01-01

    Full Text Available Intrinsically disordered proteins (IDPs are an emerging phenomenon. They may have a high degree of flexibility in their polypeptide chains, which lack a stable 3D structure. Although several biological functions of IDPs have been proposed, their general function is not known. The only finding related to their function is the genetically conserved YSK2 motif present in plant dehydrins. These proteins were shown to be IDPs with the YSK2 motif serving as a core region for the dehydrins’ cryoprotective activity. Here we examined the cryoprotective activity of randomly selected IDPs toward the model enzyme lactate dehydrogenase (LDH. All five IDPs that were examined were in the range of 35–45 amino acid residues in length and were equally potent at a concentration of 50 μg/mL, whereas folded proteins, the PSD-95/Dlg/ZO-1 (PDZ domain, and lysozymes had no potency. We further examined their cryoprotective activity toward glutathione S-transferase as an example of the other enzyme, and toward enhanced green fluorescent protein as a non-enzyme protein example. We further examined the lyophilization protective activity of the peptides toward LDH, which revealed that some IDPs showed a higher activity than that of bovine serum albumin (BSA. Based on these observations, we propose that cryoprotection is a general feature of IDPs. Our findings may become a clue to various industrial applications of IDPs in the future.

  15. An efficient heuristic method for active feature acquisition and its application to protein-protein interaction prediction

    Directory of Open Access Journals (Sweden)

    Thahir Mohamed

    2012-11-01

    Full Text Available Abstract Background Machine learning approaches for classification learn the pattern of the feature space of different classes, or learn a boundary that separates the feature space into different classes. The features of the data instances are usually available, and it is only the class-labels of the instances that are unavailable. For example, to classify text documents into different topic categories, the words in the documents are features and they are readily available, whereas the topic is what is predicted. However, in some domains obtaining features may be resource-intensive because of which not all features may be available. An example is that of protein-protein interaction prediction, where not only are the labels ('interacting' or 'non-interacting' unavailable, but so are some of the features. It may be possible to obtain at least some of the missing features by carrying out a few experiments as permitted by the available resources. If only a few experiments can be carried out to acquire missing features, which proteins should be studied and which features of those proteins should be determined? From the perspective of machine learning for PPI prediction, it would be desirable that those features be acquired which when used in training the classifier, the accuracy of the classifier is improved the most. That is, the utility of the feature-acquisition is measured in terms of how much acquired features contribute to improving the accuracy of the classifier. Active feature acquisition (AFA is a strategy to preselect such instance-feature combinations (i.e. protein and experiment combinations for maximum utility. The goal of AFA is the creation of optimal training set that would result in the best classifier, and not in determining the best classification model itself. Results We present a heuristic method for active feature acquisition to calculate the utility of acquiring a missing feature. This heuristic takes into account the change in

  16. Activity-based protein profiling reveals broad activity of the nerve agent sarin

    NARCIS (Netherlands)

    Tuin, A.W.; Mol, M.A.E.; Berg, R.M. van den; Fidder, A.; Marel, G.A. van der; Overkleeft, H.S.; Noort, D.

    2009-01-01

    Elucidation of noncholinesterase protein targets of organophosphates, and nerve agents in particular, may reveal additional mechanisms for their high toxicity as well as clues for novel therapeutic approaches toward intoxications with these agents. Within this framework, we here describe the

  17. Is increased dietary protein necessary or beneficial for individuals with a physically active lifestyle?

    Science.gov (United States)

    Lemon, P W

    1996-04-01

    For most of the 20th century, scientists have believed that protein needs are not altered by physical exercise. In contrast, athletes are typically convinced that additional dietary protein can significantly enhance exercise performance. Until recently, the opinion of the athletes has been largely unsubstantiated in the scientific literature. However, since the 1970s, an increasing number of studies have appeared that indicate dietary protein needs are elevated in individuals who are regularly physically active. Together, these data suggest that the RDA for those who engage in regular endurance exercise should be about 1.2-1.4 g protein/kg body mass/d (150-175% of the current RDA) and 1.7-1.8 g protein/kg body mass/d (212-225% of the current RDA) for strength exercisers. Fortunately, the typical North American diet contains protein near these quantities, so most individuals who decide to begin an exercise program will obtain sufficient protein as long as their diet is mixed and they are careful to consume adequate energy. Populations at greatest risk for consuming insufficient protein include any group that restricts energy intake (those on diets) or high quality protein sources (vegetarians) as well as any group that has a requirement higher than normal due to another existing condition (growing individuals). Future studies should focus on these groups. Moreover, few exercise performance measures have been made, so any negative effect of insufficient dietary protein on athletic success needs to be determined. Supplementation of several individual amino acids may be beneficial for physically active individuals, but considerable potential risk is also present. Intake of large quantities of individual amino acids is not recommended until much more information is available.

  18. The rate of synthesis and decomposition of tissue proteins in hypokinesia and increased muscular activity

    Science.gov (United States)

    Fedorov, I. V.; Chernyy, A. V.; Fedorov, A. I.

