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Sample records for antifreeze protein activity

  1. Protein-water dynamics in antifreeze protein III activity

    Science.gov (United States)

    Xu, Yao; Bäumer, Alexander; Meister, Konrad; Bischak, Connor G.; DeVries, Arthur L.; Leitner, David M.; Havenith, Martina

    2016-03-01

    We combine Terahertz absorption spectroscopy (THz) and molecular dynamics (MD) simulations to investigate the underlying molecular mechanism for the antifreeze activity of one class of antifreeze protein, antifreeze protein type III (AFP-III) with a focus on the collective water hydrogen bond dynamics near the protein. After summarizing our previous work on AFPs, we present a new investigation of the effects of cosolutes on protein antifreeze activity by adding sodium citrate to the protein solution of AFP-III. Our results reveal that for AFP-III, unlike some other AFPs, the addition of the osmolyte sodium citrate does not affect the hydrogen bond dynamics at the protein surface significantly, as indicated by concentration dependent THz measurements. The present data, in combination with our previous THz measurements and molecular simulations, confirm that while long-range solvent perturbation is a necessary condition for the antifreeze activity of AFP-III, the local binding affinity determines the size of the hysteresis.

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

    Science.gov (United States)

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

    2014-05-01

    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 at concentrations above 270 μmol.

  3. 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

    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...

  4. Snow-mold-induced apoplastic proteins in winter rye leaves lack antifreeze activity

    Science.gov (United States)

    Hiilovaara-Teijo; Hannukkala; Griffith; Yu; Pihakaski-Maunsbach

    1999-10-01

    During cold acclimation, winter rye (Secale cereale L.) plants secrete antifreeze proteins that are similar to pathogenesis-related (PR) proteins. In this experiment, the secretion of PR proteins was induced at warm temperatures by infection with pink snow mold (Microdochium nivale), a pathogen of overwintering cereals. A comparison of cold-induced and pathogen-induced proteins showed that PR proteins accumulated in the leaf apoplast to a greater level in response to cold. The PR proteins induced by cold and by snow mold were similar when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and examined by immunoblotting. Both groups of PR proteins contained glucanase-like, chitinase-like, and thaumatin-like proteins, and both groups exhibited similar levels of glucanase and chitinase activities. However, only the PR proteins induced by cold exhibited antifreeze activity. Our findings suggest that the cold-induced PR proteins may be isoforms that function as antifreeze proteins to modify the growth of ice during freezing while also providing resistance to the growth of low-temperature pathogens in advance of infection. Both functions of the cold-induced PR proteins may improve the survival of overwintering cereals.

  5. 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.

  6. 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.

  7. Antifreeze (glyco)protein mimetic behavior of poly(vinyl alcohol): detailed structure ice recrystallization inhibition activity study.

    Science.gov (United States)

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

    2013-05-13

    This manuscript reports a detailed study on the ability of poly(vinyl alcohol) to act as a biomimetic surrogate for antifreeze(glyco)proteins, with a focus on the specific property of ice-recrystallization inhibition (IRI). Despite over 40 years of study, the underlying mechanisms that govern the action of biological antifreezes are still poorly understood, which is in part due to their limited availability and challenging synthesis. Poly(vinyl alcohol) (PVA) has been shown to display remarkable ice recrystallization inhibition activity despite its major structural differences to native antifreeze proteins. Here, controlled radical polymerization is used to synthesize well-defined PVA, which has enabled us to obtain the first quantitative structure-activity relationships, to probe the role of molecular weight and comonomers on IRI activity. Crucially, it was found that IRI activity is "switched on" when the polymer chain length increases from 10 and 20 repeat units. Substitution of the polymer side chains with hydrophilic or hydrophobic units was found to diminish activity. Hydrophobic modifications to the backbone were slightly more tolerated than side chain modifications, which implies an unbroken sequence of hydroxyl units is necessary for activity. These results highlight that, although hydrophobic domains are key components of IRI activity, the random inclusion of addition hydrophobic units does not guarantee an increase in activity and that the actual polymer conformation is important.

  8. Dendrimer-Linked Antifreeze Proteins Have Superior Activity and Thermal Recovery.

    Science.gov (United States)

    Stevens, Corey A; Drori, Ran; Zalis, Shiran; Braslavsky, Ido; Davies, Peter L

    2015-09-16

    By binding to ice, antifreeze proteins (AFPs) depress the freezing point of a solution and inhibit ice recrystallization if freezing does occur. Previous work showed that the activity of an AFP was incrementally increased by fusing it to another protein. Even larger increases in activity were achieved by doubling the number of ice-binding sites by dimerization. Here, we have combined the two strategies by linking multiple outward-facing AFPs to a dendrimer to significantly increase both the size of the molecule and the number of ice-binding sites. Using a heterobifunctional cross-linker, we attached between 6 and 11 type III AFPs to a second-generation polyamidoamine (G2-PAMAM) dendrimer with 16 reactive termini. This heterogeneous sample of dendrimer-linked type III constructs showed a greater than 4-fold increase in freezing point depression over that of monomeric type III AFP. This multimerized AFP was particularly effective at ice recrystallization inhibition activity, likely because it can simultaneously bind multiple ice surfaces. Additionally, attachment to the dendrimer has afforded the AFP superior recovery from heat denaturation. Linking AFPs together via polymers can generate novel reagents for controlling ice growth and recrystallization. PMID:26267368

  9. Dendrimer-Linked Antifreeze Proteins Have Superior Activity and Thermal Recovery.

    Science.gov (United States)

    Stevens, Corey A; Drori, Ran; Zalis, Shiran; Braslavsky, Ido; Davies, Peter L

    2015-09-16

    By binding to ice, antifreeze proteins (AFPs) depress the freezing point of a solution and inhibit ice recrystallization if freezing does occur. Previous work showed that the activity of an AFP was incrementally increased by fusing it to another protein. Even larger increases in activity were achieved by doubling the number of ice-binding sites by dimerization. Here, we have combined the two strategies by linking multiple outward-facing AFPs to a dendrimer to significantly increase both the size of the molecule and the number of ice-binding sites. Using a heterobifunctional cross-linker, we attached between 6 and 11 type III AFPs to a second-generation polyamidoamine (G2-PAMAM) dendrimer with 16 reactive termini. This heterogeneous sample of dendrimer-linked type III constructs showed a greater than 4-fold increase in freezing point depression over that of monomeric type III AFP. This multimerized AFP was particularly effective at ice recrystallization inhibition activity, likely because it can simultaneously bind multiple ice surfaces. Additionally, attachment to the dendrimer has afforded the AFP superior recovery from heat denaturation. Linking AFPs together via polymers can generate novel reagents for controlling ice growth and recrystallization.

  10. Structure of solvation water around the active and inactive regions of a type III antifreeze protein and its mutants of lowered activity

    Science.gov (United States)

    Grabowska, Joanna; Kuffel, Anna; Zielkiewicz, Jan

    2016-08-01

    Water molecules from the solvation shell of the ice-binding surface are considered important for the antifreeze proteins to perform their function properly. Herein, we discuss the problem whether the extent of changes of the mean properties of solvation water can be connected with the antifreeze activity of the protein. To this aim, the structure of solvation water of a type III antifreeze protein from Macrozoarces americanus (eel pout) is investigated. A wild type of the protein is used, along with its three mutants, with antifreeze activities equal to 54% or 10% of the activity of the native form. The solvation water of the ice-binding surface and the rest of the protein are analyzed separately. To characterize the structure of solvation shell, parameters describing radial and angular characteristics of the mutual arrangement of the molecules were employed. They take into account short-distance (first hydration shell) or long-distance (two solvation shells) effects. The obtained results and the comparison with the results obtained previously for a hyperactive antifreeze protein from Choristoneura fumiferana lead to the conclusion that the structure and amino acid composition of the active region of the protein evolved to achieve two goals. The first one is the modification of the properties of the solvation water. The second one is the geometrical adjustment of the protein surface to the specific crystallographic plane of ice. Both of these goals have to be achieved simultaneously in order for the protein to perform its function properly. However, they seem to be independent from one another in a sense that very small antifreeze activity does not imply that properties of water become different from the ones observed for the wild type. The proteins with significantly lower activity still modify the mean properties of solvation water in a right direction, in spite of the fact that the accuracy of the geometrical match with the ice lattice is lost because of the

  11. Coarse grained simulation reveals antifreeze properties of hyperactive antifreeze protein from Antarctic bacterium Colwellia sp.

    Science.gov (United States)

    Nguyen, Hung; Van, Thanh Dac; Le, Ly

    2015-10-01

    The novel hyperactive antifreeze protein (AFP) of Antarctic sea ice bacterium Colwellia sp. provides a target for studying the protection of psychrophilic microgoranisms against freezing environment. Interestingly, the Colwellia sp. hyperactive antifreeze protein (ColAFP) was crystallized without the structural dynamic characteristics. Here, the result indicated, through coarse grained simulation of ColAFP under various subfreezing temperature, that ColAFP remains active at temperature of equal and greater than 275 K (∼2 °C). Extensive simulation analyses also revealed the adaptive mechanism of ColAFP in subfreezing environment. Our result provides a structural dynamic understanding of the ColAFP.

  12. Expression, purification and activity determination of the beetle tenebrio molitor antifreeze protein afp84c in escherichia coli

    International Nuclear Information System (INIS)

    Summary: A cDNA encoding antifreeze protein (AFP84c) was cloned by RT-PCR from the larva of the yellow mealworm Tenebrio molitor. The coding fragment of 252 bp encodes a protein of 84 amino acid residues and was fused to the expression vectors pMAL-c2X and pMAL-p2X. The expression plasmids pMAL-c2X-afp84c and pMAL-p2X-afp84c were constructed and transformed into Escherischia coli strains TBI, respectively. Strategy of optimization of induction conditions were used for expression of the highly disulfide-bonded beta-helix-contained protein with the activity of antifreeze in pMALTM expression system. The target fusion protein was released from the cytoplasm and periplasm by sonication and cold osmotic shock procedure respectively. Recombinant AFP84c was purified by amylose affinity column. The purified target protein displayed a single band in SDS-PAGE. Expressed AFP84c exhibits to increase low temperature resistance of bacteria. (author)

  13. Antifreeze proteins enable plants to survive in freezing conditions

    Indian Academy of Sciences (India)

    Ravi Gupta; Renu Deswal

    2014-12-01

    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 gymnosperms, dicots and monocots. Biochemical characterization of plant antifreeze activity, as determined by the high ice recrystallization inhibition (IRI) activities and low thermal hysteresis (TH) of AFPs, showed that their main function is inhibition of ice crystal growth rather than the lowering of freezing temperatures. However, recent studies showed that antifreeze activity with higher TH also exists in plants. Calcium and hormones like ethylene and jasmonic acid have been shown to regulate plant antifreeze activity. Recent studies have shown that plant AFPs bind to both prism planes and basal planes of ice crystals by means of two flat ice binding sites. Plant AFPs have been postulated to evolve from the OsLRR-PSR gene nearly 36 million years ago. In this review, we present the current scenario of plant AFP research in order to understand the possible potential of plant AFPs in generation of freezing-tolerant crops.

  14. Plant Antifreeze Proteins and Their Expression Regulatory Mechanism

    Institute of Scientific and Technical Information of China (English)

    Lin Yuan-zhen; Lin Shan-zhi; Zhang Zhi-yi; Zhang Wei; Liu Wen-feng

    2005-01-01

    Low temperature is one of the major limiting environmental factors which constitutes the growth, development,productivity and distribution of plants. Over the past several years, the proteins and genes associated with freezing resistance of plants have been widely studied. The recent progress of domestic and foreign research on plant antifreeze proteins and the identification and characterization of plant antifreeze protein genes, especially on expression regulatory mechanism of plant antifreeze proteins are reviewed in this paper. Finally, some unsolved problems and the trend of research in physiological functions and gene expression regulatory mechanism of plant antifreeze proteins are discussed.

  15. 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.

  16. Dynamical mechanism of antifreeze proteins to prevent ice growth

    CERN Document Server

    Kutschan, B; Thoms, S

    2014-01-01

    The fascinating ability of algae, insects and fishes to survive at temperatures below normal freezing is realized by antifreeze proteins (AFPs). Antifreeze proteins (AFPs) are surface-active molecules and interact with the diffusive water/ice interface preventing a complete solidification. A new dynamical mechanism is proposed how these proteins inhibit the freezing of water. We apply a Ginzburg-Landau type approach to describe the phase separation in the two-component system (ice, AFP). The free energy density involves two fields: one for the ice phase with low AFP concentration, and one for the liquid water with high AFP concentration. The time evolution of the ice reveals microstructures as a result of phase separation in the presence of AFPs. We observe a faster clustering of pre-ice structure connected with a locking of grain size by the action of AFP which is an essentially dynamical process. The adsorption of additional water molecules are inhibited and the further growth of ice grains are stopped. The...

  17. DSC Study on the Thermal Hysteresis Activity of Plant Antifreeze Proteins%沙冬青抗冻蛋白热滞活性的DSC研究

    Institute of Scientific and Technical Information of China (English)

    周晓蕾; 陈滔滔; 王保怀; 李芝芬; 费云标; 魏令波; 高素琴

    2001-01-01

    Differential scanning calorimetry (DSC) was used to measure thethermal hysteresis activity(THA) of plant antifreeze proteins(AFPs). The results reveal that DSC is a good method to screen and study AFPs. In the sixteen components extracted from Ammopipanthus mongolicus leaves, one(P3S1) was found to have apparent thermal hysteresis activity by DSC. As the amount of ice nuclei in the sample decreased, the THA of P3S1 increased from 0.01 ℃ to 0.65 ℃ . It is notable that the two-peak thermal hysteresis effect was observed. Two endothermic peaks appeared in the melting process of P3S1, while the freezing peak also consisted of two peaks. The peaks appeared antecedently showed larger thermal effect. This phenomenon shows P3S1 has two different kinds of interaction with water and ice crystal. It is probably an important property of a class of AFPs.

  18. Identification and Evaluation of Cryoprotective Peptides from Chicken Collagen: Ice-Growth Inhibition Activity Compared to That of Type I Antifreeze Proteins in Sucrose Model Systems.

    Science.gov (United States)

    Du, Lihui; Betti, Mirko

    2016-06-29

    The ability of chicken collagen peptides to inhibit the growth of ice crystals was evaluated and compared to that of fish antifreeze proteins (AFPs). This ice inhibition activity was assessed using a polarized microscope by measuring ice crystal dimensions in a sucrose model system with and without collagen peptides after seven thermal cycles. The system was stabilized at -25 °C and cycled between -16 and -12 °C. Five candidate peptides with ice inhibition activity were identified using liquid chromatography and tandem mass spectrometry and were then synthesized. Their ice inhibition capacity was compared to that of type I AFPs in a 23% sucrose model system. Specific collagen peptides with certain amino acid sequences reduced the extent of ice growth by approximately 70% at a relatively low concentration (1 mg/mL). These results suggest that specific collagen peptides may act in a noncolligative manner, inhibiting ice crystal growth like type I AFPs, but less efficiently. PMID:27293017

  19. 几种昆虫抗冻蛋白的研究概况%Research overview on several insect antifreeze proteins

    Institute of Scientific and Technical Information of China (English)

    李亚平; 刘丽娟

    2012-01-01

    昆虫抗冻蛋白具有很强的抗冻活性,其结构与其他生物不同.本文分别从结构、基因克隆、抗冻活性等方面对几种昆虫的抗冻蛋白进行综述,并对昆虫抗冻蛋白的发展前景进行了展望.%Insect antifreeze proteins have special structures and have stronger antifreeze activity than antifreeze proteins of other organisms. In this paper, the special properties of insect antifreeze proteins are reviewed. New advances on gene cloning and antifreeze activity are introduced in detail and the development perspectives of insect antifreeze proteins are prospected.

  20. Thermodynamic Properties of Linear Protein Solutions: an Application to Type Ⅰ Antifreeze Protein Solutions

    Institute of Scientific and Technical Information of China (English)

    LI Li-fen; LIANG Xi-xia; LI Qian-zhong

    2012-01-01

    A statistical thermodynamic theory of linear protein solutions was proposed with the aid of a lattice model and applied to type Ⅰ antifreeze protein(AFPI) solutions.The numerical results for several AFPI solutions show that the Gibbs function of the solution has a minimum at a certain protein concentration,but the protein chemical potential increases with increasing the concentration.The influences of temperature and protein chain length on the AFPI chemical potential were also discussed.The evaluation for the colligative depression of the freezing point confirms that the antifreeze action should be recognized as non-colligative.The theoretical deduction for the concentration dependence of the thermal hysteresis activity coincides qualitatively with the previous experimental and theoretical results.

  1. Antifreeze proteins in the Antarctic springtail, Gressittacantha terranova.

    Science.gov (United States)

    Hawes, T C; Marshall, C J; Wharton, D A

    2011-08-01

    Antarctic springtails are exemplars of extreme low temperature adaptation in terrestrial arthropods. This paper represents the first examination of such adaptation in the springtail, Gressittacantha terranova. Acclimatization state was measured in field-fresh samples over a 22-day period at the beginning of the austral summer. No evidence of temperature tracking was observed. Mean temperature of crystallization (T(c)) for all samples was -20.67 ± 0.32°C and the lowest T(c) recorded was -32.62°C. Ice affinity purification was used to collect antifreeze proteins (AFPs) from springtail homogenate. The purified ice fraction demonstrated both thermal hysteresis activity and recrystallisation inhibition. Growth-melt observations revealed that ice crystals grow normal to the c-axis (basal plane). Reverse-phased HPLC produce one clearly resolved peak (P1) and one compound peak (P2). Mass spectrometry identified the molecular mass of P1 as 8,599 Da. The P1 protein was also the most prominent in P2, although additional peptides of 6-7 KDa were also prominent. The main AFP of the Antarctic springtail, G. terranova has been isolated, although like other AFP-expressing arthropods, it shows evidence of expressing a family of AFPs. PMID:21399953

  2. Comparison of backbone dynamics of the type III antifreeze protein and antifreeze-like domain of human sialic acid synthase

    International Nuclear Information System (INIS)

    Antifreeze proteins (AFPs) are found in a variety of cold-adapted (psychrophilic) organisms to promote survival at subzero temperatures by binding to ice crystals and decreasing the freezing temperature of body fluids. The type III AFPs are small globular proteins that consist of one α-helix, three 310-helices, and two β-strands. Sialic acids play important roles in a variety of biological functions, such as development, recognition, and cell adhesion and are synthesized by conserved enzymatic pathways that include sialic acid synthase (SAS). SAS consists of an N-terminal catalytic domain and a C-terminal antifreeze-like (AFL) domain, which is similar to the type III AFPs. Despite having very similar structures, AFL and the type III AFPs exhibit very different temperature-dependent stability and activity. In this study, we have performed backbone dynamics analyses of a type III AFP (HPLC12 isoform) and the AFL domain of human SAS (hAFL) at various temperatures. We also characterized the structural/dynamic properties of the ice-binding surfaces by analyzing the temperature gradient of the amide proton chemical shift and its correlation with chemical shift deviation from random coil. The dynamic properties of the two proteins were very different from each other. While HPLC12 was mostly rigid with a few residues exhibiting slow motions, hAFL showed fast internal motions at low temperature. Our results provide insight into the molecular basis of thermostability and structural flexibility in homologous psychrophilic HPLC12 and mesophilic hAFL proteins

  3. Comparison of backbone dynamics of the type III antifreeze protein and antifreeze-like domain of human sialic acid synthase

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Yong-Geun [Gyeongsang National University, Department of Chemistry and Research Institute of Natural Science (Korea, Republic of); Park, Chin-Ju [Gwangju Institute of Science and Technology, Division of Liberal Arts and Sciences and Department of Chemistry (Korea, Republic of); Kim, Hee-Eun; Seo, Yeo-Jin; Lee, Ae-Ree; Choi, Seo-Ree; Lee, Shim Sung; Lee, Joon-Hwa, E-mail: joonhwa@gnu.ac.kr [Gyeongsang National University, Department of Chemistry and Research Institute of Natural Science (Korea, Republic of)

    2015-02-15

    Antifreeze proteins (AFPs) are found in a variety of cold-adapted (psychrophilic) organisms to promote survival at subzero temperatures by binding to ice crystals and decreasing the freezing temperature of body fluids. The type III AFPs are small globular proteins that consist of one α-helix, three 3{sub 10}-helices, and two β-strands. Sialic acids play important roles in a variety of biological functions, such as development, recognition, and cell adhesion and are synthesized by conserved enzymatic pathways that include sialic acid synthase (SAS). SAS consists of an N-terminal catalytic domain and a C-terminal antifreeze-like (AFL) domain, which is similar to the type III AFPs. Despite having very similar structures, AFL and the type III AFPs exhibit very different temperature-dependent stability and activity. In this study, we have performed backbone dynamics analyses of a type III AFP (HPLC12 isoform) and the AFL domain of human SAS (hAFL) at various temperatures. We also characterized the structural/dynamic properties of the ice-binding surfaces by analyzing the temperature gradient of the amide proton chemical shift and its correlation with chemical shift deviation from random coil. The dynamic properties of the two proteins were very different from each other. While HPLC12 was mostly rigid with a few residues exhibiting slow motions, hAFL showed fast internal motions at low temperature. Our results provide insight into the molecular basis of thermostability and structural flexibility in homologous psychrophilic HPLC12 and mesophilic hAFL proteins.