    1978-01-01

    During hypokinesia and physical loading (swimming) of rats, the radioactivity of skeletal muscle, liver, kidney, heart, and blood proteins was determined after administration of radioactive amino acids. Tissue protein synthesis decreased during hypokinesia, and decomposition increased. Both synthesis and decomposition increased during physical loading, but anabolic processes predominated in the total tissue balance. The weights of the animals decreased in hypokinesia and increased during increased muscle activity.

  19. Physiological cerebrovascular remodeling in response to chronic mild hypoxia: a role for activated protein C

    OpenAIRE

    Burnier, Laurent; Boroujerdi, Amin; Fernández, Jose A.; Welser-Alves, Jennifer V.; Griffin, John H.; Milner, Richard

    2016-01-01

    Activated protein C (APC) is a serine protease that promotes favorable changes in vascular barrier integrity and post-ischemic angiogenic remodeling in animal models of ischemic stroke, and its efficacy is currently being investigated in clinical ischemic stroke trials. Interestingly, application of sub-clinical chronic mild hypoxia (CMH) (8% O2) also promotes angiogenic remodeling and increased tight junction protein expression, suggestive of enhanced blood-brain barrier (BBB) integrity, tho...

  20. Effects of prenatal protein malnutrition on the electrical cerebral activity during development.

    Science.gov (United States)

    De Frías, V; Varela, O; Oropeza, J J; Bisiacchi, B; Alvarez, A

    2010-10-04

    Early protein restriction during the prenatal period has significant repercussions on the ontogeny and development of the central nervous system. The present study investigates whether early prenatal protein malnutrition could alter the electrical cerebral activity of the progeny. We used Sprague-Dawley female rats of 200 g randomly divided into three groups: a control group that received a diet with 25% of the protein content (lactalbumin), the experimental group, that received a diet with 6% of the protein content and the rehabilitated group that initially received a diet with 6% of the protein content, then switched to a diet with 25% of the protein content after the weaning period (P20D) up to 60 days of life (P60D). Reduction of the protein content from 25% to 6% of lactalbumin in the diet of pregnant rats produces impairment in the electrical cerebral activity in the progeny at P20D and at P60D. The power spectral analysis for each one of the electroencephalograms revealed that prenatal protein malnutrition in rats produced a significant reduction of the alpha (8-13 Hz) and the beta bands (13-30 Hz) and a significant increase of the theta (4-8 Hz), and delta bands (1-4 Hz), at two different stages of life (P20D and P60D). Similar results were obtained for the rehabilitated group. These results indicate that early malnutrition in life affects the ontogeny of the electrical cerebral activity. This insult probably disrupts the establishment of cortical neural circuits during the critical period of brain development. The rehabilitation period did not revert the impairment in the electrical cerebral activity produced by malnutrition. We used one-way ANOVA analysis, followed by Tukey test (*p<0.001). (c) 2010 Elsevier Ireland Ltd. All rights reserved.

  1. Effect of polyols on the conformational stability and biological activity of a model protein lysozyme

    OpenAIRE

    Singh, Somnath; Singh, Jagdish

    2003-01-01

    The purpose of this study was to investigate the stabilizing action of polyols against various protein degradation mechanisms (eg, aggregation, deamidation, oxidation), using a model protein lysozyme. Differential scanning calorimeter (DSC) was used to measure the thermodynamic parameters, mid point transition temperature and calorimetric enthalpy, in order to evaluate conformational stability. Enzyme activity assay was used to corroborate the DSC results. Mannitol, sucrose, lactose, glycerol...

  2. GILT expression in B cells diminishes cathepsin S steady-state protein expression and activity

    OpenAIRE

    Phipps-Yonas, Hannah; Semik, Vikki; Hastings, Karen Taraszka

    2012-01-01

    MHC class II-restricted Ag processing requires protein degradation in the endocytic pathway for the activation of CD4+ T cells. Gamma-interferon-inducible lysosomal thiol reductase (GILT) facilitates Ag processing by reducing protein disulfide bonds in this compartment. Lysosomal cysteine protease cathepsin S (CatS) contains disulfide bonds and mediates essential steps in MHC class II-restricted processing, including proteolysis of large polypeptides and cleavage of the invariant chain. We so...

  3. 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.

  4. Antroquinonol blocks Ras and Rho signaling via the inhibition of protein isoprenyltransferase activity in cancer cells.

    Science.gov (United States)

    Ho, Ching-Liang; Wang, Jui-Ling; Lee, Cheng-Chung; Cheng, Hsiu-Yi; Wen, Wu-Che; Cheng, Howard Hao-Yu; Chen, Miles Chih-Ming

    2014-10-01

    Antroquinonol is the smallest anticancer molecule isolated from Antrodia camphorata thus far. The ubiquinone-like structure of Antroquinonol exhibits a broad spectrum of activity against malignancies in vivo and in vitro. However, the mechanism of action of Antroquinonol remains unclear. Here, we provide evidence that Antroquinonol plays a role in the inhibition of Ras and Ras-related small GTP-binding protein functions through the inhibition of protein isoprenyl transferase activity in cancer cells. Using cell line-based assays, we found that the inactive forms of Ras and Rho proteins were significantly elevated after treatment with Antroquinonol. We also demonstrated that Antroquinonol binds directly to farnesyltransferase and geranylgeranyltransferase-I, which are key enzymes involved in activation of Ras-related proteins, and inhibits enzymes activities in vitro. Furthermore, a molecular docking analysis illustrated that the isoprenoid moiety of Antroquinonol binds along the hydrophobic cavity of farnesyltransferase similar to its natural substrate, farnesyl pyrophosphate. In contrast, the ring structure of Antroquinonol lies adjacent to the Ras-CAAX motif-binding site on farnesyltransferase. The molecular docking study also showed a reasonable correlation with the IC50 values of Antroquinonol analogues. We also found that the levels of LC3B-II and the autophagosome-associated LC3 form were also significantly increased in H838 after Antroquinonol administration. In conclusion, Antroquinonol inhibited Ras and Ras-related GTP-binding protein activation through inhibition of protein isoprenyl transferase activity, leading to activation of autophagy and associated mode of cell death in cancer cells. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  5. Structure and allosteric effects of low-molecular-weight activators on the protein kinase PDK1

    DEFF Research Database (Denmark)

    Hindie, Valerie; Stroba, Adriana; Zhang, Hua

    2009-01-01

    Protein phosphorylation transduces a large set of intracellular signals. One mechanism by which phosphorylation mediates signal transduction is by prompting conformational changes in the target protein or interacting proteins. Previous work described an allosteric site mediating phosphorylation...... and in solution using a fluorescence-based assay and deuterium exchange experiments. Our results indicate that the binding of the compound produces local changes at the target site, the PIF binding pocket, and also allosteric changes at the ATP binding site and the activation loop. Altogether, we present...

  6. A rapid and simple assay for growth hormone-binding protein activity in human plasma

    International Nuclear Information System (INIS)

    Baumann, G.; Shaw, M.A.; Amburn, K.

    1988-01-01

    The newly discovered circulating growth hormone binding proteins dictate a re-evaluation of the state of GH in plasma in health and disease as the binding proteins are known to affect GH metabolism and action. We describe a rapid and simple GH-binding assay that allows determination of free and complexed plasma GH, as well as GH-binding protein activity as an index of GH-binding protein levels, with relative ease. The method is based on incubation of plasma with 125 I-GH and separation of bound from free GH on small DEAE-cellulose columns; it can be used on a large scale for routine determinations. The results obtained by this method are comparable to those obtained with the previously used slow and more cumbersome gel filtration technique. Initial data obtained in normal subject and certain disease states show that the bound fraction of plasma GH is similar in men, women and children, is unaffected by pregnancy or acute infection, but is marginally decreased in liver cirrhosis. In acromegaly, binding protein activity also appears normal when allowance is made for partial saturation of the binding proteins by the high prevailing GH levels. The technique we describe should facilitate investigations of normal and abnormal regulation of the GH binding proteins. (author)

  7. Synaptic vesicle proteins under conditions of rest and activation: analysis by 2-D difference gel electrophoresis.

    Science.gov (United States)

    Burré, Jacqueline; Beckhaus, Tobias; Corvey, Carsten; Karas, Michael; Zimmermann, Herbert; Volknandt, Walter

    2006-09-01

    Synaptic vesicles are organelles of the nerve terminal that secrete neurotransmitters by fusion with the presynaptic plasma membrane. Vesicle fusion is tightly controlled by depolarization of the plasma membrane and a set of proteins that may undergo post-translational modifications such as phosphorylation. In order to identify proteins that undergo modifications as a result of synaptic activation, we induced massive exocytosis and analysed the synaptic vesicle compartment by benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-PAGE and difference gel electrophoresis (DIGE) followed by MALDI-TOF-MS. We identified eight proteins that revealed significant changes in abundance following nerve terminal depolarization. Of these, six were increased and two were decreased in abundance. Three of these proteins were phosphorylated as detected by Western blot analysis. In addition, we identified an unknown synaptic vesicle protein whose abundance increased on synaptic activation. Our results demonstrate that depolarization of the presynaptic compartment induces changes in the abundance of synaptic vesicle proteins and post-translational protein modification.

  8. Use of Different Proteases to Obtain Flaxseed Protein Hydrolysates with Antioxidant Activity.

    Science.gov (United States)