  4. Expression of a Carrot Antifreeze Protein Gene in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    Ma Xinyu; Shen Xin; Lu Cunfu

    2003-01-01

    The recombinant expression vectorpET43. lb-AFP, which contains full encoding region of a carrot 36 kD antifreeze protein (AFP) gene was constructed. The recombinant was transformed into expression host carrying T7 RNA polymerase gene (DE3 lysogen) and induced by 1 mmol. L-1 IPTG (isopropyl-β-D-thiogalactoside) to express 110 kD polypeptide of AFP fusion protein.The analysis of product solubility revealed that pET43. 1b-AFP was predominately soluble, and the expressed amount reached the maximum after the IPTG treatment for 3 h.

  5. 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

    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...... 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 and it is...... structurally unique in several aspects. In contrast to the high content of disulfide bond-formation observed in other coleopteran AFPs, RiAFP contains only a single such bond. Six internal repeat segments of a thirteen residue repeat pattern is irregularly spaced apart throughout its sequence. The central part...

  6. Computational simulations on the fish-type-Ⅱ antifreeze protein-ice-solvent system

    Institute of Scientific and Technical Information of China (English)

    LIU Kai; WANG Yan; TAN Hongwei; CHEN Guangju; TONG Zhenhe

    2007-01-01

    Based on the computational simulation with the vacuum environment for the fish-type-Ⅱ antifreeze proteinice-solvent (water)system,the multi-complex system of the antifreeze protein-ice-water has been constructed and calculated.We have studied the interaction of such proteinice system with water solvent through the dynamics simulation with 350 ps.By employing the Molecular Dynamics simulation and semi-empirical method calculation,we have further investigated the interface properties of the antifreeze protein and ice crystal combined system.Consequently,a water solvent affects significantly the properties of this combined system.

  7. Unusual dynamic properties of water near the ice-binding plane of hyperactive antifreeze protein

    Energy Technology Data Exchange (ETDEWEB)

    Kuffel, Anna; Czapiewski, Dariusz; Zielkiewicz, Jan, E-mail: jaz@chem.pg.gda.pl [Department of Chemistry, Gdansk University of Technology, Narutowicza 11/12, 80–233 Gdansk (Poland)

    2015-10-07

    The dynamical properties of solvation water of hyperactive antifreeze protein from Choristoneura fumiferana (CfAFP) are analyzed and discussed in context of its antifreeze activity. The protein comprises of three well-defined planes and one of them binds to the surface of ice. The dynamical properties of solvation water around each of these planes were analyzed separately; the results are compared with the dynamical properties of solvation water of ice around its two crystallographic planes: basal and prism. Three main conclusions are inferred from our investigations. The first one is that the solvation shell of CfAFP does not seem to be particularly far-ranged, at least not beyond what is usually observed for proteins that do not interact with ice. Therefore, it does not appear to us that the antifreeze activity is enhanced by a long-ranged retardation of water mobility. Also the correlation between the collective mobility of water and the collective mobility of protein atoms highly resembles the one measured for the protein that does not interact with ice. Our second conclusion is that the dynamical properties of solvation water of CfAFP are non-uniform. The dynamics of solvation water of ice-binding plane is, in some respects, different from the dynamics of solvation water of the two remaining planes. The feature that distinguishes the dynamics of solvation water of the three planes is the activation energy of diffusion process. The third conclusion is that—from the three analyzed solvation shells of CfAFP—the dynamical properties of solvation water of the ice-binding plane resemble the most the properties of solvation water of ice; note, however, that these properties still clearly differ from the dynamic properties of solvation water of ice.

  8. Unusual dynamic properties of water near the ice-binding plane of hyperactive antifreeze protein

    International Nuclear Information System (INIS)

    The dynamical properties of solvation water of hyperactive antifreeze protein from Choristoneura fumiferana (CfAFP) are analyzed and discussed in context of its antifreeze activity. The protein comprises of three well-defined planes and one of them binds to the surface of ice. The dynamical properties of solvation water around each of these planes were analyzed separately; the results are compared with the dynamical properties of solvation water of ice around its two crystallographic planes: basal and prism. Three main conclusions are inferred from our investigations. The first one is that the solvation shell of CfAFP does not seem to be particularly far-ranged, at least not beyond what is usually observed for proteins that do not interact with ice. Therefore, it does not appear to us that the antifreeze activity is enhanced by a long-ranged retardation of water mobility. Also the correlation between the collective mobility of water and the collective mobility of protein atoms highly resembles the one measured for the protein that does not interact with ice. Our second conclusion is that the dynamical properties of solvation water of CfAFP are non-uniform. The dynamics of solvation water of ice-binding plane is, in some respects, different from the dynamics of solvation water of the two remaining planes. The feature that distinguishes the dynamics of solvation water of the three planes is the activation energy of diffusion process. The third conclusion is that—from the three analyzed solvation shells of CfAFP—the dynamical properties of solvation water of the ice-binding plane resemble the most the properties of solvation water of ice; note, however, that these properties still clearly differ from the dynamic properties of solvation water of ice

  9. 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. PMID:26085662

  10. Towards a green hydrate inhibitor: imaging antifreeze proteins on clathrates.

    Directory of Open Access Journals (Sweden)

    Raimond Gordienko

    Full Text Available The formation of hydrate plugs in oil and gas pipelines is a serious industrial problem and recently there has been an increased interest in the use of alternative hydrate inhibitors as substitutes for thermodynamic inhibitors like methanol. We show here that antifreeze proteins (AFPs possess the ability to modify structure II (sII tetrahydrofuran (THF hydrate crystal morphologies by adhering to the hydrate surface and inhibiting growth in a similar fashion to the kinetic inhibitor poly-N-vinylpyrrolidone (PVP. The effects of AFPs on the formation and growth rate of high-pressure sII gas mix hydrate demonstrated that AFPs are superior hydrate inhibitors compared to PVP. These results indicate that AFPs may be suitable for the study of new inhibitor systems and represent an important step towards the development of biologically-based hydrate inhibitors.

  11. The inhibition of ice nucleators by insect antifreeze proteins is enhanced by glycerol and citrate.

    Science.gov (United States)

    Duman, J G

    2002-02-01

    Antifreeze proteins depress the freezing point of water while not affecting the melting point, producing a characteristic difference in freezing and melting points termed thermal hysteresis. Larvae of the beetle Dendroides canadensis accumulate potent antifreeze proteins (DAFPs) in their hemolymph and gut, but to achieve high levels of thermal hysteresis requires enhancers, such as glycerol. DAFPs have previously been shown to inhibit the activity of bacterial and hemolymph protein ice nucleators, however, the effect was not large and therefore the effectiveness of the DAFPs in promoting supercooling of the larvae in winter was doubtful. However, this study demonstrates that DAFPs, in combination with the thermal hysteresis enhancers glycerol (1 M) or citrate (0.5 M), eliminated the activity of hemolymph protein ice nucleators and Pseudomonas syringae ice-nucleating active bacteria, and lowered the supercooling points (nucleation temperatures) of aqueous solutions containing these ice nucleators to those of water or buffer alone. This shows that the DAFPs, along with glycerol, play a critical role in promoting hemolymph supercooling in overwintering D. canadensis. Also, DAFPs in combination with enhancers may be useful in applications which require inhibition of ice nucleators. PMID:11916110

  12. A root bond between ice and antifreeze protein.

    Science.gov (United States)

    Hawes, Timothy C

    2016-10-01

    It has always been assumed that a three-dimensional protein structure is essential to antifreeze protein (AFP) ice interactions. Using a 9 kDa AFP isolated from the springtail, Gomphiocephalus hodgsoni, it was found that the bond between ice and protein is maintained independent of higher order protein structure. GomplyAFP9 remained bound to ice after denaturing by a range of agents (boiling, extreme pH, DTT, ethanol, urea). Thermal hysteresis was minimal (0.03-0.04 °C), but not lost. Crystal faceting and growth occurred normal to the c-axis, indicating the protein binds primarily to sites along the a-axis. These observations lend additional support to the hypothesis of irreversible binding. More significantly, they suggest that binding to ice and functional hysteresis may be achieved independently (i.e. are different operations). These results are consistent with the view that there is a root bond with ice and it is achieved via an amino acid derived interface that bonds to water molecules in aqueous solutions. PMID:27542583

  13. 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

    2015-10-01

    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.

  14. 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

    2015-10-01

    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. PMID:26371748

  15. Fluorescence microscopy studies of the hyperactive antifreeze protein from an insect

    Science.gov (United States)

    Pertaya, N.; di Prinzio, C. L.; Wilen, L.; Thomson, E.; Wettlaufer, J. S.; Marshall, C. B.; Davies, P. L.; Braslavsky, I.

    2006-03-01

    Antifreeze proteins (AFPs) protect animals from freezing by binding to extracellular ice and inhibiting its growth. Since the initial discovery of AFPs in fish, non-homologous types have been found in insects, plants, bacteria, fungi, and vertebrates. Different AFP types have diverse structures and varied activities. For example, AFPs produced by insects are much more active in inhibiting ice crystal growth compared to most AFPs found in fish or plants. By putting a fluorescent tag on an insect AFP we were able to visualize AFP binding to ice, to determine the ice crystal surfaces to which the AFP adheres, and to follow the kinetics of AFP binding to ice. We expect this approach will contribute to a better understanding of the mechanism of AFP activity and in particular the hyperactivity of insect AFPs.

  16. A study of the growth rates and growth habits of ice crystals in a solution of antifreeze (glyco) proteins

    Science.gov (United States)

    Li, Qianzhong; Luo, Liaofu

    1996-12-01

    The mechanism of the antifreeze glycoprotein/antifreeze protein interaction on the surface of ice is analyzed. The theory of ice crystal growth in an AF(G)P solution is presented. A quantitative calculation of the growth rates for gain growth has been obtained. The anisotropic growth habits and growth rates of ice crystals in an AF(G)P solution are explained.

  17. 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.

  18. Recent Advances in Research of Antifreeze Proteins%抗冻蛋白研究进展

    Institute of Scientific and Technical Information of China (English)

    李金耀; 马纪; 张富春

    2005-01-01

    Many overwintering organisms produce antifreeze proteins (AFPs) that can be adsorbed onto the surface of ice crystals and modify their growth. These proteins show great diversity in structures, and they have been found in a variety of organisms. AFPs from insects have higher thermal hysteresis activity than other organisms. Recent studies revealed the structures of AFPs and put forward different ice-binding models. No mechanism, however, can apply to all antifreeze proteins and the molecular interaction between AFPs and ice are not accurately resolved. AFPs can be applied extensively to agriculture, aquaculture and low temperature storage of organs, tissues, as well as cells. To confer transgenic plant cold resistance application of AFPs is essential, while the expression and regulation of antifreeze gene need to be elucidated.%很多越冬的生物会产生抗冻蛋白,这些抗冻蛋白能够吸附到冰晶的表面改变冰晶形态并抑制冰晶的生长.抗冻蛋白在很多生物体内都被发现,不同的抗冻蛋白结构差异非常大.目前的一些研究揭示了几种抗冻蛋白的结构,并提出了抗冻蛋白与冰晶的结合模型,但是还没有一种机制能解释所有抗冻蛋白的作用机理.抗冻蛋白能被广泛的应用到农业、水产业和低温储藏器官、组织和细胞,利用转基因技术提高植物的抗冻性具有重要应用价值.而抗冻蛋白基因的表达调控则有待进一步阐明.

  19. 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...

  20. Research Progress on Insect Antifreeze Proteins%昆虫抗冻蛋白研究进展

    Institute of Scientific and Technical Information of China (English)

    万军; 朱兴友; 张洋

    2012-01-01

    近年来昆虫抗冻蛋白(AFPs)的研究取得了较快的发展。本文综述了昆虫抗冻蛋白的发现过程、结构特点、表达规律、抗冻机制及相关的昆虫基因工程简况。%In recent years, the research of insect antifreeze proteins has developed rapidly. The discovery process, structural characteristics, expression laws and antifreeze mechanism of insect antifreeze proteins, as well as the related insect gene engineering research were reviewed in this paper.

  1. Synthesis of Cyclic Antifreeze Glycopeptide and Glycopeptoids and Their Ice Recrystallization Inhibition Activity

    Energy Technology Data Exchange (ETDEWEB)

    Ahn, Mija; Murugan, Ravichandran N.; Bang, Jeong Kyu; Kim, Hak Jun [Korea Basic Science Institute, Daejeon (Korea, Republic of); Shin, Song Yub [Chousn Univ., Gwangju (Korea, Republic of); Kim, Eunjung; Lee, Jun Hyuck [Korea Polar Research Institute, Incheon (Korea, Republic of)

    2012-11-15

    Until now, few groups reported the antifreeze activity of cyclic glycopeptides; however, the tedious synthetic procedure is not amenable to study the intensive structure activity relationship. A series of N-linked cyclic glycopeptoids and glycopeptide have been prepared to evaluate antifreeze activity as a function of peptide backbone cyclization and methyl stereochemical effect on the rigid Thr position. This study has combined the cyclization protocol with solid phase peptide synthesis and obtained significant quantities of homogeneous cyclic glycopeptide and glycopeptoids. Analysis of antifreeze activity revealed that our cyclic peptide demonstrated RI activity while cyclic glycopeptoids showed no RI activity. These results suggest that the subtle changes in conformation and Thr orientation dramatically influence RI activity of N-linked glycopeptoids.

  2. Antifreeze proteins govern the precipitation of trehalose in a freezing-avoiding insect at low temperature.

    Science.gov (United States)

    Wen, Xin; Wang, Sen; Duman, John G; Arifin, Josh Fnu; Juwita, Vonny; Goddard, William A; Rios, Alejandra; Liu, Fan; Kim, Soo-Kyung; Abrol, Ravinder; DeVries, Arthur L; Henling, Lawrence M

    2016-06-14

    The remarkable adaptive strategies of insects to extreme environments are linked to the biochemical compounds in their body fluids. Trehalose, a versatile sugar molecule, can accumulate to high levels in freeze-tolerant and freeze-avoiding insects, functioning as a cryoprotectant and a supercooling agent. Antifreeze proteins (AFPs), known to protect organisms from freezing by lowering the freezing temperature and deferring the growth of ice, are present at high levels in some freeze-avoiding insects in winter, and yet, paradoxically are found in some freeze-tolerant insects. Here, we report a previously unidentified role for AFPs in effectively inhibiting trehalose precipitation in the hemolymph (or blood) of overwintering beetle larvae. We determine the trehalose level (29.6 ± 0.6 mg/mL) in the larval hemolymph of a beetle, Dendroides canadensis, and demonstrate that the hemolymph AFPs are crucial for inhibiting trehalose crystallization, whereas the presence of trehalose also enhances the antifreeze activity of AFPs. To dissect the molecular mechanism, we examine the molecular recognition between AFP and trehalose crystal interfaces using molecular dynamics simulations. The theory corroborates the experiments and shows preferential strong binding of the AFP to the fast growing surfaces of the sugar crystal. This newly uncovered role for AFPs may help explain the long-speculated role of AFPs in freeze-tolerant species. We propose that the presence of high levels of molecules important for survival but prone to precipitation in poikilotherms (their body temperature can vary considerably) needs a companion mechanism to prevent the precipitation and here present, to our knowledge, the first example. Such a combination of trehalose and AFPs also provides a novel approach for cold protection and for trehalose crystallization inhibition in industrial applications. PMID:27226297

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

    Science.gov (United States)

    Friis, Dennis S; Johnsen, Johannes L; Kristiansen, Erlend; Westh, Peter; Ramløv, Hans

    2014-06-01

    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, 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 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 of the ice binding motifs; high melting temperature AFPs (high disulfide content, TxT motifs), low melting temperature but high refolding capability AFPs (one disulfide bridge, TxTxTxT motifs) and irreversibly unfolded AFPs at low temperatures (no disulfide bridges, TxTxTxTxT motifs). The property of being able to cope with high temperature exposures may appear peculiar for proteins which strictly have their effect at subzero temperatures. Different aspects of this are discussed.

  4. 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. PMID:27492241

  5. An Investigation of Freezing of Supercooled Water on Anti-Freeze Protein Modified Surfaces

    Institute of Scientific and Technical Information of China (English)

    Thibaut V J Charpentier; Anne Neville; Paul Millner; Rob Hewson; Ardian Morina

    2013-01-01

    This work investigates how functionalization ofaluminium surfaces with natural type Ⅲ Anti-Freeze Protein (AFP) affects the mechanism of heterogeneous ice nucleation.First the bulk ice nucleation properties of distilled water and aqueous solution of AFP were evaluated by differential scanning calorimetry.Then the modified surface was characterized by Secondary Ions Mass Spectroscopy (SIMS),Fourier Transform InfraRed (FTIR) spectroscopy and contact angle measurement.Freezing experiments were then conducted in which water droplets underwent a slow controlled cooling.This study shows that compared to uncoated aluminium,the anti-freeze proteins functionalized surfaces exhibit a higher and narrower range of freezing temperature.It was found that these proteins that keep living organisms from freezing in cold environment act in the opposite way once immobilized on surfaces by promoting ice nucleation.Some suggestions regarding the mechanism of action of the observed phenomena were proposed based on the Classical Nucleation Theory (CNT).

  6. Antifreeze Activity of Xylomannan from the Mycelium and Fruit Body of Flammulina velutipes.

    Science.gov (United States)

    Kawahara, Hidehisa; Matsuda, Yoshiyuki; Sakaguchi, Takuya; Arai, Naoki; Koide, Yoshihide

    2016-01-01

    An identified class of antifreeze, a xylomannan-based thermal hysteresis (TH)-producing glycolipid, has been discovered from diverse taxa, including plants, insects, and amphibians. We isolated xylomannan from the mycelium and fruit body of the basidiomycete Flammulina velutipes using successive hot extraction with water, 2% and 25% aqueous KOH, and gel filtration chromatography. The xylomannan from the fruit body had a recrystallization inhibiting (RI) activity (RI=0.44) at 0.5 mg/mL. The dried weight yield of the fruit body (7.7×10(-2)%, w/w) was higher than that of the mycelium. Although the purified xylomannan from both soures were composed of mannose and xylose in a 2 : 1 molar ratio, the molecular weight of the xylomannan from the mycelium and fruit body was 320,000 and 240,000, respectively. The RI activity of mycelial xylomannan was higher than that from the fruit body (RI=0.57) at 45 µg/mL. Although this RI activity was able to remain constant after exposure to various conditions, we confirmed that the decrease of RI activity was stimulated by the decrease of molecular weight that was caused by heating during the alkaline condition. The survival rate of the CHO cells at -20℃ for two days increased to 97% due to the addition of 20 µg/mL of purified xylomannan. This was the first report to indicate that xylomannan from the mycelium of Flammulina velutipes had a high level of ice recrystallization inhibiting activity like antifreeze proteins from plants and had rhe potential to become a new material for cell storage. PMID:27667520

  7. Adsorption thermodynamics of two-domain antifreeze proteins: theory and Monte Carlo simulations.

    Science.gov (United States)

    Narambuena, Claudio F; Sanchez Varretti, Fabricio O; Ramirez-Pastor, Antonio J

    2016-09-21

    In this paper we develop the statistical thermodynamics of two-domain antifreeze proteins adsorbed on ice. We use a coarse-grained model and a lattice network in order to represent the protein and ice, respectively. The theory is obtained by combining the exact analytical expression for the partition function of non-interacting linear k-mers adsorbed in one dimension, and its extension to higher dimensions. The total and partial adsorption isotherms, and the coverage and temperature dependence of the Helmholtz free energy and configurational entropy are given. The formalism reproduces the classical Langmuir equation, leads to the exact statistical thermodynamics of molecules adsorbed in one dimension, and provides a close approximation for two-dimensional systems. Comparisons with analytical data obtained using the modified Langmuir model (MLM) and Monte Carlo simulations in the grand canonical ensemble were performed in order to test the validity of the theoretical predictions. In the MC calculations, the different mechanisms proposed in the literature to describe the adsorption of two-domain antifreeze proteins on ice were analyzed. Indistinguishable results were obtained in all cases, which verifies the thermodynamic equivalence of these mechanisms and allows the choice of the most suitable mechanism for theoretical studies of equilibrium properties. Even though a good qualitative agreement is obtained between MLM and MC data, it is found that the new theoretical framework offers a more accurate description of the phenomenon of adsorption of two-domain antifreeze proteins. PMID:27539563

  8. The response of watercress (nasturtium officinale) to vacuum impregnation: Effect of an antifreeze protein type I

    OpenAIRE

    Rui M.S. Cruz; Vieira, Margarida C.; Silva, Cristina L. M.