    Karamać, Magdalena; Kosińska-Cagnazzo, Agnieszka; Kulczyk, Anna

    2016-06-29

    The antioxidant activity of flaxseed protein hydrolysates obtained using five different enzymes was evaluated. Proteins were isolated from flaxseed cake and were separately treated with papain, trypsin, pancreatin, Alcalase and Flavourzyme. The degree of hydrolysis (DH) was determined as the percentage of cleaved peptide bonds using a spectrophotometric method with o-phthaldialdehyde. The distribution of the molecular weights (MW) of the hydrolysis products was profiled using Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) and size exclusion-high performance liquid chromatography (SE-HPLC) separations. The antioxidant activities of the protein isolate and hydrolysates were probed for their radical scavenging activity using 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonate) radical cation (ABTS(•+)) and photochemiluminescence (PCL-ACL) assays, and for their ferric reducing antioxidant power (FRAP) and ability to bind Fe(2+). The hydrolysates were more effective as antioxidants than the protein isolate in all systems. The PCL-ACL values of the hydrolysates ranged from 7.2 to 35.7 μmol Trolox/g. Both the FRAP and ABTS(•+) scavenging activity differed among the hydrolysates to a lower extent, with the ranges of 0.20-0.24 mmol Fe(2+)/g and 0.17-0.22 mmol Trolox/g, respectively. The highest chelating activity (71.5%) was noted for the pancreatin hydrolysate. In general, the hydrolysates obtained using Alcalase and pancreatin had the highest antioxidant activity, even though their DH (15.4% and 29.3%, respectively) and the MW profiles of the peptides varied substantially. The O₂(•-) scavenging activity and the ability to chelate Fe(2+) of the Flavourzyme hydrolysate were lower than those of the Alcalase and pancreatin hydrolysates. Papain was the least effective in releasing the peptides with antioxidant activity. The study showed that the type of enzyme used for flaxseed protein hydrolysis determines the antioxidant activity

  9. TGEV nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p53 signaling.

    Science.gov (United States)

    Ding, Li; Huang, Yong; Du, Qian; Dong, Feng; Zhao, Xiaomin; Zhang, Wenlong; Xu, Xingang; Tong, Dewen

    2014-03-07

    Our previous studies showed that TGEV infection could induce cell cycle arrest and apoptosis via activation of p53 signaling in cultured host cells. However, it is unclear which viral gene causes these effects. In this study, we investigated the effects of TGEV nucleocapsid (N) protein on PK-15 cells. We found that TGEV N protein suppressed cell proliferation by causing cell cycle arrest at the S and G2/M phases and apoptosis. Characterization of various cellular proteins that are involved in regulating cell cycle progression demonstrated that the expression of N gene resulted in an accumulation of p53 and p21, which suppressed cyclin B1, cdc2 and cdk2 expression. Moreover, the expression of TGEV N gene promoted translocation of Bax to mitochondria, which in turn caused the release of cytochrome c, followed by activation of caspase-3, resulting in cell apoptosis in the transfected PK-15 cells following cell cycle arrest. Further studies showed that p53 inhibitor attenuated TGEV N protein induced cell cycle arrest at S and G2/M phases and apoptosis through reversing the expression changes of cdc2, cdk2 and cyclin B1 and the translocation changes of Bax and cytochrome c induced by TGEV N protein. Taken together, these results demonstrated that TGEV N protein might play an important role in TGEV infection-induced p53 activation and cell cycle arrest at the S and G2/M phases and apoptosis occurrence. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Identification of Adenyl Cyclase Activity in a Disease Resistance Protein in Arabidopsis thaliana

    KAUST Repository

    Hussein, Rana

    2012-11-01

    Cyclic nucleotide, cAMP, is an important signaling molecule in animals and plants. However, in plants the enzymes that synthesize this second messenger, adenyl cyclases (ACs), remain elusive. Given the physiological importance of cAMP in signaling, particularly in response to biotic and abiotic stresses, it is thus important to identify and characterize ACs in higher plants. Using computational approaches, a disease resistance protein from Arabidopsis thaliana, At3g04220 was found to have an AC catalytic center motif. In an attempt to prove that this candidate has adenyl cyclases activity in vitro, the coding sequence of the putative AC catalytic domain of this protein was cloned and expressed in E. coli and the recombinant protein was purified. The nucleotide cyclase activity of the recombinant protein was examined using cyclic nucleotide enzyme immunoassays. In parallel, the expression of At3g04220 was measured in leaves under three different stress conditions in order to determine under which conditions the disease resistance protein could function. Results show that the purified recombinant protein has Mn2+ dependent AC activity in vitro, and the expression analysis supports a role for At3g04220 and cAMP in plant defense.

  11. Free Fatty Acids Inhibit Protein Tyrosine Phosphatase 1B and Activate Akt

    Directory of Open Access Journals (Sweden)

    Eisuke Shibata

    2013-09-01

    Full Text Available Background/Aims: Accumulating evidence has suggested that free fatty acids (FFAs interact with protein kinases and protein phosphatases. The present study examined the effect of FFAs on protein phosphatases and Akt. Methods: Activities of protein phosphatase 1 (PP1, protein phosphatase 2A (PP2A, and protein tyrosine phosphatase 1B (PTP1B were assayed under the cell-free conditions. Phosphorylation of Akt was monitored in MSTO-211H human malignant pleural mesothelioma cells without and with knocking-down phosphatidylinositol 3 kinase (PI3K or 3-phosphoinositide-dependent protein kinase-1 (PDK1. Results: In the cell-free assay, unsaturated FFAs (uFFAs such as oleic, linoleic and linolenic acid and saturated FFAs (sFFAs such as stearic, palmitic, myristic, and behenic acid markedly reduced PTP1B activity, with the potential for uFFAs greater than that for sFFAs. All the investigated sFFAs inhibited PP2A activity, but otherwise no inhibition was obtained with uFFAs. Both uFFAs and sFFAs had no effect on PP1 activity. Oleic acid phosphorylated Akt both on Thr308 and Ser473, while stearic acid phosphorylated Akt on Thr308 alone. The effects of oleic and stearic acid on Akt phosphorylation were abrogated by the PI3K inhibitor wortmannin or the PDK1 inhibitor BX912 and also by knocking-down PI3K or PDK1. Conclusion: The results of the present study indicate that uFFAs and sFFAs could activate Akt through a pathway along a PI3K/PDK1/Akt axis in association with PTP1B inhibition.

  12. Cloning, Overexpression andin vitroAntifungal Activity ofZea MaysPR10 Protein.

    Science.gov (United States)

    Zandvakili, Niloofar; Zamani, Mohammadreza; Motallebi, Mostafa; Moghaddassi Jahromi, Zahra

    2017-03-01

    Plants have various defense mechanisms such as production of antimicrobial peptides, particularly pathogenesis related proteins (PR proteins). PR10 family is an essential member of this group, with antifungal, antibacterial and antiviral activities. The goal of this study is to assess the antifungal activity of maize PR10 against some of fungal phytopathogens. Zea mays PR10 gene (TN-05-147) was cloned from genomic DNA and cDNA and overexpressed in Escherichia coli . The existence of a 77- bp intron and two exons in PR10 was confi rmed by comparing the genomic and cDNA sequences. The PR10 cDNA was cloned in pET26b (+) expression vector and transformed into E. coli strain Rosetta DE3 in order to express PR10 recombinant protein. Expression of the recombinant protein was checked by western analysis. Recombinant PR10 appeared as insoluble inclusion bodies and thus solubilized and refolded. PR10 was isolated using Ni- NTA column. The activity of the refolded protein was confi rmed by DNA degradation test. The antifungal activity of PR10 was assessed using radial diff usion, disc diff usion and spore germination. The hemolytic assay was performed to investigate the biosafety of recombinant PR10. Recombinant maize PR10 exerted broad spectrum antifungal activity against Botrytis cinerea, Sclerotinia sclerotiorum, Fusarium oxysporum, Verticillium dahlia and Alternaria solani . Hemolysis biosafety test indicated that the protein is not poisonous to mammalian cells. Maize PR10 has the potential to be used as the antifungal agent against diff erent fungal phytopathogens. Therefore, this protein can be used in order to produce antifungal agents and fungi resistance transgenic plants.

  13. Regulation of the activity of the dual-function DnaA protein in Caulobacter crescentus.

    Directory of Open Access Journals (Sweden)

    Carmen Fernandez-Fernandez

    Full Text Available DnaA is a conserved essential bacterial protein that acts as the initiator of chromosomal replication as well as a master transcriptional regulator in Caulobacter crescentus. Thus, the intracellular levels of active DnaA need to be tightly regulated during the cell cycle. Our previous work suggested that DnaA may be regulated at the level of its activity by the replisome-associated protein HdaA. Here, we describe the construction of a mutant DnaA protein [DnaA(R357A]. The R357 residue in the AAA+ domain of the C. crescentus DnaA protein is equivalent to the R334 residue of the E. coli DnaA protein, which is required for the Regulatory Inactivation of DnaA (RIDA. We found that the expression of the DnaA(R357A mutant protein in C. crescentus, but not the expression of the wild-type DnaA protein at similar levels, causes a severe phenotype of over-initiation of chromosomal replication and that it blocks cell division. Thus, the mutant DnaA(R357A protein is hyper-active to promote the initiation of DNA replication, compared to the wild-type DnaA protein. DnaA(R357A could not replace DnaA in vivo, indicating that the switch in DnaA activity once chromosomal replication has started may be an essential process in C. crescentus. We propose that the inactivation of DnaA is the main mechanism ensuring that chromosomal replication starts only once per cell cycle. We further observed that the R357A substitution in DnaA does not promote the activity of DnaA as a direct transcriptional activator of four important genes, encoding HdaA, the GcrA master cell cycle regulator, the FtsZ cell division protein and the MipZ spatial regulator of cell division. Thus, the AAA+ domain of DnaA may play a role in temporally regulating the bifunctionality of DnaA by reallocating DnaA molecules from initiating DNA replication to transcribing genes within the unique DnaA regulon of C. crescentus.

  14. Structural Basis for Receptor Activity-Modifying Protein-Dependent Selective Peptide Recognition by a G Protein-Coupled Receptor.

    Science.gov (United States)

    Booe, Jason M; Walker, Christopher S; Barwell, James; Kuteyi, Gabriel; Simms, John; Jamaluddin, Muhammad A; Warner, Margaret L; Bill, Roslyn M; Harris, Paul W; Brimble, Margaret A; Poyner, David R; Hay, Debbie L; Pioszak, Augen A

    2015-06-18

    Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind related GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. The structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  15. Classification, expression pattern, and E3 ligase activity assay of rice U-box-containing proteins.