    2009-01-01

    The setting up of methodologies that reduce the size of ice crystals and reduce or inhibit the recrystallisation phenomena could have an extraordinary significance in the final quality of frozen products and consequently bring out new market opportunities. In this work, the effect of an antifreeze protein type I (AFP-I), by vacuum impregnation (VI), on frozen watercress was studied. The VI pressure, samples’ weight, Hunter Lab colour, scanning electron microscopy (SEM), and a wilting test ...

  9. Antifreeze proteins govern the precipitation of trehalose in a freezing-avoiding insect at low temperature

    OpenAIRE

    WEN, Xin; Wang, Sen; Duman, John G.; Arifin, Josh Fnu; Juwita, Vonny; Goddard, William A.; Rios, Alejandra; Liu, Fan; Kim, Soo-Kyung; Abrol, Ravinder; DeVries, Arthur L.; Henling, Lawrence M.

    2016-01-01

    The remarkable adaptive strategies of insects to extreme environments are linked to the biochemical compounds in their body fluids. Trehalose, a versatile sugar molecule, can accumulate to high levels in freeze-tolerant and freeze-avoiding insects, functioning as a cryoprotectant and a supercooling agent. Antifreeze proteins (AFPs), known to protect organisms from freezing by lowering the freezing temperature and deferring the growth of ice, are present at high levels in some freeze-avoiding ...

  10. A low molecular weight peptide from snow mold with epitopic homology to the winter flounder antifreeze protein.

    Science.gov (United States)

    Newsted, W J; Polvi, S; Papish, B; Kendall, E; Saleem, M; Koch, M; Hussain, A; Cutler, A J; Georges, F

    1994-01-01

    Evidence for a small size protein (ca. 3500 kDa) exhibiting epitopic homology to the Atlantic winter flounder antifreeze protein (AFP) is found in the snow molds Coprinus psychromorbidus, Myriosclerotinia borealis, and Typhula incarnata. The protein shows strong cross-reactivity with antisera specific for the flounder AFP. Preliminary studies suggest that the protein is synthesized in response to lowering the culture temperature, and that it is membrane associated and, therefore, may function in an analogous capacity to the fish AFP. Also, the protein is shown to have antifreeze properties as determined by nuclear magnetic resonance microimaging experiments.

  11. 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 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, 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 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...

  12. 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.

  13. Antifreeze proteins in the primary urine of larvae of the beetle Dendroides canadensis.

    Science.gov (United States)

    Nickell, Philip K; Sass, Sandra; Verleye, Dawn; Blumenthal, Edward M; Duman, John G

    2013-05-01

    To avoid freezing while overwintering beneath the bark of fallen trees, Dendroides canadensis (Coleoptera: Pyrochroidae) larvae produce a family of antifreeze proteins (DAFPs) that are transcribed in specific tissues and have specific compartmental fates. DAFPs and associated thermal hysteresis activity (THA) have been shown previously in hemolymph and midgut fluid, but the presence of DAFPs has not been explored in primary urine, a potentially important site that can contain endogenous ice-nucleating compounds that could induce freezing. A maximum mean THA of 2.65±0.33°C was observed in primary urine of winter-collected D. canadensis larvae. THA in primary urine increased significantly through autumn, peaked in the winter and decreased through spring to levels of 0.2-0.3°C in summer, in a pattern similar to that of hemolymph and midgut fluid. THA was also found in hindgut fluid and excreted rectal fluid, suggesting that these larvae not only concentrate AFPs in the hindgut, but also excrete AFPs from the rectal cavity. Based on dafp transcripts isolated from Malpighian tubule epithelia, cDNAs were cloned and sequenced, identifying the presence of transcripts encoding 24 DAFP isoforms. Six of these Malpighian tubule DAFPs were known previously, but 18 are new. We also provide functional evidence that DAFPs can inhibit ice nucleators present in insect primary urine. This is potentially critical because D. canadensis larvae die if frozen, and therefore ice formation in any body fluid, including the urine, would be lethal.

  14. Antifreeze protein from freeze-tolerant grass has a beta-roll fold with an irregularly structured ice-binding site.

    Science.gov (United States)

    Middleton, Adam J; Marshall, Christopher B; Faucher, Frédérick; Bar-Dolev, Maya; Braslavsky, Ido; Campbell, Robert L; Walker, Virginia K; Davies, Peter L

    2012-03-01

    The grass Lolium perenne produces an ice-binding protein (LpIBP) that helps this perennial tolerate freezing by inhibiting the recrystallization of ice. Ice-binding proteins (IBPs) are also produced by freeze-avoiding organisms to halt the growth of ice and are better known as antifreeze proteins (AFPs). To examine the structural basis for the different roles of these two IBP types, we have solved the first crystal structure of a plant IBP. The 118-residue LpIBP folds as a novel left-handed beta-roll with eight 14- or 15-residue coils and is stabilized by a small hydrophobic core and two internal Asn ladders. The ice-binding site (IBS) is formed by a flat beta-sheet on one surface of the beta-roll. We show that LpIBP binds to both the basal and primary-prism planes of ice, which is the hallmark of hyperactive AFPs. However, the antifreeze activity of LpIBP is less than 10% of that measured for those hyperactive AFPs with convergently evolved beta-solenoid structures. Whereas these hyperactive AFPs have two rows of aligned Thr residues on their IBS, the equivalent arrays in LpIBP are populated by a mixture of Thr, Ser and Val with several side-chain conformations. Substitution of Ser or Val for Thr on the IBS of a hyperactive AFP reduced its antifreeze activity. LpIBP may have evolved an IBS that has low antifreeze activity to avoid damage from rapid ice growth that occurs when temperatures exceed the capacity of AFPs to block ice growth while retaining the ability to inhibit ice recrystallization.

  15. Molecular cloning, sequence analysis and homology modeling of the first caudata amphibian antifreeze-like protein in axolotl (Ambystoma mexicanum).

    Science.gov (United States)

    Zhang, Songyan; Gao, Jiuxiang; Lu, Yiling; Cai, Shasha; Qiao, Xue; Wang, Yipeng; Yu, Haining

    2013-08-01

    Antifreeze proteins (AFPs) refer to a class of polypeptides that are produced by certain vertebrates, plants, fungi, and bacteria and which permit their survival in subzero environments. In this study, we report the molecular cloning, sequence analysis and three-dimensional structure of the axolotl antifreeze-like protein (AFLP) by homology modeling of the first caudate amphibian AFLP. We constructed a full-length spleen cDNA library of axolotl (Ambystoma mexicanum). An EST having highest similarity (∼42%) with freeze-responsive liver protein Li16 from Rana sylvatica was identified, and the full-length cDNA was subsequently obtained by RACE-PCR. The axolotl antifreeze-like protein sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 93 amino acids. The calculated molecular mass and the theoretical isoelectric point (pl) of this mature protein were 10128.6 Da and 8.97, respectively. The molecular characterization of this gene and its deduced protein were further performed by detailed bioinformatics analysis. The three-dimensional structure of current AFLP was predicted by homology modeling, and the conserved residues required for functionality were identified. The homology model constructed could be of use for effective drug design. This is the first report of an antifreeze-like protein identified from a caudate amphibian. PMID:23915159

  16. Molecular cloning, sequence analysis and homology modeling of the first caudata amphibian antifreeze-like protein in axolotl (Ambystoma mexicanum).

    Science.gov (United States)

    Zhang, Songyan; Gao, Jiuxiang; Lu, Yiling; Cai, Shasha; Qiao, Xue; Wang, Yipeng; Yu, Haining

    2013-08-01

    Antifreeze proteins (AFPs) refer to a class of polypeptides that are produced by certain vertebrates, plants, fungi, and bacteria and which permit their survival in subzero environments. In this study, we report the molecular cloning, sequence analysis and three-dimensional structure of the axolotl antifreeze-like protein (AFLP) by homology modeling of the first caudate amphibian AFLP. We constructed a full-length spleen cDNA library of axolotl (Ambystoma mexicanum). An EST having highest similarity (∼42%) with freeze-responsive liver protein Li16 from Rana sylvatica was identified, and the full-length cDNA was subsequently obtained by RACE-PCR. The axolotl antifreeze-like protein sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 93 amino acids. The calculated molecular mass and the theoretical isoelectric point (pl) of this mature protein were 10128.6 Da and 8.97, respectively. The molecular characterization of this gene and its deduced protein were further performed by detailed bioinformatics analysis. The three-dimensional structure of current AFLP was predicted by homology modeling, and the conserved residues required for functionality were identified. The homology model constructed could be of use for effective drug design. This is the first report of an antifreeze-like protein identified from a caudate amphibian.

  17. Role of ice nucleation and antifreeze activities in pathogenesis and growth of snow molds.

    Science.gov (United States)

    Snider, C S; Hsiang, T; Zhao, G; Griffith, M

    2000-04-01

    ABSTRACT We examined the ability of snow molds to grow at temperatures from -5 to 30 degrees C and to influence the growth of ice through assays for ice nucleation and antifreeze activities. Isolates of Coprinus psychromorbidus (low temperature basidiomycete variant), Microdochium nivale, Typhula phacorrhiza, T. ishikariensis, T. incarnata, and T. canadensis all grew at -5 degrees C, whereas Sclerotinia borealis and S. homoeocarpa did not grow at temperatures below 4 degrees C. The highest threshold ice nucleation temperature was -7 degrees C. Because snow molds are most damaging to their hosts at temperatures above this, our results imply that the pathogenesis of these fungi is not dependent on ice nucleation activity to cause freeze-wounding of host plants. All snow molds that grew at subzero temperatures also exhibited antifreeze activity in the growth medium and in the soluble and insoluble hyphal fractions, with the exception of M. nivale and one isolate of T. canadensis. The lack of high ice nucleation activity combined with the presence of antifreeze activity in all fungal fractions indicates that snow molds can moderate their environment to inhibit or modify intra- and extracellular ice formation, which helps explain their ability to grow at subzero temperatures under snow cover.

  18. 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.

  19. Patrones electroforéticos de proteínas y actividad anticongelante en el apoplasto de la hoja de la especie andina tropical Senecio niveoaureus PROTEIN ELECTROPHORETIC PATTERNS AND ANTIFREEZING ACTIVITY IN THE LEAF APOPLAST OF THE TROPICAL ANDEAN SPECIES Senecio niveoaureus

    Directory of Open Access Journals (Sweden)

    F ÁLVAREZFLÓREZ

    2006-06-01

    Full Text Available Las plantas de alta montaña tienen diferentes adaptaciones para sobrevivir a cambios drásticos de temperatura, especialmente a condiciones de congelamiento. En plantas de invierno, la supervivencia a temperaturas bajas está relacionada con la capacidad de las células para producir proteínas específicas de bajo peso molecular (proteínas anticongelantes y exportarlas al apoplasto. Para establecer si plantas tropicales de alta montaña sobreviven las temperaturas bajas a través del mismo mecanismo, se colectaron hojas de plantas de Senecio niveoaureus durante 24 horas y a dos alturas 3.300 y 3.600 msnm en el Páramo de Palacio, Chingaza, Colombia. Se observaron proteínas apoplásticas de pesos moleculares entre 3512 kDa. Los patrones electroforéticos fueron diferentes dependiendo de la altura y la hora de muestreo, sin embargo, se observaron variaciones en el patrón de bandeo que no pueden ser atribuidas ni a la temperatura ni al gradiente altitudinal únicamente. Se detectó actividad anticongelante en el apoplasto de hojas de S. niveoaureus, siendo este el primer reporte en especies tropicales de alta montaña.Tropical high mountain plants have different adaptations to survive extreme daily temperature fluctuations and specially freezing night conditions. In winter plant species, survival to low temperatures is related to the ability of the cell to produce specific low molecular weight proteins (antifreezing proteins and to export them to the apoplast. In order to see if high mountain tropical plants survive to low temperatures through the same mechanism we collected, during a 24 hourperiod, leaves from Senecio niveoaureus growing at 3,300 and 3,600 m.o.s.l, in the Páramo de Palacio, Chingaza, Colombia. Leaf apoplast proteins had MW between 3512 kDa. Electrophoretic patterns were different depending on the altitude and the time of sampling. However the observed variations could not be linked to changes in temperature or to the

  20. 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.

  1. Transcriptomic and proteomic analyses on the supercooling ability and mining of antifreeze proteins of the Chinese white wax scale insect.

    Science.gov (United States)

    Yu, Shu-Hui; Yang, Pu; Sun, Tao; Qi, Qian; Wang, Xue-Qing; Chen, Xiao-Ming; Feng, Ying; Liu, Bo-Wen

    2016-06-01

    The Chinese white wax scale insect, Ericerus pela, can survive at extremely low temperatures, and some overwintering individuals exhibit supercooling at temperatures below -30°C. To investigate the deep supercooling ability of E. pela, transcriptomic and proteomic analyses were performed to delineate the major gene and protein families responsible for the deep supercooling ability of overwintering females. Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that genes involved in the mitogen-activated protein kinase, calcium, and PI3K-Akt signaling pathways and pathways associated with the biosynthesis of soluble sugars, sugar alcohols and free amino acids were dominant. Proteins responsible for low-temperature stress, such as cold acclimation proteins, glycerol biosynthesis-related enzymes and heat shock proteins (HSPs) were identified. However, no antifreeze proteins (AFPs) were identified through sequence similarity search methods. A random forest approach identified 388 putative AFPs in the proteome. The AFP gene ep-afp was expressed in Escherichia coli, and the expressed protein exhibited a thermal hysteresis activity of 0.97°C, suggesting its potential role in the deep supercooling ability of E. pela.

  2. Transcriptomic and proteomic analyses on the supercooling ability and mining of antifreeze proteins of the Chinese white wax scale insect.

    Science.gov (United States)

    Yu, Shu-Hui; Yang, Pu; Sun, Tao; Qi, Qian; Wang, Xue-Qing; Chen, Xiao-Ming; Feng, Ying; Liu, Bo-Wen

    2016-06-01

    The Chinese white wax scale insect, Ericerus pela, can survive at extremely low temperatures, and some overwintering individuals exhibit supercooling at temperatures below -30°C. To investigate the deep supercooling ability of E. pela, transcriptomic and proteomic analyses were performed to delineate the major gene and protein families responsible for the deep supercooling ability of overwintering females. Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that genes involved in the mitogen-activated protein kinase, calcium, and PI3K-Akt signaling pathways and pathways associated with the biosynthesis of soluble sugars, sugar alcohols and free amino acids were dominant. Proteins responsible for low-temperature stress, such as cold acclimation proteins, glycerol biosynthesis-related enzymes and heat shock proteins (HSPs) were identified. However, no antifreeze proteins (AFPs) were identified through sequence similarity search methods. A random forest approach identified 388 putative AFPs in the proteome. The AFP gene ep-afp was expressed in Escherichia coli, and the expressed protein exhibited a thermal hysteresis activity of 0.97°C, suggesting its potential role in the deep supercooling ability of E. pela. PMID:26799455

  3. A 9 kDa antifreeze protein from the Antarctic springtail, Gomphiocephalus hodgsoni.

    Science.gov (United States)

    Hawes, T C; Marshall, C J; Wharton, D A

    2014-08-01

    A 9 kDA antifreeze protein (AFP) was isolated and purified from the Antarctic springtail, Gomphiocephalus hodgsoni. By combining selective sampling procedures and a modified ice affinity purification protocol it was possible to directly isolate a single AFP protein without recourse to chromatographic separation techniques. Mass spectrometry identified a single 9 kDa component in the purified ice fraction. Intramolecular disulphide bonding was suggested by the presence of 12 cysteine residues. The specific amino acid composition is unique, particularly with regard to the presence of histidine (11.5%). But it also shows noticeable commonalities with insect AFPs in the abundance of cysteine (13.8%), while simultaneously hinting, through the presence of glycine (11.5%), that the metabolic building blocks of AFPs in Collembola may have a phylogenetically-determined component. PMID:25025820

  4. Improvement of texture properties and flavor of frozen dough by carrot (Daucus carota) antifreeze protein supplementation.

    Science.gov (United States)

    Zhang, Chao; Zhang, Hui; Wang, Li; Gao, Hong; Guo, Xiao Na; Yao, Hui Yuan

    2007-11-14

    The effects of concentrated carrot protein (CCP) containing 15.4% (w/w) carrot (Daucus carota) antifreeze protein on texture properties of frozen dough and volatile compounds of crumb were studied. CCP supplementation lowered the freezable water content of the dough, resulting in some beneficial effects including holding loaf volume steadily and making the dough softer and steadier during frozen storage. Furthermore, SPME-GC-MS analysis showed CCP supplementation did not give any negative influences on volatile compounds of crumb and gave a pleasant aroma felt like Michelia alba DC from trans-caryophyllene simultaneously. Combining our previous results that CCP supplementation improves the fermentation capacity of the frozen dough, CCP could be used as a beneficial additive for frozen dough processing. PMID:17935294

  5. 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...

  6. The Surface of Ice in the presence of Antifreeze Proteins studied by Atomic Force Microscopy

    Science.gov (United States)

    Zepeda, Salvador; Orme, Christine; Yeh, Yin

    2002-03-01

    The surface of ice has been a topic of interest for centuries. In particular, the surface structure and properties have been explored with the advent of new surface techniques. Several groups have convincingly shown a surface transition layer to exist between the solid-vapor interface as well as the solid-liquid interface. In addition, the characteristics of this region may be directly correlated with growth morphologies of ice. Certain peptide molecules have the ability to significantly alter the growth morphology of an ice crystal. Do these molecules simply disrupt this transition region? Or do they anchor themselves deep into it reaching the bulk-ice phase? And is there a similar mechanism by which they function? We use AFM to study the morphological changes to the true ice surface due to the presence antifreeze proteins. We will discuss the implications of our results on the longstanding debate to the above questions.

  7. Evolution of Type II Antifreeze Protein Genes in Teleost Fish: A Complex Scenario Involving Lateral Gene Transfers and Episodic Directional Selection

    OpenAIRE

    Ulf Sorhannus

    2012-01-01

    I examined hypotheses about lateral transfer of type II antifreeze protein (AFP) genes among “distantly” related teleost fish. The effects of episodic directional selection on amino acid evolution were also investigated. The strict consensus results showed that the type II AFP and type II antifreeze-like protein genes were transferred from Osmerus mordax to Clupea harengus, from the ancestral lineage of the Brachyopsis rostratus—Hemitripterus americanus clade to the ancestor of the Hypomesus ...

  8. Antifreeze and cryoprotective activities of ice-binding collagen peptides from pig skin.

    Science.gov (United States)

    Cao, Hui; Zhao, Ying; Zhu, Yu Bing; Xu, Fei; Yu, Jing Song; Yuan, Min

    2016-03-01

    A novel "hyperactive" ice-binding peptide from porcine collagen was prepared by alkaline protease hydrolysis and a series of column chromatography separations, and then its antifreeze and cryoprotective properties were reported. Using differential scanning calorimetry (DSC), the thermal hysteresis (TH) of ice-binding collagen peptides was closely related to their concentration and crystal fraction. Collagen hydrolysates with maximal TH were obtained by hydrolysis at pH 8.0, DH 15.0%, and 5% alkaline protease at 55°C. After purification by column chromatography, the AP-3 ice-binding collagen peptide (GLLGPLGPRGLL) with 1162.8Da molecular weights exhibited the highest TH (5.28°C), which can be classified as "hyperactive". Recrystallisation and melt-resistance of ice cream were improved by AP-3 ice-binding collagen peptide at 0.2% (w/v) in a similar manner to natural antifreeze proteins. Moreover, the addition of AP-3 collagen peptides in ice cream greatly elevated the glass transition temperature (Tg) to -17.64°C. PMID:26471678

  9. Expression of a Carrot 36 kD Antifreeze Protein Gene Improves Cold Stress Tolerance in Transgenic Tobacco

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Antifreeze proteins (AFPs) enable organisms to survive under cold conditions, and have great potential in improving cold tolerance of cold-sensitive plants. In order to determine whether expression of the carrot 36 kD antifreeze protein gene confers improved cold-resistant properties to plant tissues, we tried to obtain transgenic tobacco plants which expressed the antifreeze protein. Cold, salt, and drought induced promoter Prd29A was cloned using PCR from Arabidopsis. Two plant expression vectors based on pBI121 were constructed with CaMV35S:AFP and Prd29A:AFP. Tobacco plantlets were transformed by Agrobacterium-medicated transformation. PCR and Southern blotting demonstrated that the carrot 36 kD afp gene was successfully integrated into the genomes of transformed plantlets. The expression of the afp gene in transgenic plants led to improved tolerance to cold stress.However, the use of the strong constitutive 35S cauliflower mosaic virus (CaMV) promoter to drive expression of afp also resulted in growth retardation under normal growing conditions. In contrast, the expression of afp driven by the stress-inducible Prd29A promoter from Arabidopsis gave rise to minimal effects on plant growth while providing an increased tolerance to cold stress condition (2℃). The results demonstrated the prospect of using Prd29A-AFP transgenic plants in cold-stressed conditions that will in turn benefit agriculture.

  10. Structural Basis for the Inhibition of Gas Hydrates by α-Helical Antifreeze Proteins.

    Science.gov (United States)

    Sun, Tianjun; Davies, Peter L; Walker, Virginia K

    2015-10-20

    Kinetic hydrate inhibitors (KHIs) are used commercially to inhibit gas hydrate formation and growth in pipelines. However, improvement of these polymers has been constrained by the lack of verified molecular models. Since antifreeze proteins (AFPs) act as KHIs, we have used their solved x-ray crystallographic structures in molecular modeling to explore gas hydrate inhibition. The internal clathrate water network of the fish AFP Maxi, which extends to the protein's outer surface, is remarkably similar to the {100} planes of structure type II (sII) gas hydrate. The crystal structure of this water web has facilitated the construction of in silico models for Maxi and type I AFP binding to sII hydrates. Here, we have substantiated our models with experimental evidence of Maxi binding to the tetrahydrofuran sII model hydrate. Both in silico and experimental evidence support the absorbance-inhibition mechanism proposed for KHI binding to gas hydrates. Based on the Maxi crystal structure we suggest that the inhibitor adsorbs to the gas hydrate lattice through the same anchored clathrate water mechanism used to bind ice. These results will facilitate the rational design of a next generation of effective green KHIs for the petroleum industry to ensure safe and efficient hydrocarbon flow. PMID:26488661

  11. 胡萝卜与黄粉虫抗冻融合基因在拟南芥中的表达与抗冻性分析%Expression and Antifreeze Feature Analysis of the Daucus carota and Tentbrio molitor Fusion Antifreeze Protein Gene in Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    张振华; 陈介南; 卢孟柱; 章怀云; 刘伯斌

    2012-01-01

    抗冻蛋白(antifreeze protein,AFP)是一类能控制冰晶生长和抑制冰晶之间发生重结晶的蛋白质,能在低温结冰条件下保护生物体不受伤害.抗冻蛋白具有2种明显不同的抗冻活性——热滞(thermal hysteresis,TH)活性和重结晶抑制(recrystallizationinhibition,RI)活性.鱼类和昆虫抗冻蛋白的特征是高TH低RI活性,而植物抗冻蛋白的特征是低TH高RI活性.为了获得高TH、高RI活性的抗冻蛋白(antifreeze protein,AFP),克隆得到胡萝卜抗冻蛋白基因(DcAFP)和黄粉虫抗冻蛋白基因(TmAFP)cDNA 全序列.通过重叠延伸PCR,将2个基因前后串联构建融合基因Dc- TmAFP和Tm -DcAFP,并构建了植物表达载体,通过农杆菌介导转化模式植物拟南芥,成功获得4个基因的转基因拟南芥植株.对转基因拟南芥植株进行抗冻试验处理,并进行了存活率统计和叶片黄化率统计分析.分析结果表明,不同功能的抗冻蛋白对植物的抗冻性有不同的帮助,热滞效应高效的黄粉虫抗冻蛋白对植物降低冰点方面效果大,而在植物遭受较长时间冻害时,抑制重结晶效应高效的胡萝卜抗冻蛋白对植物质外体中流体保持稳定性作用较大,从而提高植物的耐冻能力.%Antifreeze proteins (AFPs) are a family of proteins capable of protecting organisms from damage in freezing or sub-freezing conditions by controlling the growth of ice and inhibiting the recrystallization between ice granules, which are termed thermal hysteresis (TH) activity and recrystallization inhibition (RI) activity respectively. In comparison with the characteristic of high TH and low RI of fish AFPs and insect AFPs, antifreeze proteins of plants was low TH and high RI activity. In order to obtain antifreeze proteins with high TH and high RI activity, the complete cDNA of antifreeze protein genes (DcAFP and TmAFP) were cloned from Daucus carota and Tentbrio molitor. The two fusion antifreeze protein genes, Dc

  12. Determining the ice-binding planes of antifreeze proteins by fluorescence-based ice plane affinity.

    Science.gov (United States)

    Basu, Koli; Garnham, Christopher P; Nishimiya, Yoshiyuki; Tsuda, Sakae; Braslavsky, Ido; Davies, Peter

    2014-01-15

    Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

  13. Structure and evolutionary origin of Ca(2+-dependent herring type II antifreeze protein.

    Directory of Open Access Journals (Sweden)

    Yang Liu

    Full Text Available In order to survive under extremely cold environments, many organisms produce antifreeze proteins (AFPs. AFPs inhibit the growth of ice crystals and protect organisms from freezing damage. Fish AFPs can be classified into five distinct types based on their structures. Here we report the structure of herring AFP (hAFP, a Ca(2+-dependent fish type II AFP. It exhibits a fold similar to the C-type (Ca(2+-dependent lectins with unique ice-binding features. The 1.7 A crystal structure of hAFP with bound Ca(2+ and site-directed mutagenesis reveal an ice-binding site consisting of Thr96, Thr98 and Ca(2+-coordinating residues Asp94 and Glu99, which initiate hAFP adsorption onto the [10-10] prism plane of the ice lattice. The hAFP-ice interaction is further strengthened by the bound Ca(2+ through the coordination with a water molecule of the ice lattice. This Ca(2+-coordinated ice-binding mechanism is distinct from previously proposed mechanisms for other AFPs. However, phylogenetic analysis suggests that all type II AFPs evolved from the common ancestor and developed different ice-binding modes. We clarify the evolutionary relationship of type II AFPs to sugar-binding lectins.

  14. Production of a recombinant type 1 antifreeze protein analogue by L. lactis and its applications on frozen meat and frozen dough.

    Science.gov (United States)

    Yeh, Chuan-Mei; Kao, Bi-Yu; Peng, Hsuan-Jung

    2009-07-22

    In this study, a novel recombinant type I antifreeze protein analogue (rAFP) was produced and secreted by Lactococcus lactis, a food-grade microorganism of major commercial importance. Antifreeze proteins are potent cryogenic protection agents for the cryopreservation of food and pharmaceutical materials. A food-grade expression and fermentation system (BSE- and antibiotic-free) for the production and secretion of high levels of rAFP was developed. Lyophilized, crude rAFP produced by L. lactis was tested in a frozen meat and frozen dough processing model. The frozen meat treated with the antifreeze protein showed less drip loss, less protein loss, and a high score on juiciness by sensory evaluation. Frozen dough treated with the rAFP showed better fermentation capacity than untreated frozen dough. Breads baked from frozen dough treated with rAFP acquired the same consumer acceptance as fresh bread. PMID:19545118

  15. Crystal structure and mutational analysis of Ca2+-independent type II antifreeze protein from longsnout poacher, Brachyopsis rostratus.

    Science.gov (United States)

    Nishimiya, Yoshiyuki; Kondo, Hidemasa; Takamichi, Manabu; Sugimoto, Hiroshi; Suzuki, Mamoru; Miura, Ai; Tsuda, Sakae

    2008-10-10

    We recently found that longsnout poacher (Brachyosis rostratus) produces a Ca(2+)-independent type II antifreeze protein (lpAFP) and succeeded in expressing recombinant lpAFP using Phichia pastoris. Here, we report, for the first time, the X-ray crystal structure of lpAFP at 1.34 A resolution. The lpAFP structure displayed a relatively planar surface, which encompasses two loop regions (Cys86-Lys89 and Asn91-Cys97) and a short beta-strand (Trp109-Leu112) with three unstructured segments (Gly57-Ile58, Ala103-Ala104, and Pro113-His118). Electrostatic calculation of the protein surface showed that the relatively planar surface was divided roughly into a hydrophobic area (composed of the three unstructured segments lacking secondary structure) and a hydrophilic area (composed of the loops and beta-strand). Site-directed mutation of Ile58 with Phe at the center of the hydrophobic area decreased activity significantly, whereas mutation of Leu112 with Phe at an intermediate area between the hydrophobic and hydrophilic areas retained complete activity. In the hydrophilic area, a peptide-swap mutant in the loops retained 60% activity despite simultaneous mutations of eight residues. We conclude that the epicenter of the ice-binding site of lpAFP is the hydrophobic region, which is centered by Ile58, in the relatively planar surface. We built an ice-binding model for lpAFP on the basis of a lattice match of ice and constrained water oxygen atoms surrounding the hydrophobic area in the lpAFP structure. The model in which lpAFP has been docked to a secondary prism (2-1-10) plane, which is different from the one determined for Ca(2+)-independent type II AFP from sea raven (11-21), appears to explain the results of the mutagenesis analysis. PMID:18674542

  16. 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.

  17. In silico characterization of antifreeze proteins using computational tools and servers

    Indian Academy of Sciences (India)

    K Sivakumar; S Balaji; Gangaradhakrishnan

    2007-09-01

    In this paper, seventeen different fish Antifreeze Proteins (AFPs) retrieved from Swiss-Prot database are analysed and characterized using In silico tools. Primary structure analysis shows that most of the AFPs are hydrophobic in nature due to the high content of non-polar residues. The presence of 11 cysteines in the rainbow smelt fish and sea raven fish AFPs infer that these proteins may form disulphide (SS) bonds, which are regarded as a positive factor for stability. The aliphatic index computed by Ex-Pasy’s ProtParam infers that AFPs may be stable for a wide range of temperature. Secondary structure analysis shows that most of the fish AFPs have predominant α-helical structures and rest of the AFPs have mixed secondary structure. The very high coil structural content of rainbow smelt fish and sea raven fish AFPs are due to the rich content of more flexible glycine and hydrophobic proline amino acids. Proline has a special property of creating kinks in polypetide chains and disrupting ordered secondary structure. SOSUI server predicts one transmembrane region in winter flounder fish and atlantic cod and two transmembrane regions in yellowtail flounder fish AFP. The predicted transmembrane regions were visualized and analysed using helical wheel plots generated by EMBOSS pepwheel tool. The presence of disulphide (SS) bonds in the AFPs Q01758 and P05140 are predicted by CYS_REC tool and also identified from the three-dimensional structure using Rasmol tool. The disulphide bonds identified from the three-dimensional structure using the Rasmol tool might be correct as the evaluation parameters are within the acceptable limits for the modelled 3D structures.

  18. Modeling the Influence of Antifreeze Proteins on Three-Dimensional Ice Crystal Melt Shapes using a Geometric Approach

    CERN Document Server

    Liu, Jun Jie; Dolev, Maya Bar; Celik, Yeliz; Wettlaufer, J S; Braslavsky, Ido

    2012-01-01

    The melting of pure axisymmetric ice crystals has been described previously by us within the framework of so-called geometric crystal growth. Nonequilibrium ice crystal shapes evolving in the presence of hyperactive antifreeze proteins (hypAFPs) are experimentally observed to assume ellipsoidal geometries ("lemon" or "rice" shapes). To analyze such shapes we harness the underlying symmetry of hexagonal ice Ih and extend two-dimensional geometric models to three-dimensions to reproduce the experimental dissolution process. The geometrical model developed will be useful as a quantitative test of the mechanisms of interaction between hypAFPs and ice.

  19. 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

  20. Antifreeze glycopeptide diastereomers

    Directory of Open Access Journals (Sweden)

    Lilly Nagel

    2012-10-01

    Full Text Available Antifreeze glycopeptides (AFGPs are a special class of biological antifreeze agents, which possess the property to inhibit ice growth in the body fluids of arctic and antarctic fish and, thus, enable life under these harsh conditions. AFGPs are composed of 4–55 tripeptide units -Ala-Ala-Thr- glycosylated at the threonine side chains. Despite the structural homology among all the fish species, divergence regarding the composition of the amino acids occurs in peptides from natural sources. Although AFGPs were discovered in the early 1960s, the adsorption mechanism of these macromolecules to the surface of the ice crystals has not yet been fully elucidated. Two AFGP diastereomers containing different amino acid configurations were synthesized to study the influence of amino acid stereochemistry on conformation and antifreeze activity. For this purpose, peptides containing monosaccharide-substituted allo-L- and D-threonine building blocks were assembled by solid-phase peptide synthesis (SPPS. The retro-inverso AFGP analogue contained all amino acids in D-configuration, while the allo-L-diastereomer was composed of L-amino acids, like native AFGPs, with replacement of L-threonine by its allo-L-diastereomer. Both glycopeptides were analyzed regarding their conformational properties, by circular dichroism (CD, and their ability to inhibit ice recrystallization in microphysical experiments.

  1. Research Progress in Antifreeze Proteins and Application in Food Industry%抗冻蛋白的研究进展及其在食品工业中的应用

    Institute of Scientific and Technical Information of China (English)

    汪少芸; 赵珺; 吴金鸿; 陈琳

    2011-01-01

    抗冻蛋白是一类具有热滞效应、冰晶形态效应和重结晶抑制效应的蛋白质,因其特殊的结构和功能,抗冻蛋白引起了研究人员的极大兴趣.探讨了近年来抗冻蛋白的研究进展,介绍了目前已知的抗冻蛋白的来源、特性、测定方法、基因结构及在食品工业中的应用.抗冻蛋白对冷冻食品有显著的品质改良功能,是未来冷冻食品工业中极具潜力的抗冻添加剂.%Antifreeze proteins (AFPs) are the thermal hysteresis proteins that have the ability to modify the growth and inhibit the recrystallization of the ice. Antifreeze proteins aroused great interests of many researchers due to its special structure and functions. In this article, the recent advance in antifreeze protein was reviewed, and the types, properties, measurements, gene structures of antifreeze protein, and its applications in food industry were introduced. The application trials indicated that antifreeze protein could significantly improve the qualities of frozen foods, which suggested the potential food additives of antifreeze protein in future frozen food industry.

  2. Ice-binding site of snow mold fungus antifreeze protein deviates from structural regularity and high conservation.

    Science.gov (United States)

    Kondo, Hidemasa; Hanada, Yuichi; Sugimoto, Hiroshi; Hoshino, Tamotsu; Garnham, Christopher P; Davies, Peter L; Tsuda, Sakae

    2012-06-12

    Antifreeze proteins (AFPs) are found in organisms ranging from fish to bacteria, where they serve different functions to facilitate survival of their host. AFPs that protect freeze-intolerant fish and insects from internal ice growth bind to ice using a regular array of well-conserved residues/motifs. Less is known about the role of AFPs in freeze-tolerant species, which might be to beneficially alter the structure of ice in or around the host. Here we report the 0.95-Å high-resolution crystal structure of a 223-residue secreted AFP from the snow mold fungus Typhula ishikariensis. Its main structural element is an irregular β-helix with six loops of 18 or more residues that lies alongside an α-helix. β-Helices have independently evolved as AFPs on several occasions and seem ideally structured to bind to several planes of ice, including the basal plane. A novelty of the β-helical fold is the nonsequential arrangement of loops that places the N- and C termini inside the solenoid of β-helical coils. The ice-binding site (IBS), which could not be predicted from sequence or structure, was located by site-directed mutagenesis to the flattest surface of the protein. It is remarkable for its lack of regularity and its poor conservation in homologs from psychrophilic diatoms and bacteria and other fungi.

  3. Molecular and quantum mechanical studies on the monomer recognition of a highly-regular β-helical antifreeze protein

    Institute of Scientific and Technical Information of China (English)

    YANG; Zuoyin; JIA; Zongchao; LIU; Ruozhuang; CHEN; Guangj

    2004-01-01

    The possible interaction models for an antifreeze protein from Tenebrio molitar (TmAFP) have been systematically studied using the methods of molecular mechanics, molecular dynamics and quantum chemistry. It is hoped that these approaches would provide insights into the nature of interaction between protein monomers through sampling a number of interaction possibilities and evaluating their interaction energies between two monomers in the course of recognition. The results derived from the molecular mechanics indicate that monomer's β-sheets would be involved in interaction area and the side chains on two β-faces can match each other at the two-dimensional level. The results from molecular mechanics and ONIOM methods show that the strongest interaction energy could be gained through the formation of H-bonds when the two β-sheets are involved in the interaction model. Furthermore, the calculation of DFT and analysis of van der Waals bond charge density confirm further that recognition between the two TCTs mainly depends on inter-molecular hydroxyls. Therefore, our results demonstrate that during the course of interaction the most favorable association of TmAFPs is via their β-sheets.

  4. 白菜型冬油菜质外体抗冻蛋白研究%Study on apoplast anti-freeze proteins in winter turnip rape (Brassica rape L.)

    Institute of Scientific and Technical Information of China (English)

    杨刚; 刘林波; 杨建胜; 方园; 张娟; 史鹏辉; 孙万仓; 刘自刚; 曾秀存; 武军艳; 方彦; 李学才; 陈奇

    2016-01-01

    The objective of this paper was to lay the basis for studying cold resistance of winter rapeseed. The anti-freeze activities of apoplast proteins were determined in the ‘Longyou 6’ winter rape leaves and roots under cold vernalization. The apoplast proteins were separated by SDS-PAGE and high expression proteins identified in MALDI-TOF/TOF mass spectrometry under field and pot experiments. The results showed that apoplast protein content of ‘Longyou 6’ leaves increased significantly (P < 0.05) after cold acclimation in an artificial climate chamber, reaching 92.31 µg•g-1(FW) on the fifth day, which represented an increase of 246.12% over CK. Apoplast protein content after 10-15 days of cold acclimation dropped compared with that after 5 days, but was still significantly higher than that of CK (P < 0.05). Apoplast protein content continued to increase with increasing cold acclimation time from 20 to 25 days (P < 0.05). Apoplast protein content decreased significantly with after 10 days of de-acclimation. In the process of cold acclimation, apoplast protein content of ‘Longyou 6’ leaves significantly accumulated. However, it decreased significantly after de-acclimation. Obviously, apoplast proteins of‘Longyou 6’ winter rape belonged to low temperature induced proteins. Anti-freeze activity detection analysis suggested that apoplast proteins had re-crystallization inhibition activity. Mass spectrometry identification revealed a variety of proteins with unclear functions along with β-1-3-glucanase consistent anti-freeze proteins reported in winter rye. The class glucanase detected by mass spectrometry was found to have weaker ice crystal forms due to modification effect with reclamation and anti-freeze activity test. The test suggested that this class glucanase was a low activity anti-freeze protein. Many anti-freeze proteins were synthesized and secreted by winter rape in apoplast of leaves and roots under low temperature stress. The proteins

  5. Solution structures, dynamics, and ice growth inhibitory activity of peptide fragments derived from an antarctic yeast protein.