    Science.gov (United States)

    Zeng, Li-Rong; Park, Chan Ho; Venu, R C; Gough, Julian; Wang, Guo-Liang

    2008-09-01

    Ubiquitin ligases play a central role in determining the specificity of the ubiquitination system by selecting a myriad of appropriate candidate proteins for modification. The U-box is a recently identified, ubiquitin ligase activity-related protein domain that shows greater presence in plants than in other organisms. In this study, we identified 77 putative U-box proteins from the rice genome using a battery of whole genome analysis algorithms. Most of the U-box protein genes are expressed, as supported by the identification of their corresponding expressed sequence tags (ESTs), full-length cDNAs, or massively parallel signature sequencing (MPSS) tags. Using the same algorithms, we identified 61 U-box proteins from the Arabidopsis genome. The rice and Arabidopsis U-box proteins were classified into nine major classes based on their domain compositions. Comparison between rice and Arabidopsis U-box proteins indicates that the majority of rice and Arabidopsis U-box proteins have the same domain organizations. The inferred phylogeny established the homology between rice and Arabidopsis U-box/ARM proteins. Cell death assay using the rice protoplast system suggests that one rice U-box gene, OsPUB51, might act as a negative regulator of cell death signaling. In addition, the selected U-box proteins were found to be functional E3 ubiquitin ligases. The identification and analysis of rice U-box proteins hereby at the genomic level will help functionally characterize this class of E3 ubiquitin ligase in the future.

  16. A Role for Protein Phosphatase 2A in Regulating p38 Mitogen Activated Protein Kinase Activation and Tumor Necrosis Factor-Alpha Expression during Influenza Virus Infection

    Directory of Open Access Journals (Sweden)

    Anna H. Y. Law

    2013-04-01

    Full Text Available Influenza viruses of avian origin continue to pose pandemic threats to human health. Some of the H5N1 and H9N2 virus subtypes induce markedly elevated cytokine levels when compared with the seasonal H1N1 virus. We previously showed that H5N1/97 hyperinduces tumor necrosis factor (TNF-alpha through p38 mitogen activated protein kinase (MAPK. However, the detailed mechanisms of p38MAPK activation and TNF-alpha hyperinduction following influenza virus infections are not known. Negative feedback regulations of cytokine expression play important roles in avoiding overwhelming production of proinflammatory cytokines. Here we hypothesize that protein phosphatases are involved in the regulation of cytokine expressions during influenza virus infection. We investigated the roles of protein phosphatases including MAPK phosphatase-1 (MKP-1 and protein phosphatase type 2A (PP2A in modulating p38MAPK activation and downstream TNF-alpha expressions in primary human monocyte-derived macrophages (PBMac infected with H9N2/G1 or H1N1 influenza virus. We demonstrate that H9N2/G1 virus activated p38MAPK and hyperinduced TNF-alpha production in PBMac when compared with H1N1 virus. H9N2/G1 induced PP2A activity in PBMac and, with the treatment of a PP2A inhibitor, p38MAPK phosphorylation and TNF-alpha production were further increased in the virus-infected macrophages. However, H9N2/G1 did not induce the expression of PP2A indicating that the activation of PP2A is not mediated by p38MAPK in virus-infected PBMac. On the other hand, PP2A may not be the targets of H9N2/G1 in the upstream of p38MAPK signaling pathways since H1N1 also induced PP2A activation in primary macrophages. Our results may provide new insights into the control of cytokine dysregulation.

  17. Dengue Virus Type 2: Protein Binding and Active Replication in Human Central Nervous System Cells

    Directory of Open Access Journals (Sweden)

    Ma Isabel Salazar

    2013-01-01

    Full Text Available An increased number of dengue cases with neurological complications have been reported in recent years. The lack of reliable animal models for dengue has hindered studies on dengue virus (DENV pathogenesis and cellular tropism in vivo. We further investigate the tropism of DENV for the human central nervous system (CNS, characterizing DENV interactions with cell surface proteins in human CNS cells by virus overlay protein binding assays (VOPBA and coimmunoprecipitations. In VOPBA, three membrane proteins (60, 70, and 130 kDa from the gray matter bound the entire virus particle, whereas only a 70 kDa protein bound in white matter. The coimmunoprecipitation assays revealed three proteins from gray matter consistently binding virus particles, one clearly distinguishable protein (~32 kDa and two less apparent proteins (100 and 130 kDa. Monoclonal anti-NS3 targeted the virus protein in primary cell cultures of human CNS treated with DENV-2, which also stained positive for NeuH, a neuron-specific marker. Thus, our results indicate (1 that DENV-2 exhibited a direct tropism for human neurons and (2 that human neurons sustain an active DENV replication as was demonstrated by the presence of the NS3 viral antigen in primary cultures of these cells treated with DENV-2.