    Directory of Open Access Journals (Sweden)

    Syed Hussinien H Shah

    Full Text Available Exotic functions of antifreeze proteins (AFP and antifreeze glycopeptides (AFGP have recently been attracted with much interest to develop them as commercial products. AFPs and AFGPs inhibit ice crystal growth by lowering the water freezing point without changing the water melting point. Our group isolated the Antarctic yeast Glaciozyma antarctica that expresses antifreeze protein to assist it in its survival mechanism at sub-zero temperatures. The protein is unique and novel, indicated by its low sequence homology compared to those of other AFPs. We explore the structure-function relationship of G. antarctica AFP using various approaches ranging from protein structure prediction, peptide design and antifreeze activity assays, nuclear magnetic resonance (NMR studies and molecular dynamics simulation. The predicted secondary structure of G. antarctica AFP shows several α-helices, assumed to be responsible for its antifreeze activity. We designed several peptide fragments derived from the amino acid sequences of α-helical regions of the parent AFP and they also showed substantial antifreeze activities, below that of the original AFP. The relationship between peptide structure and activity was explored by NMR spectroscopy and molecular dynamics simulation. NMR results show that the antifreeze activity of the peptides correlates with their helicity and geometrical straightforwardness. Furthermore, molecular dynamics simulation also suggests that the activity of the designed peptides can be explained in terms of the structural rigidity/flexibility, i.e., the most active peptide demonstrates higher structural stability, lower flexibility than that of the other peptides with lower activities, and of lower rigidity. This report represents the first detailed report of downsizing a yeast AFP into its peptide fragments with measurable antifreeze activities.

  6. 抗冻蛋白应用前景及基因工程表达研究进展%Researches Advances in Application of Antifreeze Protein and Its Gene Engineering Expression

    Institute of Scientific and Technical Information of China (English)

    蔡文萍; 马纪

    2012-01-01

    抗冻蛋白(antifreeze protein,AFP)又称为热滞蛋白,是一类抑制冰晶生长的蛋白,它具有三个基本特征:热滞效应(thermal hysteresis activity,THA)、冰晶形态效应和重结晶抑制效应(frecrystallization inhibition,RI),抗冻蛋白是一类广泛存在于鱼类、植物、真菌、昆虫中的蛋白,不同生物的AFPs的化学结构、理化性质、空间构型各不相同,并且同类生物之间的AFPs同源性也不高,不存在相似性序列或结构模式,说明它们可能是在不同的有机体中独立进化而来,没有共同的演化规律.随着研究的深入,抗冻蛋白可广泛应用于医学农学食品工业等领域,起到冷冻保护剂,食品添加剂的作用,前人已经对抗冻蛋白的结构、生化性质和抗冻机制进行了阐述,研究主要对抗冻蛋白应用方面及基因工程的表达作了系统综述,为抗冻蛋白的深入研究奠定基础.%Antifreeze protein (AFP) , also known as thermal hysteresis protein, It is a class of proteinThat inhibiting the growth of ice crystals. It has three basic characteristics; thermal hysteresis effect (THA), the effect of ice crystals form, recrystallization inhibitory effect (RI), To date, AFPs have been widely found in a variety of organisms, such as fish, insects, plants, bacteria and fungi, isolated from a number of fish, plants , bacteria, fungi and arthropods . Different biological AFPs chemical structure, physical and chemical properties, spatial configuration of each are not identical, and similar biological between AFPs homology is not high, there is no similar sequence or structural patterns, indicating that they may be in different organisms evolved independently, there is no common evolution law. Along with the development of research, Antifreeze protein can be widely used in agriculture, medicine food sciences and industry, It played a role in the cryoprotectant and food additives. The biochemical characteristics, as well as antifreeze

  7. Bacterial Ice Crystal Controlling Proteins

    OpenAIRE

    Lorv, Janet S. H.; Rose, David R; Glick, Bernard R.

    2014-01-01

    Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. R...

  8. Saccharide antifreeze compositions

    Science.gov (United States)

    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. Identification of antifreeze proteins and their functional residues by support vector machine and genetic algorithms based on n-peptide compositions.

    Directory of Open Access Journals (Sweden)

    Chin-Sheng Yu

    Full Text Available For the first time, multiple sets of n-peptide compositions from antifreeze protein (AFP sequences of various cold-adapted fish and insects were analyzed using support vector machine and genetic algorithms. The identification of AFPs is difficult because they exist as evolutionarily divergent types, and because their sequences and structures are present in limited numbers in currently available databases. Our results reveal that it is feasible to identify the shared sequential features among the various structural types of AFPs. Moreover, we were able to identify residues involved in ice binding without requiring knowledge of the three-dimensional structures of these AFPs. This approach should be useful for genomic and proteomic studies involving cold-adapted organisms.

  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.

  11. Validation of antifreeze properties of glutathione based on its thermodynamic characteristics and protection of baker's yeast during cryopreservation.

    Science.gov (United States)

    Zhang, Chao; Zhang, Hui; Wang, Li; Yao, Huiyuan

    2007-06-13

    The antifreeze ability of glutathione was evaluated on the basis of its thermodynamic characteristics and protection of baker's yeast during cryopreservation at -30 degrees C. The thermodynamic characteristics and protection of baker's yeast of glutathione were similar to those of known antifreeze proteins, such as carrot antifreeze protein and holly antifreeze protein. These properties included lowering the freezing point at about 0.20 degrees C non-colligatively, decreasing freezable water content, controlling the movement of free water for its strong hydrophilicity, and improving baker's yeast survival during the simulated processing of frozen dough. Therefore, glutathione was viewed to be an antifreeze protein like substance on the basis of its unique thermodynamic characteristics and protection of baker's yeast. The method combining thermodynamic characteristic analysis and protection evaluation is a new and simple way to screen new antifreeze proteins. PMID:17508758

  12. The Roles of Insects Body Cold-resistance Effects on Ice Nucleation Proteins and Insect Antifreeze Proteins%冰核蛋白和昆虫抗冻蛋白在虫体抗寒中的作用

    Institute of Scientific and Technical Information of China (English)

    周世豪; 袁哲明

    2012-01-01

    为了冰核蛋白和昆虫抗冻蛋白共同维持虫体在寒冬中的动态平衡.尝试着从冰核蛋白和昆虫抗冻蛋白的结构和功能出发,阐述两者在虫体中的抗寒作用,有助于日后更为深入的研究.%In order to the ice nucleation proteins (INPs) and the insects antifreeze proteins (AFPs) are two closely related to the body of insects, intervening of antagonism and synergy have maintained the dynamical equilibrium in the body of insects in winter. This paper expounds on two of the body in insect role, further cold more in-depth research.

  13. Bacterial ice crystal controlling proteins.

    Science.gov (United States)

    Lorv, Janet S H; Rose, David R; Glick, Bernard R

    2014-01-01

    Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. Rather than direct binding, it is probable that these protein classes create an ice surface prior to ice crystal surface adsorption. Function is differentiated by molecular size of the protein. This paper reviews the similar and different aspects of bacterial antifreeze and ice nucleation proteins, the role of these proteins in freezing tolerance, prevalence of these proteins in psychrophiles, and current mechanisms of protein-ice interactions. PMID:24579057

  14. 印楝Azadirachta Indica A.Juss的冷驯化与抗冻蛋白的研究%On Cold-Acclimation and Antifreeze Proteins of Azadirachta Indica A.Juss

    Institute of Scientific and Technical Information of China (English)

    王颖; 杨光伟

    2012-01-01

    The micropropagation system has been established through plant tissue culture technology with traditional herbal plant Azadirachta Indica A. Juss as material before cold acclimation process is carried out; in vivo antifreeze proteins of neem have been investigated and analyzed. The main results are as follows: ①After cold acclimation, total amount of neem protein increases and several new proteins produce. However, with the cold treatment for too long a time, amount of the neem cold-induced proteins decrease, and even some of them are degradated. ②The stability of antifreeze proteinfrom neem is related to the time of cold treatment in cold acclimation. The shortest time for antifreeze protein production is two weeks in 5℃ , and the cold resistant limit of neem is in 5℃ for 20 days. When the temperature drops below 0℃ , AFPs are accumulated in the first days of cold treatment(0~ 15 d). However, while the treatment time i.s prolonged, Antifreeze protein are degradated and dismissed on the 30th day. ③ Purified antifreeze protein is obtained, and the relative molecular mass is around 36 KD.%采用木本植物材料——印楝,通过组织培养建立快繁体系,然后对其进行冷驯化处理,并分析检测印楝植物体内抗冻蛋白.主要结果如下:①冷驯化处理后印楝的总蛋白一些表现为量的增加同时会有新的蛋白产生.但脱驯化或处理时间过长时,抗冻蛋白在量的表达上会有逐渐减少或消失的现象.②在对印楝的冷驯化中,发现不同的温度处理后蛋白稳定存在的时间不同.抗冻蛋白出现的最早时期为5℃处理2周左右,印楝能耐受的稳定最低温为5℃,所持续的最长时间约为20 d.在0℃低温处理后,虽然在处理初期(0~15 d)也有抗冻蛋白的产生,但随处理时间的延长,这种差异逐渐减少,在处理30 d时完全消失.③得到了分离纯化的抗冻蛋白,其相对分子质量约为3.6×104.

  15. Inhibition of Gas Hydrate Nucleation and Growth: Efficacy of an Antifreeze Protein from the Longhorn BeetleRhagium mordax

    DEFF Research Database (Denmark)

    Perfeldt, Christine Malmos; Chua, Pei Cheng; Daraboina, Nagu;

    2014-01-01

    showed inhibition of hydrates compared to amino acids (l-valine and l-threonine) and the protein bovine serum albumin (BSA). This indicates that proteins or amino acids do not generally inhibit hydrate formation. The promising performance of RmAFP1 as a new green kinetic hydrate inhibitor could further...

  16. Latent Ice Recrystallization Inhibition Activity in Nonantifreeze Proteins: Ca2+-Activated Plant Lectins and Cation-Activated Antimicrobial Peptides.

    Science.gov (United States)

    Mitchell, Daniel E; Gibson, Matthew I

    2015-10-12

    Organisms living in polar regions have evolved a series of antifreeze (glyco) proteins (AFGPs) to enable them to survive by modulating the structure of ice. These proteins have huge potential for use in cellular cryopreservation, ice-resistant surfaces, frozen food, and cryosurgery, but they are limited by their relatively low availability and questions regarding their mode of action. This has triggered the search for biomimetic materials capable of reproducing this function. The identification of new structures and sequences capable of inhibiting ice growth is crucial to aid our understanding of these proteins. Here, we show that plant c-type lectins, which have similar biological function to human c-type lectins (glycan recognition) but no sequence homology to AFPs, display calcium-dependent ice recrystallization inhibition (IRI) activity. This IRI activity can be switched on/off by changing the Ca2+ concentration. To show that more (nonantifreeze) proteins may exist with the potential to display IRI, a second motif was considered, amphipathicity. All known AFPs have defined hydrophobic/hydrophilic domains, rationalizing this choice. The cheap, and widely used, antimicrobial Nisin was found to have cation-dependent IRI activity, controlled by either acid or addition of histidine-binding ions such as zinc or nickel, which promote its amphipathic structure. These results demonstrate a new approach in the identification of antifreeze protein mimetic macromolecules and may help in the development of synthetic mimics of AFPs.

  17. 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.

  18. Ice-active proteins from New Zealand snow tussocks, Chionochloa macra AND C. rigida.

    Science.gov (United States)

    Wharton, D A; Selvanesan, L; Marshall, C J

    2010-01-01

    The ice active protein profile of New Zealand snow tussocks Chionochloa macra and C. rigida consisted of ice nucleation activity but no antifreeze or recrystallization inhibition activity. The ice nucleation activity was similar in the two species, despite them being collected at different altitudes and at different times. The activity is intrinsic to the plant and is associated with the surface of the leaves. Snow tussocks collect water from fog. Nucleation sites on the surface of their leaves may aid the efficiency of this process. PMID:20919453

  19. Influence of frozen dough processing conditions on the activity of antifreeze yeast in fermented dough%冷冻工艺条件对发酵面团中抗冻酵母活性的影响

    Institute of Scientific and Technical Information of China (English)

    周璐; 孙冰玉; 石彦国; 梁金钟

    2011-01-01

    研究了冷冻面团的冷冻终结温度、冻藏温度、冻藏时间对抗冻酵母活性的影响,得出最有利于抗冻酵母活性保持的冷冻工艺条件:冷冻终结温度-18℃,冻藏温度应与冷冻终结温度保持一致,贮存时间不超过30d为宜。%The effects of the end of freezing temperature,freeze preservation temperature and freeze preservation time of frozen dough on the activity of antifreeze yeast were researched.The most beneficial freezing process conditions to keep the activity of antifreeze yeast were found.The end of freezing temperature was-18℃,freeze preservation temperature should be consistent with the end of the freezing temperature.And it's better to keep the freeze preservation time less than 30 days.

  20. Structure and collective dynamics of hydrated anti-freeze protein type III from 180 K to 298 K by X-ray diffraction and inelastic X-ray scattering

    Science.gov (United States)

    Yoshida, Koji; Baron, Alfred Q. R.; Uchiyama, Hiroshi; Tsutsui, Satoshi; Yamaguchi, Toshio

    2016-04-01

    We investigated hydrated antifreeze protein type III (AFP III) powder with a hydration level h (=mass of water/mass of protein) of 0.4 in the temperature range between 180 K and 298 K using X-ray diffraction and inelastic X-ray scattering (IXS). The X-ray diffraction data showed smooth, largely monotonic changes between 180 K and 298 K without freezing water. Meanwhile, the collective dynamics observed by IXS showed a strong change in the sound velocity at 180 K, after being largely temperature independent at higher temperatures (298-220 K). We interpret this change in terms of the dynamic transition previously discussed using other probes including THz IR absorption spectroscopy and incoherent elastic and quasi-elastic neutron scattering. This finding suggests that the dynamic transition of hydrated proteins is observable on the subpicosecond time scale as well as nano- and pico-second scales, both in collective dynamics from IXS and single particle dynamics from neutron scattering. Moreover, it is most likely that the dynamic transition of hydrated AFP III is not directly correlated with its hydration structure.

  1. Preparation and application of antifreeze proteins extracted from winter wheat bran%冬小麦抗冻蛋白制备及其在汤圆中的应用研究

    Institute of Scientific and Technical Information of China (English)

    夏露; 张超; 王立; 张晖

    2009-01-01

    研究了冬小麦麸皮抗冻蛋白的制备方法及其在速冻汤圆中的应用.研究确定冬小麦麸皮抗冻蛋白的提取工艺为:pH8.0,提取时间3h,液料比5:1,该条件下小麦麸皮水溶性蛋白质提取率达到38%,其中含抗冻蛋白1.6%.抗冻蛋白粗品在汤圆中的应用实验结果显示,2.5%的蛋白添加量对汤圆的品质有明显的改善效果.%The preparation and application of antifreeze proteins (AFPs) were studied.The extraction process of AFPs from winter wheat bran was optimized as following, water/material ratio 5:1, pH 8.0, extraction time 3h.The extraction rate of soluble protein from winter wheat bran was 38%, and the AFPs content in the extraction (crude AFPs) was 1.6%.The application experiment of AFP in rice dumpling showed that the quality of rice dumpling would be improved by adding with 2.5% crude AFPs.

  2. Effect of Antifreeze Peptide Pretreatment on Ice Crystal Size, Drip Loss, Texture, and Volatile Compounds of Frozen Carrots.

    Science.gov (United States)

    Kong, Charles H Z; Hamid, Nazimah; Liu, Tingting; Sarojini, Vijayalekshmi

    2016-06-01

    Ice crystal formation is of primary concern to the frozen food industry. In this study, the effects of antifreeze peptides (AFPs) on ice crystal formation were assessed in carrot during freezing and thawing. Three synthetic analogues based on naturally occurring antifreeze peptides were used in this study. The AFPs exhibited modification of ice crystal morphology, confirming their antifreeze activity in vitro. The ability of the synthetic AFPs to minimize drip loss and preserve color, structure, texture, and volatiles of frozen carrot was evaluated using the techniques of SEM, GC-MS, and texture analysis. The results prove the potential of these AFPs to preserve the above characteristics in frozen carrot samples. PMID:27138051

  3. Cloning and sequencing of antifreeze protein gene inDaucus carota var \\%sativus\\% Hoffm Deutschl%胡萝卜var sativus Hoffm Deutschl抗冻蛋白基因的克隆及测序

    Institute of Scientific and Technical Information of China (English)

    尹明安; 崔鸿文; 樊代明; 郭立

    2001-01-01

    Antifreeze protein gene (afp) in three native carrot cultivars(Daucus carota var \\%sativus\\% Hoffm Deutschl),Wuzhong carrot in Ningxia,H uaxian carrot in Shaanxi and Hanzhong carrot in Shaanxi,was cloned by PCR (polym erase chain reaction).Wuzhong carrots afp was sequenced and its sequence w as compared with that of Daucus carota var \\%autumn\\% King from British.Ther e were 35 different bases between two varieties in 1004 sequenced nucleotides,among which there were 20 nonsense mutations and 15 sense mutations.Based on sense mutations homology was 98.5%.%以宁夏吴忠胡萝卜、陕西华县胡萝卜、陕西汉中胡萝卜3个地方品种为材料,用PCR方法克隆了中国胡萝卜var\\%sativus\\%HoffmDeutschl的抗冻蛋白基因\\%afp\\%,测定了宁夏吴忠胡萝卜\\%afp\\%的核苷酸序列,和英国胡萝卜var\\%autumn\\%King\\%afp\\%序列对比,在所测1004个核苷酸中,有35个碱基不同,其中无义突变20个,有义突变15个。按有义突变计,同源性为\\{98.5%\\}

  4. Observation on the modifying activity of the protein from Elytrzgia repens rhizome for ice crystal

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    In winter, spring and summer, the rhizome of wild Elytrzgia repens of Heilongjiang Province was selected to extract the soluble which whole protein and the apoplastic protein, and analyzed by SDS-PAGE. The result indicated that there were two specific polypeptides in two types protein from winter; their relative molecular weight were identified as 52 ku and 26 ku by analyzing software; the apoplastic protein from winter had the ability of modifing the growth of ice crystal which appeared hexagonal in shape observed with the phase-contrast photomicroscope. So the apoplastic protein from winter has the antifreeze characters and the 52 ku protein is more likely the antifreeze protein.

  5. Ice-active proteins and cryoprotectants from the New Zealand alpine cockroach, Celatoblatta quinquemaculata.

    Science.gov (United States)

    Wharton, D A; Pow, B; Kristensen, M; Ramløv, H; Marshall, C J

    2009-01-01

    Celatoblatta quinquemaculata is moderately freezing tolerant. We have investigated low and high molecular weight compounds that may be associated with its survival. Glycerol and trehalose were identified as potential cryoprotectants, with trehalose at the higher concentration. Trehalose was at its highest concentration in late autumn, during the periods sampled. Water contents declined with time and were significantly lower in late autumn than in late summer. No thermal hysteresis activity was detected in haemolymph or in extracts of the head, muscles and the fat body. Extracts of the Malpighian tubules showed an hexagonal crystal growth form, as did those of the gut tissue and gut contents. The gut tissue had high levels of thermal hysteresis (approximately 2 degrees C) and the gut contents somewhat lower levels (approximately 0.6 degrees C). Recrystallization inhibition activity mirrored that of thermal hysteresis, with activity absent in the haemolymph or fat body cells but present in the gut tissues and contents. Activity was reduced by heating and was associated with a molecule >14kDa in size. These findings suggest an antifreeze protein is involved. In fed animals, ice nucleation is likely to start in the gut. Gut cells have a much greater resistance to freezing than do fat body or Malpighian tubule cells. The antifreeze protein may enable this tissue to survive freezing stress by inhibiting recrystallization. PMID:18955061

  6. Solution conformation of C-linked antifreeze glycoprotein analogues and modulation of ice recrystallization.

    Science.gov (United States)

    Tam, Roger Y; Rowley, Christopher N; Petrov, Ivan; Zhang, Tianyi; Afagh, Nicholas A; Woo, Tom K; Ben, Robert N

    2009-11-01

    Antifreeze glycoproteins (AFGPs) are a unique class of proteins that are found in many organisms inhabiting subzero environments and ensure their survival by preventing ice growth in vivo. During the last several years, our laboratory has synthesized functional C-linked AFGP analogues (3 and 5) that possess custom-tailored antifreeze activity suitable for medical, commercial, and industrial applications. These compounds are potent inhibitors of ice recrystallization and do not exhibit thermal hysteresis. The current study explores how changes in the length of the amide-containing side chain between the carbohydrate moiety and the polypeptide backbone in 5 influences ice recrystallization inhibition (IRI) activity. Analogue 5 (n = 3, where n is the number of carbons in the side chain) was a potent inhibitor of ice recrystallization, while 4, 6, and 7 (n = 4, 2, and 1, respectively) exhibited no IRI activity. The solution conformation of the polypeptide backbone in C-linked AFGP analogues 4-7 was examined using circular dichroism (CD) spectroscopy. The results suggested that all of the analogues exhibit a random coil conformation in solution and that the dramatic increase in IRI activity observed with 5 is not due to a change in long-range solution conformation. Variable-temperature (1)H NMR studies on truncated analogues 26-28 failed to elucidate the presence of persistent intramolecular bonds between the amide in the side chain and the peptide backbone. Molecular dynamics simulations performed on these analogues also failed to show persistent intramolecular hydrogen bonds. However, the simulations did indicate that the side chain of IRI-active analogue 26 (n = 3) adopts a unique short-range solution conformation in which it is folded back onto the peptide backbone, orienting the more hydrophilic face of the carbohydrate moiety away from the bulk solvent. In contrast, the solution conformation of IRI-inactive analogues 25, 27, and 28 had fully extended side chains

  7. Characterization of a family of ice-active proteins from the Ryegrass, Lolium perenne.

    Science.gov (United States)

    Kumble, Krishnanand D; Demmer, Jerome; Fish, Steven; Hall, Claire; Corrales, Sofia; DeAth, Angela; Elton, Clare; Prestidge, Ross; Luxmanan, Selvanesan; Marshall, Craig J; Wharton, David A

    2008-12-01

    Five genes coding for ice-active proteins were identified from an expressed sequence tag database of Lolium perenne cDNA libraries. Each of the five genes were characterized by the presence of an N-terminal signal peptide, a region enriched in hydrophilic amino acids and a leucine-rich region in four of the five genes that is homologous with the receptor domain of receptor-like protein kinases of plants. The C-terminal region of all five genes contains sequence homologous with Lolium and Triticum ice-active proteins. Of the four ice-active proteins (IAP1, IAP2, IAP3 and IAP5) cloned, three could be expressed in Escherichia coli and recovered in a functional form in order to study their ice activity. All three ice-active proteins had recrystallization inhibition activity but showed no detectable antifreeze or ice nucleation activity at the concentration tested. IAP2 and IAP5 formed distinct hexagonal-shaped crystals in the nanolitre osmometer as compared to the weakly hexagonal crystals produced by IAP3. PMID:18835384

  8. Novel dimeric β-helical model of an ice nucleation protein with bridged active sites

    Directory of Open Access Journals (Sweden)

    Walker Virginia K

    2011-09-01

    Full Text Available Abstract Background Ice nucleation proteins (INPs allow water to freeze at high subzero temperatures. Due to their large size (>120 kDa, membrane association, and tendency to aggregate, an experimentally-determined tertiary structure of an INP has yet to be reported. How they function at the molecular level therefore remains unknown. Results Here we have predicted a novel β-helical fold for the INP produced by the bacterium Pseudomonas borealis. The protein uses internal serine and glutamine ladders for stabilization and is predicted to dimerize via the burying of a solvent-exposed tyrosine ladder to make an intimate hydrophobic contact along the dimerization interface. The manner in which PbINP dimerizes also allows for its multimerization, which could explain the aggregation-dependence of INP activity. Both sides of the PbINP structure have tandem arrays of amino acids that can organize waters into the ice-like clathrate structures seen on antifreeze proteins. Conclusions Dimerization dramatically increases the 'ice-active' surface area of the protein by doubling its width, increasing its length, and presenting identical ice-forming surfaces on both sides of the protein. We suggest that this allows sufficient anchored clathrate waters to align on the INP surface to nucleate freezing. As PbINP is highly similar to all known bacterial INPs, we predict its fold and mechanism of action will apply to these other INPs.

  9. Separating proteins with activated carbon.

    Science.gov (United States)

    Stone, Matthew T; Kozlov, Mikhail

    2014-07-15

    Activated carbon is applied to separate proteins based on differences in their size and effective charge. Three guidelines are suggested for the efficient separation of proteins with activated carbon. (1) Activated carbon can be used to efficiently remove smaller proteinaceous impurities from larger proteins. (2) Smaller proteinaceous impurities are most efficiently removed at a solution pH close to the impurity's isoelectric point, where they have a minimal effective charge. (3) The most efficient recovery of a small protein from activated carbon occurs at a solution pH further away from the protein's isoelectric point, where it is strongly charged. Studies measuring the binding capacities of individual polymers and proteins were used to develop these three guidelines, and they were then applied to the separation of several different protein mixtures. The ability of activated carbon to separate proteins was demonstrated to be broadly applicable with three different types of activated carbon by both static treatment and by flowing through a packed column of activated carbon. PMID:24898563

  10. Separating proteins with activated carbon.

    Science.gov (United States)

    Stone, Matthew T; Kozlov, Mikhail

    2014-07-15

    Activated carbon is applied to separate proteins based on differences in their size and effective charge. Three guidelines are suggested for the efficient separation of proteins with activated carbon. (1) Activated carbon can be used to efficiently remove smaller proteinaceous impurities from larger proteins. (2) Smaller proteinaceous impurities are most efficiently removed at a solution pH close to the impurity's isoelectric point, where they have a minimal effective charge. (3) The most efficient recovery of a small protein from activated carbon occurs at a solution pH further away from the protein's isoelectric point, where it is strongly charged. Studies measuring the binding capacities of individual polymers and proteins were used to develop these three guidelines, and they were then applied to the separation of several different protein mixtures. The ability of activated carbon to separate proteins was demonstrated to be broadly applicable with three different types of activated carbon by both static treatment and by flowing through a packed column of activated carbon.

  11. Preparation of Food-based Antifreeze Peptides and Research on the Ice Crystal Inhibition%食品源抗冻多肽的制备及冰晶抑制作用研究

    Institute of Scientific and Technical Information of China (English)

    洪晶; 汪少芸; 吴金鸿; 饶平凡

    2013-01-01

    Objective:Antifreeze protein is becoming a popular research point because it could inhibit ice crystal growth, reduce damage of cell membranes and maintain products' quality during food during storage and handling. Methods:This paper reports that gelatin peptides of a certain molecular size range with compact-packed structural domain derived from Papain hydrolysis of bovine gelatin are able to inhibit recrystallization of ice crystals in ice cream mix and show natural antifreeze activity. Results:The optimum conditions for producing antifreeze peptides were hydrolysis at pH 7.0 for 30 min at 37℃ and an Papain to gelatin ratio of 1 :10. The gelatin peptides were fractionated on size exclusion (Sephadex C-SO) and ion exchange (sulfopropyl-Sephadex C-2S) columns, and the molecular mass distribution of the antifreeze peptide fractions was determined by matrixassisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. The gelatin peptide fractions in the molecular mass range of 700~1 318 u strongly inhibited ice recrystallization in ice cream mix. Conclusion:The highly effective antifreeze peptide on ice crystal inhibition shows specific rules during cold-heat-stage cycles, the key approach is how to control hydrolysis conditions. It probably exists the surface hydropholic-complementary interaction between antifreeze peptide and ice molecules.%目的:因抗冻蛋白具有控制冰晶生长,减少细胞损伤及保持产品原有组织结构、质地和品质的特点和突出意义而成为研究的热点.方法:以食品源的食用明胶为原材料,通过控制木瓜蛋白酶的切割条件,将活性多肤切割为具有特定的肽链长度和结构组成,从而使抗冻活性得以高效实现.结果:酶切多肽抗冻活性的实现受酶/底物比、酶解时间、酶解温度等条件的影响.优化的酶解条件为:pH 7.0,酶/底物配比1:10;酶解时间30 min;酶解温度37℃.通过Sephadex G-50和Sephadex C-25色谱分离

  12. Prediction of Antifreeze Critical Strength of Infant Age Concrete

    Institute of Scientific and Technical Information of China (English)

    LIU Jun; LIU Runqing

    2008-01-01

    The rule of infant age concrete strength development under low temperature and complex affecting factors is researched. An efficient and reliable mathematical forecast model is set up to predict the infant age concrete antifreeze critical strength under low temperature at construction site. On the basis of the revision of concrete equivalent coefficient under complex influencing factors, least-squares curve-fitting method is applied to approximate the concrete strength under standard curing and the forecast formula of concrete compressive strength could be obtained under natural temperature condition by various effects. When the amounts of donble-doped are 10% fly ashes and 4% silica fumes as cement replacement, the antifreeze critical strength changes form 3.5-4.1MPa under different low temperature curing. The equivalent coefficient correction formula of concrete under low temperature affected by various factors could be obtained. The obtainede quivalent coefficient is suitable for calculating the strength which is between 10% to 40% of standard strength and the curing temperature from 5-20 ℃. The forecast value of concrete antifreeze critical strength under low temperature could be achieved by combining the concrete antifreeze critical strength value with the compressive strength forecast of infant age concrete under low temperature. Then the theory for construction quality control under low temperature is provided.

  13. 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.

  14. Effect of Anti-freezing Admixtures on Alkali-silica Reaction in Mortars

    Institute of Scientific and Technical Information of China (English)

    LIU Junzhe; LI Yushun; LV Lihua

    2005-01-01

    The influence of anti-freezing admixture on the alkali aggregate reaction in mortar was analyzed with accelerated methods. It is confirmed that the addition of sodium salt ingredients of anti-freezing admixture accelerates the alkali silica reaction to some extent, whereas calcium salt ingredient of anti-freezing admixture reduces the expansion of alkali silica reaction caused by high alkali cement. It is found that the addition of the fly ash considerably suppresses the expansion of alkali silica reaction induced by the anti-freezing admixtures.

  15. Degradation of Activated Protein Kinases by Ubiquitination

    OpenAIRE

    Lu, Zhimin; Hunter, Tony

    2009-01-01

    Protein kinases are important regulators of intracellular signal transduction pathways and play critical roles in diverse cellular functions. Once a protein kinase is activated, its activity is subsequently downregulated through a variety of mechanisms. Accumulating evidence indicates that the activation of protein kinases commonly initiates their downregulation via the ubiquitin/proteasome pathway. Failure to regulate protein kinase activity or expression levels can cause human diseases.

  16. Physiological roles of mitogen-activated-protein-kinase-activated p38-regulated/activated protein kinase

    Institute of Scientific and Technical Information of China (English)

    Sergiy; Kostenko; Gianina; Dumitriu; Kari; Jenssen; Lgreid; Ugo; Moens

    2011-01-01

    Mitogen-activated protein kinases(MAPKs)are a family of proteins that constitute signaling pathways involved in processes that control gene expression,cell division, cell survival,apoptosis,metabolism,differentiation and motility.The MAPK pathways can be divided into conventional and atypical MAPK pathways.The first group converts a signal into a cellular response through a relay of three consecutive phosphorylation events exerted by MAPK kinase kinases,MAPK kinase,and MAPK.Atypical MAPK pathways are not organized into this three-tiered cascade.MAPK that belongs to both conventional and atypical MAPK pathways can phosphorylate both non-protein kinase substrates and other protein kinases.The latter are referred to as MAPK-activated protein kinases.This review focuses on one such MAPK-activated protein kinase,MAPK-activated protein kinase 5(MK5)or p38-regulated/activated protein kinase(PRAK).This protein is highly conserved throughout the animal kingdom and seems to be the target of both conventional and atypical MAPK pathways.Recent findings on the regulation of the activity and subcellular localization,bona fide interaction partners and physiological roles of MK5/PRAK are discussed.

  17. Activity assay of membrane transport proteins

    Institute of Scientific and Technical Information of China (English)

    Hao Xie

    2008-01-01

    Membrane transport proteins are integral membrane proteins and considered as potential drug targets. Activity assay of transport proteins is essential for developing drugs to target these proteins. Major issues related to activity assessment of transport proteins include availability of transporters,transport activity of transporters, and interactions between ligands and transporters. Researchers need to consider the physiological status of proteins (bound in lipid membranes or purified), availability and specificity of substrates, and the purpose of the activity assay (screening, identifying, or comparing substrates and inhibitors) before choosing appropriate assay strategies and techniques. Transport proteins bound in vesicular membranes can be assayed for transporting substrate across membranes by means of uptake assay or entrance counterflow assay. Alternatively, transport proteins can be assayed for interactions with ligands by using techniques such as isothermal titration calorimetry, nuclear magnetic resonance spectroscopy, or surface plasmon resonance. Other methods and techniques such as fluorometry, scintillation proximity assay, electrophysiological assay, or stopped-flow assay could also be used for activity assay of transport proteins. In this paper the major strategies and techniques for activity assessment of membrane transport proteins are reviewed.

  18. Activity-Based Protein Profiling of Microbes

    Energy Technology Data Exchange (ETDEWEB)

    Sadler, Natalie C.; Wright, Aaron T.

    2015-02-01

    Activity-Based Protein Profiling (ABPP) in conjunction with multimodal characterization techniques has yielded impactful findings in microbiology, particularly in pathogen, bioenergy, drug discovery, and environmental research. Using small molecule chemical probes that react irreversibly with specific proteins or protein families in complex systems has provided insights in enzyme functions in central metabolic pathways, drug-protein interactions, and regulatory protein redox, for systems ranging from photoautotrophic cyanobacteria to mycobacteria, and combining live cell or cell extract ABPP with proteomics, molecular biology, modeling, and other techniques has greatly expanded our understanding of these systems. New opportunities for application of ABPP to microbial systems include: enhancing protein annotation, characterizing protein activities in myriad environments, and reveal signal transduction and regulatory mechanisms in microbial systems.

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

    Science.gov (United States)

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

    2014-09-01

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

  20. Antifreeze Peptides and Glycopeptides, and Their Derivatives: Potential Uses in Biotechnology

    Directory of Open Access Journals (Sweden)

    Hyun-Cheol Kim

    2013-06-01

    Full Text Available Antifreeze proteins (AFPs and glycoproteins (AFGPs, collectively called AF(GPs, constitute a diverse class of proteins found in various Arctic and Antarctic fish, as well as in amphibians, plants, and insects. These compounds possess the ability to inhibit the formation of ice and are therefore essential to the survival of many marine teleost fishes that routinely encounter sub-zero temperatures. Owing to this property, AF(GPs have potential applications in many areas such as storage of cells or tissues at low temperature, ice slurries for refrigeration systems, and food storage. In contrast to AFGPs, which are composed of repeated tripeptide units (Ala-Ala-Thrn with minor sequence variations, AFPs possess very different primary, secondary, and tertiary structures. The isolation and purification of AFGPs is laborious, costly, and often results in mixtures, making characterization difficult. Recent structural investigations into the mechanism by which linear and cyclic AFGPs inhibit ice crystallization have led to significant progress toward the synthesis and assessment of several synthetic mimics of AFGPs. This review article will summarize synthetic AFGP mimics as well as current challenges in designing compounds capable of mimicking AFGPs. It will also cover our recent efforts in exploring whether peptoid mimics can serve as structural and functional mimics of native AFGPs.

  1. Hydrogen peroxide activates activator protein-1 and mitogen-activated protein kinases in pancreatic stellate cells.

    Science.gov (United States)

    Kikuta, Kazuhiro; Masamune, Atsushi; Satoh, Masahiro; Suzuki, Noriaki; Satoh, Kennichi; Shimosegawa, Tooru

    2006-10-01

    Activated pancreatic stellate cells (PSCs) are implicated in the pathogenesis of pancreatic inflammation and fibrosis, where oxidative stress is thought to play a key role. Reactive oxygen species such as hydrogen peroxide (H(2)O(2)) may act as a second messenger to mediate the actions of growth factors and cytokines. But the role of reactive oxygen species in the activation and regulation of cell functions in PSCs remains largely unknown. We here examined the effects of H(2)O(2) on the activation of signal transduction pathways and cell functions in PSCs. PSCs were isolated from the pancreas of male Wistar rats, and used in their culture-activated, myofibroblast-like phenotype unless otherwise stated. Activation of transcription factors was examined by electrophoretic mobility shift assay and luciferase assay. Activation of mitogen-activated protein (MAP) kinases was assessed by Western blotting using anti-phosphospecific antibodies. The effects of H(2)O(2) on proliferation, alpha(1)(I)procollagen gene expression, and monocyte chemoattractant protein-1 production were evaluated. The effect of H(2)O(2) on the transformation of freshly isolated PSCs in culture was also assessed. H(2)O(2) at non-cytotoxic concentrations (up to 100 microM) induced oxidative stress in PSCs. H(2)O(2) activated activator protein-1, but not nuclear factor kappaB. In addition, H(2)O(2) activated three classes of MAP kinases: extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 MAP kinase. H(2)O(2) induced alpha(1)(I)procollagen gene expression but did not induce proliferation or monocyte chemoattractant protein-1 production. H(2)O(2) did not initiate the transformation of freshly isolated PSCs to myofibroblast-like phenotype. Specific activation of these signal transduction pathways and collagen gene expression by H(2)O(2) may play a role in the pathogenesis of pancreatic fibrosis.

  2. 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. PMID:26651017

  3. Computational Introduction of Catalytic Activity into Proteins.

    Science.gov (United States)

    Bertolani, Steve J; Carlin, Dylan Alexander; Siegel, Justin B

    2016-01-01

    Recently, there have been several successful cases of introducing catalytic activity into proteins. One method that has been used successfully to achieve this is the theozyme placement and enzyme design algorithms implemented in Rosetta Molecular Modeling Suite. Here, we illustrate how to use this software to recapitulate the placement of catalytic residues and ligand into a protein using a theozyme, protein scaffold, and catalytic constraints as input. PMID:27094294

  4. 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. PMID:25529728

  5. Synaptic vesicle proteins and active zone plasticity

    Directory of Open Access Journals (Sweden)

    Robert J Kittel

    2016-04-01

    Full Text Available Neurotransmitter is released from synaptic vesicles at the highly specialized presynaptic active zone. The complex molecular architecture of active zones mediates the speed, precision and plasticity of synaptic transmission. Importantly, structural and functional properties of active zones vary significantly, even for a given connection. Thus, there appear to be distinct active zone states, which fundamentally influence neuronal communication by controlling the positioning and release of synaptic vesicles. Vice versa, recent evidence has revealed that synaptic vesicle components also modulate organizational states of the active zone.The protein-rich cytomatrix at the active zone (CAZ provides a structural platform for molecular interactions guiding vesicle exocytosis. Studies in Drosophila have now demonstrated that the vesicle proteins Synaptotagmin-1 (Syt1 and Rab3 also regulate glutamate release by shaping differentiation of the CAZ ultrastructure. We review these unexpected findings and discuss mechanistic interpretations of the reciprocal relationship between synaptic vesicles and active zone states, which has heretofore received little attention.

  6. DNA-based control of protein activity.

    Science.gov (United States)

    Engelen, W; Janssen, B M G; Merkx, M

    2016-03-01

    DNA has emerged as a highly versatile construction material for nanometer-sized structures and sophisticated molecular machines and circuits. The successful application of nucleic acid based systems greatly relies on their ability to autonomously sense and act on their environment. In this feature article, the development of DNA-based strategies to dynamically control protein activity via oligonucleotide triggers is discussed. Depending on the desired application, protein activity can be controlled by directly conjugating them to an oligonucleotide handle, or expressing them as a fusion protein with DNA binding motifs. To control proteins without modifying them chemically or genetically, multivalent ligands and aptamers that reversibly inhibit their function provide valuable tools to regulate proteins in a noncovalent manner. The goal of this feature article is to give an overview of strategies developed to control protein activity via oligonucleotide-based triggers, as well as hurdles yet to be taken to obtain fully autonomous systems that interrogate, process and act on their environments by means of DNA-based protein control. PMID:26812623

  7. 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

    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...... inducers. Binding of HCV core to MAPKAPK3 was confirmed by in vitro pulldown assay and further verified by coimmunoprecipitation assay. HCV core protein interacted with MAPKAPK3 through amino acid residues 41 to 75 of core and the N-terminal half of kinase domain of MAPKAPK3. In addition, both RNA...