  18. Multimodular biosensors reveal a novel platform for activation of G proteins by growth factor receptors.

    Science.gov (United States)

    Midde, Krishna K; Aznar, Nicolas; Laederich, Melanie B; Ma, Gary S; Kunkel, Maya T; Newton, Alexandra C; Ghosh, Pradipta

    2015-03-03

    Environmental cues are transmitted to the interior of the cell via a complex network of signaling hubs. Receptor tyrosine kinases (RTKs) and trimeric G proteins are two such major signaling hubs in eukaryotes. Conventionally, canonical signal transduction via trimeric G proteins is thought to be triggered exclusively by G protein-coupled receptors. Here we used molecular engineering to develop modular fluorescent biosensors that exploit the remarkable specificity of bimolecular recognition, i.e., of both G proteins and RTKs, and reveal the workings of a novel platform for activation of G proteins by RTKs in single living cells. Comprised of the unique modular makeup of guanidine exchange factor Gα-interacting vesicle-associated protein (GIV)/girdin, a guanidine exchange factor that links G proteins to a variety of RTKs, these biosensors provide direct evidence that RTK-GIV-Gαi ternary complexes are formed in living cells and that Gαi is transactivated within minutes after growth factor stimulation at the plasma membrane. Thus, GIV-derived biosensors provide a versatile strategy for visualizing, monitoring, and manipulating the dynamic association of Gαi with RTKs for noncanonical transactivation of G proteins in cells and illuminate a fundamental signaling event regulated by GIV during diverse cellular processes and pathophysiologic states.

  19. Slow acting protein extract from fruit pulp of Momordica charantia with insulin secretagogue and insulinomimetic activities.

    Science.gov (United States)

    Yibchok-anun, Sirintorn; Adisakwattana, Sirichai; Yao, Cheng Yu; Sangvanich, Polkit; Roengsumran, Sophon; Hsu, Walter Haw

    2006-06-01

    The protein from Thai bitter gourd (Momordica charantia) fruit pulp was extracted and studied for its hypoglycemic effect. Subcutaneous administration of the protein extract (5, 10 mg/kg) significantly and markedly decreased plasma glucose concentrations in both normal and streptozotocin-induced diabetic rats in a dose-dependent manner. The onset of the protein extract-induced antihyperglycemia/hypoglycemia was observed at 4 and 6 h in diabetic and normal rats, respectively. This protein extract also raised plasma insulin concentrations by 2 fold 4 h following subcutaneous administration. In perfused rat pancreas, the protein extract (10 microg/ml) increased insulin secretion, but not glucagon secretion. The increase in insulin secretion was apparent within 5 min of administration and was persistent during 30 min of administration. Furthermore, the protein extract enhanced glucose uptake into C2C12 myocytes and 3T3-L1 adipocytes. Time course experiments performed in rat adipocytes revealed that M. charantia protein extract significantly increased glucose uptake after 4 and 6 h of incubation. Thus, the M. charantia protein extract, a slow acting chemical, exerted both insulin secretagogue and insulinomimetic activities to lower blood glucose concentrations in vivo.

  20. Study of plasma protein binding activity of isometamidium and its impact on anthelmintic activity using trypanosoma induced calf model

    Directory of Open Access Journals (Sweden)

    Suprita Sinha

    Full Text Available Aim: The objective of present study was to determine Plasma Protein Binding (PPB activity and its effect on clinical efficacy of isometamidium after intramuscular administration in calves. The binding of drugs to plasma proteins is an important factor in controlling the availability and distribution of drugs. In general, PPB reduces the free fraction of drug available for therapeutic activity, since only the non-protein bound drug is pharmacologically active. Materials and Methods: Six calves were used for PPB study and eighteen for clinical efficacy. Isometamidium was administered @ 0.5mg/kg intramuscularly as a single dose for PPB study. Equilibrium dialysis technique was used to determine the PPB activity. For clinical efficacy, infection with Trypanosoma was induced in calves of two groups, untreated control and experimental group. Infection was confirmed after 28 days by mice inoculation test. Isometamidium @ 0.5mg/kg was administered to experimental group. Haematoobiochemical and mice inoculation tests were performed after 7 days of drug administration (Day 35. Result: The percentage of PPB activity of isometamidium was 86.71 ± 0.59 to 93.03 ± 0.63% against the concentration 9.76± 0.84 to 4.39 ± 0.20 g ml-1. Higher percentage of PPB activity (>86% suggests greater duration of safety by this drug. It was found that anthelmintic activity of isometamidium was substantially affected by higher PPB. Conclusion: It was concluded that isometamidium has greater plasma protein binding capacity which did not hamper clinical efficacy of drug. [Vet World 2013; 6(7.000: 444-448

  1. Effect of dietary crude protein level on jejunal brush border enzyme activities in weaned pigs.

    Science.gov (United States)

    Opapeju, Florence Omobola; Rademacher, Meike; Nyachoti, Charles Martin

    2009-01-01

    Forty weaned pigs (7.0 ± 0.5 kg, mean ± SD) were used to determine the effects of feeding a low crude protein, amino acid-supplemented diet to piglets on the activities of jejunal brush border enzymes. Pigs were randomly allotted to two diets: a 222 g crude protein (HCP) per kg diet, or a 173 g crude protein per kg diet supplemented with amino acids (LCP). Pigs fed the HCP diet had higher small intestine weight compared with those fed the LCP diet on day 7 after weaning. Diet had no effect on the specific activities of jejunal sucrase, lactase, leucine aminopeptidase, aminopeptidase A, aminopeptidase N and dipeptidyl peptidase IV. The activities of sucrase and lactase decreased (p effect on the development of jejunal brush border enzymes.