  8. Activated protein C modulates the proinflammatory activity of dendritic cells

    Directory of Open Access Journals (Sweden)

    Matsumoto T

    2015-05-01

    Full Text Available Takahiro Matsumoto,1,2* Yuki Matsushima,1* Masaaki Toda,1 Ziaurahman Roeen,1 Corina N D'Alessandro-Gabazza,1,5 Josephine A Hinneh,1 Etsuko Harada,1,3 Taro Yasuma,4 Yutaka Yano,4 Masahito Urawa,1,5 Tetsu Kobayashi,5 Osamu Taguchi,5 Esteban C Gabazza1 1Department of Immunology, Mie University Graduate School of Medicine, Tsu, Mie Prefecture, 2BONAC Corporation, BIO Factory 4F, Fukuoka, 3Iwade Research Institute of Mycology, 4Department of Endocrinology, Diabetes and Metabolism, 5Department of Pulmonary and Critical Care Medicine, Mie University Graduate School of Medicine, Tsu, Mie Prefecture, Japan *These authors contributed equally to this work Background: Previous studies have demonstrated the beneficial activity of activated protein C in allergic diseases including bronchial asthma and rhinitis. However, the exact mechanism of action of activated protein C in allergies is unclear. In this study, we hypothesized that pharmacological doses of activated protein C can modulate allergic inflammation by inhibiting dendritic cells. Materials and methods: Dendritic cells were prepared using murine bone marrow progenitor cells and human peripheral monocytes. Bronchial asthma was induced in mice that received intratracheal instillation of ovalbumin-pulsed dendritic cells. Results: Activated protein C significantly increased the differentiation of tolerogenic plasmacytoid dendritic cells and the secretion of type I interferons, but it significantly reduced lipopolysaccharide-mediated maturation and the secretion of inflammatory cytokines in myeloid dendritic cells. Activated protein C also inhibited maturation and the secretion of inflammatory cytokines in monocyte-derived dendritic cells. Activated protein C-treated dendritic cells were less effective when differentiating naïve CD4 T-cells from Th1 or Th2 cells, and the cellular effect of activated protein C was mediated by its receptors. Mice that received adoptive transfer of activated protein C

  9. Effect of antifreeze glycoprotein 8 supplementation during vitrification on the developmental competence of bovine oocytes.

    Science.gov (United States)

    Liang, Shuang; Yuan, Bao; Kwon, Jeong-Woo; Ahn, Mija; Cui, Xiang-Shun; Bang, Jeong Kyu; Kim, Nam-Hyung

    2016-07-15

    The purpose of this study was to investigate the effect of antifreeze glycoprotein 8 (AFGP8) supplementation during vitrification on the survival, fertilization, and embryonic development of bovine oocytes and the underlying molecular mechanism(s). Survival, fertilization, early embryonic development, apoptosis, DNA double-strand breaks, reactive oxygen species levels, meiotic cytoskeleton assembly, chromosome alignment, and energy status of mitochondria were measured in the present experiments. Compared with that in the nonsupplemented group; survival, monospermy, blastocyst formation rates, and blastomere counts were significantly higher in the AFGP8-supplemented animals. Oocytes of the latter group also presented fewer double-strand breaks and lower cathepsin B and caspase activities. Rates of normal spindle organization and chromosome alignment, actin filament impairment, and mitochondrial distribution were significantly higher in the AFGP8-supplemented group. In addition, intracellular reactive oxygen species levels significantly decreased in the AFGP8-supplemented groups, maintaining a higher ΔΨm than that in the nonsupplemented group. Taken together, these results indicated that supplementation with AFGP8 during vitrification has a protective effect on bovine oocytes against chilling injury. PMID:26948296

  10. 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)

  11. Nonequilibrium antifreeze peptides and the recrystallization of ice.

    Science.gov (United States)

    Knight, C A; Wen, D; Laursen, R A

    1995-02-01

    Evidence is presented that the nonequilibrium antifreeze peptide (AFP) from winter flounder has a special ability to inhibit recrystallization in ice only when an appreciable amount of liquid is present, as is the case when the system contains salts and the temperature is not too low. In this circumstance the AFP binds to the ice surface at the ice-solution interfaces in grain boundaries, preventing migration of the solution and effectively immobilizing the boundaries. In the absence of liquid, recrystallization inhibition appears to be a common property of many peptides. This is consistent with the view that the special effects of AFPs require a structural fit onto ice, and therefore require the AFP molecules to have the mobility to achieve that fit. Since the concentration of salt required to induce the special recrystallization inhibition effects of AFPs is lower (recrystallization. The proposition that mobility is needed for AFP molecules to produce their special influence upon ice growth argues against any special effects of AFPs in devitrification.

  12. Heterologous expression of type I antifreeze peptide GS-5 in baker's yeast increases freeze tolerance and provides enhanced gas production in frozen dough.

    Science.gov (United States)

    Panadero, Joaquin; Randez-Gil, Francisca; Prieto, Jose Antonio

    2005-12-28

    The demand for frozen-dough products has increased notably in the baking industry. Nowadays, no appropriate industrial baker's yeast with optimal gassing capacity in frozen dough is, however, available, and it is unlikely that classical breeding programs could provide significant improvements of this trait. Antifreeze proteins, found in diverse organisms, display the ability to inhibit the growth of ice, allowing them to survive at temperatures below 0 degrees C. In this study a recombinant antifreeze peptide GS-5 was expressed from the polar fish grubby sculpin (Myoxocephalus aenaeus) in laboratory and industrial baker's yeast strains of Saccharomyces cerevisiae. Production of the recombinant protein increased freezing tolerance in both strains tested. Furthermore, expression of the GS-5 encoding gene enhanced notably the gassing rate and total gas production in frozen and frozen sweet doughs. These effects are unlikely to be due to reduced osmotic damage during freezing/thawing, because recombinant cells showed growth behavior similar to that of the parent under hypermosmotic stress conditions. PMID:16366681

  13. Activation and activities of the p53 tumour suppressor protein

    OpenAIRE

    Bálint, É; Vousden, K H

    2001-01-01

    The p53 tumour suppressor protein inhibits malignant progression by mediating cell cycle arrest, apoptosis or repair following cellular stress. One of the major regulators of p53 function is the MDM2 protein, and multiple forms of cellular stress activate p53 by inhibiting the MDM2-mediated degradation of p53. Mutations in p53, or disruption of the pathways that allow activation of p53, seem to be a general feature of all cancers. Here we review recent advances in our understanding of the pat...

  14. Total Cellular RNA Modulates Protein Activity.

    Science.gov (United States)

    Majumder, Subhabrata; DeMott, Christopher M; Reverdatto, Sergey; Burz, David S; Shekhtman, Alexander

    2016-08-16

    RNA constitutes up to 20% of a cell's dry weight, corresponding to ∼20 mg/mL. This high concentration of RNA facilitates low-affinity protein-RNA quinary interactions, which may play an important role in facilitating and regulating biological processes. In the yeast Pichia pastoris, the level of ubiquitin-RNA colocalization increases when cells are grown in the presence of dextrose and methanol instead of methanol as the sole carbon source. Total RNA isolated from cells grown in methanol increases β-galactosidase activity relative to that seen with RNA isolated from cells grown in the presence of dextrose and methanol. Because the total cellular RNA content changes with growth medium, protein-RNA quinary interactions can alter in-cell protein biochemistry and may play an important role in cell adaptation, critical to many physiological and pathological states. PMID:27456029

  15. Raman optical activity of proteins and glycoproteins

    Energy Technology Data Exchange (ETDEWEB)

    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 {beta}-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

  16. Mitogen-activated protein kinases in atherosclerosis

    Directory of Open Access Journals (Sweden)

    Dorota Bryk

    2014-01-01

    Full Text Available Intracellular signalling cascades, in which MAPK (mitogen-activated protein kinases intermediate, are responsible for a biological response of a cell to an external stimulus. MAP kinases, which include ERK1/2 (extracellular signalling-regulated kinase, JNK (c-Jun N-terminal kinase and p 38 MAPK, regulate the activity of many proteins, enzymes and transcription factors and thus have a wide spectrum of biological effects. Many basic scientific studies have defined numerous details of their pathway organization and activation. There are also more and more studies suggesting that individual MAP kinases probably play an important role in the pathogenesis of atherosclerosis. They may mediate inflammatory processes, endothelial cell activation, monocyte/macrophage recruitment and activation, smooth muscle cell proliferation and T-lymphocyte differentiation, all of which represent crucial mechanisms involved in pathogenesis of atherosclerosis. The specific inhibition of an activity of the respective MAP kinases may prove a new therapeutic approach to attenuate atherosclerotic plaque formation in the future. In this paper, we review the current state of knowledge concerning MAP kinase-dependent cellular and molecular mechanisms underlying atherosclerosis.

  17. Smelt was the likely beneficiary of an antifreeze gene laterally transferred between fishes

    Directory of Open Access Journals (Sweden)

    Graham Laurie A

    2012-09-01

    Full Text Available Abstract Background Type II antifreeze protein (AFP from the rainbow smelt, Osmerus mordax, is a calcium-dependent C-type lectin homolog, similar to the AFPs from herring and sea raven. While C-type lectins are ubiquitous, type II AFPs are only found in a few species in three widely separated branches of teleost fishes. Furthermore, several other non-homologous AFPs are found in intervening species. We have previously postulated that this sporadic distribution has resulted from lateral gene transfer. The alternative hypothesis, that the AFP evolved from a lectin present in a shared ancestor and that this gene was lost in most species, is not favored because both the exon and intron sequences are highly conserved. Results Here we have sequenced and annotated a 160 kb smelt BAC clone containing a centrally-located AFP gene along with 14 other genes. Quantitative PCR indicates that there is but a single copy of this gene within the smelt genome, which is atypical for fish AFP genes. The corresponding syntenic region has been identified and searched in a number of other species and found to be devoid of lectin or AFP sequences. Unlike the introns of the AFP gene, the intronic sequences of the flanking genes are not conserved between species. As well, the rate and pattern of mutation in the AFP gene are radically different from those seen in other smelt and herring genes. Conclusions These results provide stand-alone support for an example of lateral gene transfer between vertebrate species. They should further inform the debate about genetically modified organisms by showing that gene transfer between ‘higher’ eukaryotes can occur naturally. Analysis of the syntenic regions from several fishes strongly suggests that the smelt acquired the AFP gene from the herring.

  18. Synaptic Vesicle Proteins and Active Zone Plasticity.

    Science.gov (United States)

    Kittel, Robert J; Heckmann, Manfred

    2016-01-01

    Neurotransmitter is released from synaptic vesicles at the highly specialized presynaptic active zone (AZ). The complex molecular architecture of AZs mediates the speed, precision and plasticity of synaptic transmission. Importantly, structural and functional properties of AZs vary significantly, even for a given connection. Thus, there appear to be distinct AZ states, which fundamentally influence neuronal communication by controlling the positioning and release of synaptic vesicles. Vice versa, recent evidence has revealed that synaptic vesicle components also modulate organizational states of the AZ. The protein-rich cytomatrix at the active zone (CAZ) provides a structural platform for molecular interactions guiding vesicle exocytosis. Studies in Drosophila have now demonstrated that the vesicle proteins Synaptotagmin-1 (Syt1) and Rab3 also regulate glutamate release by shaping differentiation of the CAZ ultrastructure. We review these unexpected findings and discuss mechanistic interpretations of the reciprocal relationship between synaptic vesicles and AZ states, which has heretofore received little attention.

  19. [Protein kinase C activation induces platelet apoptosis].

    Science.gov (United States)

    Zhao, Li-Li; Chen, Meng-Xing; Zhang, Ming-Yi; Dai, Ke-Sheng

    2013-10-01

    Platelet apoptosis elucidated by either physical or chemical compound or platelet storage occurs wildly, which might play important roles in controlling the numbers and functions of circulated platelets, or in the development of some platelet-related diseases. However, up to now, a little is known about the regulatory mechanisms of platelet apoptosis. Protein kinase C (PKC) is highly expressed in platelets and plays central roles in regulating platelet functions. Although there is evidence indicating that PKC is involved in the regulation of apoptosis of nucleated cells, it is still unclear whether PKC plays a role in platelet apoptosis. The aim of this study was to investigate the role of PKC in platelet apoptosis. The effects of PKC on mitochondrial membrane potential (ΔΨm), phosphatidylserine (PS) exposure, and caspase-3 activation of platelets were analyzed by flow cytometry and Western blot. The results showed that the ΔΨm depolarization in platelets was induced by PKC activator in time-dependent manner, and the caspase-3 activation in platelets was induced by PKC in concentration-dependent manner. However, the platelets incubated with PKC inhibitor did not results in ΔΨm depolarization and PS exposure. It is concluded that the PKC activation induces platelet apoptosis through influencing the mitochondrial functions and activating caspase 3. The finds suggest a novel mechanism for PKC in regulating platelet numbers and functions, which has important pathophysiological implications for thrombosis and hemostasis.

  20. Pyrrolopyridine inhibitors of mitogen-activated protein kinase-activated protein kinase 2 (MK-2).

    Science.gov (United States)

    Anderson, David R; Meyers, Marvin J; Vernier, William F; Mahoney, Matthew W; Kurumbail, Ravi G; Caspers, Nicole; Poda, Gennadiy I; Schindler, John F; Reitz, David B; Mourey, Robert J

    2007-05-31

    A new class of potent kinase inhibitors selective for mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP-K2 or MK-2) for the treatment of rheumatoid arthritis has been prepared and evaluated. These inhibitors have IC50 values as low as 10 nM against the target and have good selectivity profiles against a number of kinases including CDK2, ERK, JNK, and p38. These MK-2 inhibitors have been shown to suppress TNFalpha production in U397 cells and to be efficacious in an acute inflammation model. The structure-activity relationships of this series, the selectivity for MK-2 and their activity in both in vitro and in vivo models are discussed. The observed selectivity is discussed with the aid of an MK-2/inhibitor crystal structure.

  1. 复合防冻剂的防冻机理及施工要求%On the Antifreeze Mechanism and the Construction Requirements of Antifreeze Compound

    Institute of Scientific and Technical Information of China (English)

    王超

    2011-01-01

    Concrete is the most widely used material in construction,.The durability of concrete which is strongly influenced by its frost resistance has raised great attention.Therefore,improving and developing concrete of good frost resistance has very significant economic and social benefits.Using concrete with antifreeze compound can achieve convenient construction,can conserve energy,can save concrete cost and improve the quality of concrete constructed in winter.It can not only reduce the liquid freezing point of concrete,but can promote freezing and save water as well.Therefore,understanding the antifreeze mechanism and the construction requirements of antifreeze compound can achieve better technical economic benefit.%在建筑工程中,混凝土是使用最广泛的一种材料,混凝土的耐久性受到人们的普遍关注,其中冻害性是影响混凝土耐久性的一个最重要的因素。改善并开发抗冻性能良好的混凝土具有非常重大的经济效益与社会效益。而采用掺复合防冻剂混凝土具有施工简便、节能、节约混凝土冬施费用,提高混凝土冬施质量等优点,它不仅能够降低混凝土中液相冰点,同时还具有促凝、早强和减水作用。

  2. 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.

  3. Observation of microtubule-based motor protein activity.

    Science.gov (United States)

    Sloboda, Roger D

    2015-02-01

    It is possible to detect the presence of motor proteins that have the ability to translocate particles along microtubules. The two procedures described here were developed to detect microtubule-dependent motor protein activity in cell lysates or of purified proteins. In the first procedure, latex beads bound to the putative motor protein are assayed for their ability to translocate along microtubules in an ATP-dependent fashion. If motor protein activity is present, it will bind to the beads and translocate them unidirectionally along the microtubules. In the second procedure, motor proteins induce microtubule gliding over a glass coverslip surface that is coated with active motor protein. Because the mass of a microtubule is negligible compared to that of a coverslip or slide, the microtubule glides over the glass surface when the surface is coated with active motor protein. Also included here are descriptions of assays designed to determine the directionality of movement of microtubule-based motor proteins. PMID:25646501

  4. Evaluation of anti-freeze viscosity modifier for potential external tank applications

    Science.gov (United States)

    Lynn, R. O. L.

    1981-01-01

    Viscosity modifiers and gelling agents were evaluated in combination with ethylene glycol and dimethyl sulfoxide water eutectics. Pectin and agarose are found to gel these eutectics effectively in low concentration, but the anti-freeze protection afforded by these compositions is found to be marginal in simulations of the intended applications. Oxygen vent shutters and vertical metallic surfaces were simulated, with water supplied as a spray, dropwise, and by condensation from the air.

  5. Reconstitution of Membrane Proteins into Model Membranes: Seeking Better Ways to Retain Protein Activities

    OpenAIRE

    Trevor Lithgow; Lisa Martin; Hsin-Hui Shen

    2013-01-01

    The function of any given biological membrane is determined largely by the specific set of integral membrane proteins embedded in it, and the peripheral membrane proteins attached to the membrane surface. The activity of these proteins, in turn, can be modulated by the phospholipid composition of the membrane. The reconstitution of membrane proteins into a model membrane allows investigation of individual features and activities of a given cell membrane component. However, the activity of mem...

  6. Protein C activity in dogs envenomed by Vipera palaestinae.

    Science.gov (United States)

    Hadar, Gil; Kelmer, Efrat; Segev, Gilad; Bruchim, Yaron; Aroch, Itamar

    2014-09-01

    Vipera palaestinae is responsible for most envenomations in humans and domestic animal in Israel. Its venom has pro- and anticoagulant properties. Protein C is a major natural anticoagulant, preventing excess clotting and thrombosis. This study investigated protein C activity and its prognostic value, as well as several other hemostatic analytes in dogs (Canis familiaris) accidently envenomed by V. palaestinae. Protein C activity was compared between envenomed dogs and 33 healthy control dogs. Mean protein C was lower in dogs envenomed by V. palaestinae compared to controls (12.9% vs. 22.9%, respectively; P Dogs diagnosed with consumptive coagulopathy (14%) tended to have lower protein C activity compared to others; however, their mortality did differ from that of other dogs. This is the first study assessing protein C activity in V. palaestinae victims. Decreased protein C activity in such dogs may play a role in formation of thrombosis and hemostatic derangement as well as inflammation in V. palaestinae envenomations.

  7. Human cytomegalovirus IE2 protein interacts with transcription activating factors

    Institute of Scientific and Technical Information of China (English)

    徐进平; 叶林柏

    2002-01-01

    The human cytomegalovirus (HCMV) IE86 Cdna was cloned into Pgex-2T and fusion protein GST-IE86 was expressed in E. Coli. SDS-PAGE and Western blot assay indicated that fusion protein GST-IE86 with molecular weight of 92 ku is soluble in the supernatant of cell lysate. Protein GST and fusion protein GST-IE86 were purified by affinity chromatography. The technology of co-separation and specific affinity chromatography was used to study the interactions of HCMV IE86 protein with some transcriptional regulatory proteins and transcriptional factors. The results indicated that IE86 interacts separately with transcriptional factor TFIIB and promoter DNA binding transcription trans-activating factors SP1, AP1 and AP2 to form a heterogenous protein complex. These transcriptional trans-activating factors, transcriptional factor and IE86 protein were adsorbed and retained in the affinity chromatography simultaneously. But IE86 protein could not interact with NF-Кb, suggesting that the function of IE86 protein that can interact with transcriptional factor and transcriptional trans-activating factors has no relevance to protein glycosylation. IE86 protein probably has two domains responsible for binding transcriptional trans-activating regulatory proteins and transcriptional factors respectively, thus activating the transcription of many genes. The interactions accelerated the assembly of the transcriptional initiation complexes.

  8. Activated protein C to heal pressure ulcers.

    Science.gov (United States)

    Wijewardena, Aruna; Lajevardi, Sepehr S; Vandervord, Elle; Vandervord, John; Lang, Thomas C; Fulcher, Gregory; Jackson, Christopher J

    2016-10-01

    Pressure ulcers present a major clinical challenge, are physically debilitating and place the patient at risk of serious comorbidities such as septic shock. Recombinant human activated protein C (APC) is an anticoagulant with anti-inflammatory, cytoprotective and angiogenic effects that promote rapid wound healing. Topical negative pressure wound therapy (TNP) has become widely used as a treatment modality in wounds although its efficacy has not been proven through randomised controlled trials. The aim of this study was to determine the preliminary efficacy and safety of treatment with APC for severe chronic pressure sores with and without TNP. This case presentation describes the history, management and outcome of two patients each with a severe chronic non-healing pressure ulcer that had failed to respond to conventional therapy. TNP was added to conservative management of both ulcers with no improvement seen. Then local application of small doses of APC was added to TNP and with conservative management, resulted in significant clinical improvement and rapid healing of both ulcers, displaying rapid growth of vascular granulation tissue with subsequent epithelialisation. Patients tolerated the treatment well and improvements suggested by long-term follow-up were provided. Randomised placebo-controlled double blind trials are needed to quantify the efficacy, safety, cost-effectiveness, optimal dose and quality of life changes seen from treatment with APC.