  2. Biological Activities and Applications of Dioscorins, the Major Tuber Storage Proteins of Yam

    Directory of Open Access Journals (Sweden)

    Yeh-Lin Lu

    2012-01-01

    Full Text Available Yam tubers, a common tuber crop and an important traditional Chinese medicine in Taiwan, have many bioactive substances, including phenolic compounds, mucilage polysaccharides, steroidal saponins and proteins. Among the total soluble proteins, 80% of them are dioscorins. In the past two decades, many studies showed that dioscorins exhibited biological activities both in vitro and in vivo, including the enzymatic, antioxidant, antihypertensive, immunomodulatory, lectin activities and the protecting role on airway epithelial cells against allergens in vitro. Some of these activities are survived after chemical, heating process or enzymatic digestion. Despite of lacking the intact structural information and the detail action mechanisms in the cells, yam dioscorins are potential resources for developing as functional foods and interesting targets for food protein researchers.

  3. Protein C activation during the initial phase of experimental acute pancreatitis in the rabbit

    DEFF Research Database (Denmark)

    Ottesen, L H; Bladbjerg, E-M; Osman, M

    2000-01-01

    situation. METHODS: ANP was introduced in 7 rabbits by infusion of chenodeoxycholic acid in the pancreatic duct. Seven rabbits served as sham-operated controls. Serial measurements of coagulation variables (prothrombin time, activated partial thromboplastin time, FVII activity, fibrinogen, tissue factor...... of the lungs or kidneys was found in 2 rabbits with ANP. CONCLUSION: An immediate activation of protein C is a specific characteristic of the haemostatic activation in ANP in rabbits. This activation has not been described previously and the possible therapeutic implications ought to be studied....

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

    Directory of Open Access Journals (Sweden)

    Qin Feng

    2013-01-01

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

  5. Therapeutic effects of cell-permeant peptides that activate G proteins downstream of growth factors.

    Science.gov (United States)

    Ma, Gary S; Aznar, Nicolas; Kalogriopoulos, Nicholas; Midde, Krishna K; Lopez-Sanchez, Inmaculada; Sato, Emi; Dunkel, Ying; Gallo, Richard L; Ghosh, Pradipta

    2015-05-19

    In eukaryotes, receptor tyrosine kinases (RTKs) and trimeric G proteins are two major signaling hubs. Signal transduction via trimeric G proteins has long been believed to be triggered exclusively by G protein-coupled receptors (GPCRs). This paradigm has recently been challenged by several studies on a multimodular signal transducer, Gα-Interacting Vesicle associated protein (GIV/Girdin). We recently demonstrated that GIV's C terminus (CT) serves as a platform for dynamic association of ligand-activated RTKs with Gαi, and for noncanonical transactivation of G proteins. However, exogenous manipulation of this platform has remained beyond reach. Here we developed cell-permeable GIV-CT peptides by fusing a TAT-peptide transduction domain (TAT-PTD) to the minimal modular elements of GIV that are necessary and sufficient for activation of Gi downstream of RTKs, and used them to engineer signaling networks and alter cell behavior. In the presence of an intact GEF motif, TAT-GIV-CT peptides enhanced diverse processes in which GIV's GEF function has previously been implicated, e.g., 2D cell migration after scratch-wounding, invasion of cancer cells, and finally, myofibroblast activation and collagen production. Furthermore, topical application of TAT-GIV-CT peptides enhanced the complex, multireceptor-driven process of wound repair in mice in a GEF-dependent manner. Thus, TAT-GIV peptides provide a novel and versatile tool to manipulate Gαi activation downstream of growth factors in a diverse array of pathophysiologic conditions.

  6. Protein immobilization on epoxy-activated thin polymer films: effect of surface wettability and enzyme loading.

    Science.gov (United States)

    Chen, Bo; Pernodet, Nadine; Rafailovich, Miriam H; Bakhtina, Asya; Gross, Richard A

    2008-12-02

    A series of epoxy-activated polymer films composed of poly(glycidyl methacrylate/butyl methacrylate/hydroxyethyl methacrylate) were prepared. Variation in comonomer composition allowed exploration of relationships between surface wettability and Candida antartica lipase B (CALB) binding to surfaces. By changing solvents and polymer concentrations, suitable conditions were developed for preparation by spin-coating of uniform thin films. Film roughness determined by AFM after incubation in PBS buffer for 2 days was less than 1 nm. The occurrence of single CALB molecules and CALB aggregates at surfaces was determined by AFM imaging and measurements of volume. Absolute numbers of protein monomers and multimers at surfaces were used to determine values of CALB specific activity. Increased film wettability, as the water contact angle of films increased from 420 to 550, resulted in a decreased total number of immobilized CALB molecules. With further increases in the water contact angle of films from 55 degrees to 63 degrees, there was an increased tendency of CALB molecules to form aggregates on surfaces. On all flat surfaces, two height populations, differing by more than 30%, were observed from height distribution curves. They are attributed to changes in protein conformation and/or orientation caused by protein-surface and protein-protein interactions. The fraction of molecules in these populations changed as a function of film water contact angle. The enzyme activity of immobilized films was determined by measuring CALB-catalyzed hydrolysis of p-nitrophenyl butyrate. Total enzyme specific activity decreased by decreasing film hydrophobicity.

  7. Evaluation of protein intake and physical activity associated with sarcopenia in the elderly