  9. 4-hydroxy-2, 3-nonenal activates activator protein-1 and mitogen-activated protein kinases in rat pancreatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    Kazuhiro Kikuta; Atsushi Masamune; Masahiro Satoh; Noriaki Suzuki; Tooru Shimosegawa

    2004-01-01

    AIM: Activated pancreatic stellate cells (PSCs) are implicated in the pathogenesis of pancreatic inflammation and fibrosis,where oxidative stress is thought to play a key role. 4-hydroxy2,3-nonenal (HNE) is generated endogenously during the process of lipid peroxidation, and has been accepted as a mediator of oxidative stress. The aim of this study was to clarify the effects of HNE on the activation of signal transduction pathways and cellular functions in PSCs.METHODS: PSCs were isolated from the pancreas of male Wistar rats after perfusion with collagenase P, and used in their culture-activated, myofibroblast-like phenotype unless otherwise stated. PSCs were treated with physiologically relevant and non-cytotoxic concentrations (up to 5 μmol/L)of HNE. Activation of transcription factors was examined by electrophoretic mobility shift assay and luciferase assay.Activation of mitogen-activated protein (MAP) kinases was assessed by Western blotting using anti-phosphospecific antibodies. Cell proliferation was assessed by measuring the incorporation of 5-bromo-2'-deoxyuridine. Production of type Ⅰ collagen and monocyte chemoattractant protein-1was determined by enzyme-linked immunosorbent assay.The effect of HNE on the transformation of freshly isolated PSCs in culture was also assessed.RESULTS: HNE activated activator protein-1, but not nuclear factor κB. In addition, HNE activated three classes of MAP kinases: extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 MAP kinase. HNE increased type Ⅰ collagen production through the activation of p38 MAP kinase and c-Jun N-terminal kinase. HNE did not alter the proliferation,or monocyte chemoattractant protein-1 production. HNE did not initiate the transformation of freshly isolated PSCs to myofibroblast-like phenotype.CONCLUSION: Specific activation of these signal transduction pathways and altered cell functions such as collagen production by HNE may play a role in the pathogenesis of pancreatic

  10. Heat dissipation guides activation in signaling proteins

    OpenAIRE

    Weber, Jeffrey K.; Shukla, Diwakar; Pande, Vijay S.

    2015-01-01

    As with their macroscopic counterparts, the moving parts of nanoscale protein machines grow hot while in operation. A portion of the energy biomolecules harness to perform meaningful work is always dissipated as heat into the surroundings. Here, we feature a methodology by which dominant dissipative trajectories can be extracted from detailed models of protein dynamics. In two important classes of signaling proteins [kinases and G-protein–coupled receptors (GPCRs)], we find that the regions o...

  11. 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.

  12. G protein activation by G protein coupled receptors: ternary complex formation or catalyzed reaction?

    Science.gov (United States)

    Roberts, David J; Waelbroeck, Magali

    2004-09-01

    G protein coupled receptors catalyze the GDP/GTP exchange on G proteins, thereby activating them. The ternary complex model, designed to describe agonist binding in the absence of GTP, is often extended to G protein activation. This is logically unsatisfactory as the ternary complex does not accumulate when G proteins are activated by GTP. Extended models taking into account nucleotide binding exist, but fail to explain catalytic G protein activation. This review puts forward an enzymatic model of G protein activation and compares its predictions with the ternary complex model and with observed receptor phenomenon. This alternative model does not merely provide a new set of formulae but leads to a new philosophical outlook and more readily accommodates experimental observations. The ternary complex model implies that, HRG being responsible for efficient G protein activation, it should be as stable as possible. In contrast, the enzyme model suggests that although a limited stabilization of HRG facilitates GDP release, HRG should not be "too stable" as this might trap the G protein in an inactive state and actually hinder G protein activation. The two models also differ completely in the definition of the receptor "active state": the ternary complex model implies that the active state corresponds to a single active receptor conformation (HRG); in contrast, the catalytic model predicts that the active receptor state is mobile, switching smoothly through various conformations with high and low affinities for agonists (HR, HRG, HRGGDP, HRGGTP, etc.).

  13. Serum paraoxonase activity and protein thiols in patients with hyperlipidemia

    Institute of Scientific and Technical Information of China (English)

    Mungli Prakash; Jeevan K Shetty; Sudeshna Tripathy; Pannuri Vikram; Manish Verma

    2009-01-01

    Objective: In the present study we evaluated the paraoxonase activity and protein thiols level in south Indian population with newly diagnosed hyperlipidemia. Methods: The study was conducted on 55 newly diagnosed hyperlipidemic pa-tients and 57 healthy controls. Serum paraoxonase activity and protein thiols were estimated by spectrophotometeric method and lipid profile by enzymatic kinetic assay method. Results: Serum paraoxonase activity, protein thiols and high density lipoprotein levels were low and total cholesterol, triglycerides and low density lipoprutein levels were high in patients with hyperlipidemia compared to healthy controls ( P < 0.01 ). Serum paranxonase activity correlated positively with protein thiols and high density lipoprotein (P<0.01). Conclusion: Decreased paraoxonase activity and protein thiols were found in patients with hyperlipi-demia. This may indicate the susceptibility of this population to accelerated atherogenesis and protein oxidation.

  14. Sulfur activation-related extracellular proteins of Acidithiobacillus ferrooxidans

    Institute of Scientific and Technical Information of China (English)

    ZHANG Cheng-gui; ZHANG Rui-yong; XIA Jin-lan; ZHANG Qian; NIE Zhen-yuan

    2008-01-01

    The fractions of the extracellular proteins of Acidithiobacillus ferrooxidans grown on two different energy substrates,elemental sulfur and ferrous sulfate,were selectively prepared with hot water treatment and distinctly shown by two-dimensional gel electrophoresis.Some protein spots with apparently higher abundance in sulfur energy substrate than in ferrous sulfate energy substrate were identified by using MALDI-TOF/TOF.Based on peptide mass fingerprints and bioinformatical analysis,the extracellular proteins were classified according to their functions as conjugal transfer protein,pilin,vacJ lipoprotein,polysaccharide deacetylase family protein,Ser/Thr protein phosphatase family protein and hypothetical proteins.Several extracellular proteins were found abundant in thiol groups and with CXXC functional motif,these proteins may be directly involved in the sulfur activation by use of their thiol group (Pr-SH) to bond the elemental sulfur.

  15. Active Nuclear Import of Membrane Proteins Revisited

    NARCIS (Netherlands)

    Laba, Justyna K; Steen, Anton; Popken, Petra; Chernova, Alina; Poolman, Bert; Veenhoff, Liesbeth M

    2015-01-01

    It is poorly understood how membrane proteins destined for the inner nuclear membrane pass the crowded environment of the Nuclear Pore Complex (NPC). For the Saccharomyces cerevisiae proteins Src1/Heh1 and Heh2, a transport mechanism was proposed where the transmembrane domains diffuse through the m

  16. Antioxidant activity of whey protein hydrolysates in milk beverage system

    OpenAIRE

    Mann, Bimlesh; Kumari, Anuradha; Kumar, Rajesh; Sharma, Rajan; Prajapati, Kishore; Mahboob, Shaik; Athira, S.

    2014-01-01

    The aim of the present study was to evaluate the antioxidant activity of flavoured milk enriched with antioxidative whey protein hydrolysates (WPHs) by radical scavenging method. Whey protein concentrate (WPC) was hydrolyzed by using three commercial proteases; flavouzyme, alcalase and corolase PP and these WPHs were analyzed for degree of hydrolysis and antioxidant activity. The antioxidant activities of these WPHs were evaluated using ABTS method. Trolox equivalent antioxidant activity of a...

  17. Antifreeze effect and mechanism of sodium poly-γ-glutamate on backers' yeast cells%γ-聚谷氨酸钠对面包酵母的抗冻作用及其机理

    Institute of Scientific and Technical Information of China (English)

    时晓剑; 缪冶炼; 卫昊; 徐虹; 王冀宁

    2012-01-01

    .27 ℃/rain, a storage temperature at -30 ℃and storage time at 6 d, the survival ratio of yeast cells was increased from 36.5% to 67.5% by adding 1% of Na-y-PGA. Na-y-PGA, glucose and glutamic acid had an antifreeze activity of 2.48, 2.03 and 1.42 respectively. The survival ratio of yeast cells (η, %) increased linearly with increasing antifreeze activity (Aa, -). The high antifreeze activity of Na-y-PGA was resulted from its high dissociation degree and the high capability of Na+ to combine water molecules.

  18. Purification, renaturation, and reconstituted protein kinase activity of the Sendai virus large (L) protein: L protein phosphorylates the NP and P proteins in vitro.

    OpenAIRE

    Einberger, H; Mertz, R; Hofschneider, P H; Neubert, W J

    1990-01-01

    Sodium dodecyl sulfate-solubilized Sendai virus large (L) protein was highly purified by a one-step procedure, using hydroxylapatite column chromatography. Monoclonal antibodies addressed to the carboxyl-terminal amino acid sequence of the L protein were used for monitoring L protein during purification. By removing sodium dodecyl sulfate from purified L protein, a protein kinase activity was successfully renatured. P and NP proteins served as its substrates. After immunoprecipitation with an...

  19. 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.

  20. Protein determination in seeds by proton activation

    Science.gov (United States)

    Morales, J. R.; Dinator, M. I.; Cerda, P.

    1989-04-01

    A proton beam of 6.6 MeV has been used to produce 11C and 13N in Araucaria Araucana seeds. Their positron decay allows determination of the N/C ratio. In seeds the nitrogen content is associated to proteins while carbon is spread in the organic material. Samples were irradiated for about 10 min with a beam intensity of 5 nA on areas of 1 mm 2. Slices of the seed were radially explored, showing a larger concentration of protein in the center.

  1. Protein determination in seeds by proton activation

    Energy Technology Data Exchange (ETDEWEB)

    Morales, J.R.; Dinator, M.I. (Chile Univ., Santiago (Chile). Lab. de Fisica); Cerda, P. (Bio-Bio Univ., Chillan (Chile))

    1989-04-01

    A proton beam of 6.6 MeV has been used to produce /sup 11/C and /sup 13/N in Araucaria Araucana seeds. Their positron decay allows determination of the N/C ratio. In seeds the nitrogen content is associated to proteins while carbon is spread in the organic material. Samples were irradiated for about 10 min with a beam intensity of 5 nA on areas of 1 mm/sup 2/. Slices of the seed were radially explored, showing a larger concentration of protein in the center. (orig.).

  2. Hydrodynamic collective effects of active proteins in biological membranes

    CERN Document Server

    Koyano, Yuki; Mikhailov, Alexander S

    2016-01-01

    Lipid bilayers forming biological membranes are known to behave as viscous 2D fluids on submicrometer scales; usually they contain a large number of active protein inclusions. Recently, it has been shown [Proc. Nat. Acad. Sci. USA 112, E3639 (2015)] that such active proteins should in- duce non-thermal fluctuating lipid flows leading to diffusion enhancement and chemotaxis-like drift for passive inclusions in biomembranes. Here, a detailed analytical and numerical investigation of such effects is performed. The attention is focused on the situations when proteins are concentrated within lipid rafts. We demonstrate that passive particles tend to become attracted by active rafts and are accumulated inside them.

  3. 脊尾白虾虾糜的制备及其抗冷冻变性工艺%Technology of shrimp surimi preparation from Exopalaemon carinicauda and its anti-freeze denaturation

    Institute of Scientific and Technical Information of China (English)

    曹文红; 赵子科; 田申; 陈良

    2015-01-01

    This paper studied the rinsing technology and anti-freeze denaturation technology of shrimp surimi made from Exopalaemon carinicauda.The effect of different rinsing conditions on gel strength and elasticity of the minced shrimp were determined and estimated.With the weighted value of the gel strength and elasticity as the indexes,the rinsing process technology was optimized with an orthogonal trial.The optimal conditions were:rinsing time 7 min,shrimp meat:water ratio 1∶9,CaCl2 concentration 0.7%.In order to understand the influence of frozen storage on the quality of the rinsed shrimp meat,trehalose,sodium lactate,sorbitol and sucrose were used as cryoprotectants,salt-soluble protein content,Ca2+-ATPase activity,total sulfhydryl content,gel strength and pH of the rinsed shrimp meat were determined and compared during a storage of 8 weeks.The results indicated that all cryoprotectants showed good anti-freeze denaturation activity on rinsed shrimp meat as compared to the control samples.The trehalose added samples had better anti-frozen effect than other cryoprotectants added samples.The results of this research show that shrimp surimi of Exopalaemon carinicauda can remain better qualities after a relatively long time storage.%以脊尾白虾为原料,研究虾糜漂洗工艺及其抗冷冻变性工艺.以凝胶强度和弹性为指标,探索不同漂洗条件对虾糜凝胶性能的影响,以凝胶强度和弹性的加权平均值为指标进行正交试验得出虾糜最佳的漂洗工艺为:漂洗时间7 min,料水比1∶9(g∶ mL),漂洗液CaCl2浓度0.7%.为研究冻藏对虾糜品质的影响,以海藻糖、乳酸钠、山梨糖醇、蔗糖等为抗冻剂,比较了添加不同抗冻剂时8周冻藏期间内虾糜的盐溶蛋白含量、Ca2-AT-Pase、总巯基含量、凝胶强度、pH值的变化规律.结果显示:不同抗冻剂均能较好地抑制脊尾白虾虾糜蛋白质的冷冻变性,海藻糖的抗冻效果优于另外两种抗冻剂.研究表

  4. 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. PMID:27630047

  5. Protein stability and enzyme activity at extreme biological temperatures

    Energy Technology Data Exchange (ETDEWEB)

    Feller, Georges, E-mail: gfeller@ulg.ac.b [Laboratory of Biochemistry, Centre for Protein Engineering, Institute of Chemistry B6a, University of Liege, B-4000 Liege (Belgium)

    2010-08-18

    Psychrophilic microorganisms thrive in permanently cold environments, even at subzero temperatures. To maintain metabolic rates compatible with sustained life, they have improved the dynamics of their protein structures, thereby enabling appropriate molecular motions required for biological activity at low temperatures. As a consequence of this structural flexibility, psychrophilic proteins are unstable and heat-labile. In the upper range of biological temperatures, thermophiles and hyperthermophiles grow at temperatures > 100 {sup 0}C and synthesize ultra-stable proteins. However, thermophilic enzymes are nearly inactive at room temperature as a result of their compactness and rigidity. At the molecular level, both types of extremophilic proteins have adapted the same structural factors, but in opposite directions, to address either activity at low temperatures or stability in hot environments. A model based on folding funnels is proposed accounting for the stability-activity relationships in extremophilic proteins. (topical review)

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

    OpenAIRE

    Jarine Amaral do EVANGELHO; Jose de J. BERRIOS; Vânia Zanella PINTO; Mariana Dias ANTUNES; Nathan Levien VANIER; Elessandra da Rosa ZAVAREZE

    2016-01-01

    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,...

  7. 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.

  8. Mitogen activated protein kinases: a role in inflammatory bowel disease?

    DEFF Research Database (Denmark)

    Broom, O J; Widjaya, B; Troelsen, J;

    2009-01-01

    Since their discovery more than 15 years ago, the mitogen activated protein kinases (MAPK) have been implicated in an ever-increasingly diverse array of pathways, including inflammatory signalling cascades. Inflammatory bowel diseases (IBD), such as ulcerative colitis and Crohn's disease...... and their related signalling proteins in influencing the progression of IBD....

  9. Dietary Protein Considerations to Support Active Aging

    OpenAIRE

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

    2014-01-01

    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 ...

  10. Coactivator p100 protein enhances histone acetyltransferase activity of CBP

    Institute of Scientific and Technical Information of China (English)

    JIE YANG; HONG BAI; Li JIE DONG; JIE SHAO; OLLI SILVENNOINEN; ZHI YAO

    2006-01-01

    Human p100 protein consists of four repeated domains of staphylococcal nuclease (SN)-like domain, as well as a tudor (TD) domain thereafter. We have previously shown that the SN-like domain of p100 interacted with STAT6 and the large subunit of RNA pol Ⅱ, resulting in the enhancement of STAT6-mediated gene transcriptional activation. Here, we show that SN-like domain also interacted with CREB binding protein (CBP) and directly enhanced the acetyl transferase activity of CBP on histone. On the other hand, overexpression of CBP alone had no ability to significantly increase STAT6-dependent transcriptional activation, however, together with p100 protein, sufficiently enhanced the activation of transcription which was in line with the previous result that p100 protein bridged STAT6 with CBP.

  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

    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...... activity in vitro. Interestingly, knocking down RACK1 increased the cellular frequency of DNA breaks. Depletion of the WRN protein in return caused a fraction of nuclear RACK1 to translocate out of the nucleus to bind and activate PKCdelta and PKCbetaII in the membrane fraction of cells. In contrast......), 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. Acquired deficiencies of protein S. Protein S activity during oral anticoagulation, in liver disease, and in disseminated intravascular coagulation.

    OpenAIRE

    D'Angelo, A.; Vigano-D'Angelo, S; Esmon, C T; Comp, P C

    1988-01-01

    Protein S is a vitamin K-dependent plasma protein which serves as the cofactor for activated protein C. Protein S circulates in both an active, free form and in an inactive complex with C4b-binding protein. To elucidate the role of protein S in disease states and during oral anticoagulation, we developed a functional assay for protein S that permits evaluation of the distribution of protein S between free and bound forms and permits determination of the specific activity of the free protein S...

  13. Protein kinase D activity controls endothelial nitric oxide synthesis

    OpenAIRE

    Aicart-Ramos, Clara; Sánchez-Ruiloba, Lucía; Gómez-Parrizas, Mónica; Zaragoza, Carlos; Iglesias, Teresa; Rodríguez-Crespo, Ignacio

    2014-01-01

    Vascular endothelial growth factor (VEGF) regulates key functions of the endothelium, such as angiogenesis or vessel repair in processes involving endothelial nitric oxide synthase (eNOS) activation. One of the effector kinases that become activated in endothelial cells upon VEGF treatment is protein kinase D (PKD). Here, we show that PKD phosphorylates eNOS, leading to its activation and a concomitant increase in NO synthesis. Using mass spectrometry, we show that the purified active kinase ...

  14. Regulatory crosstalk by protein kinases on CFTR trafficking and activity

    Science.gov (United States)

    Farinha, Carlos Miguel; Swiatecka-Urban, Agnieszka; Brautigan, David; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e. channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease.

  15. 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.

  16. Cellular reprogramming through mitogen-activated protein kinases

    Directory of Open Access Journals (Sweden)

    Justin eLee

    2015-10-01

    Full Text Available Mitogen-activated protein kinase (MAPK cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554 in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression – including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding and degradation steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes.

  17. Conserved interaction of the papillomavirus E2 transcriptional activator proteins with human and yeast TFIIB proteins.

    OpenAIRE

    Benson, J D; Lawande, R; Howley, P M

    1997-01-01

    Papillomavirus early gene expression is regulated by the virus gene-encoded E2 proteins. The best-characterized E2 protein, encoded by bovine papillomavirus type 1 (BPV-1), has been shown to interact with basal transcription factor IIB (TFIIB) and the TATA binding protein basal transcription factor (N. M. Rank and P. F. Lambert, J. Virol. 69:6323-6334, 1995). We demonstrate that the potent E2 transcriptional activator protein encoded by a gene of human PV type 16 also interacts with TFIIB in ...

  18. 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

  19. 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......-induced activation of HSL was abolished by the protein kinase C (PKC) inhibitors bisindolylmaleimide I and calphostin C and reduced 50% by the mitogen-activated protein kinase kinase (MEK) inhibitor U0126, which also completely blocked extracellular signal-regulated kinase (ERK) 1 and 2 phosphorylation. None...... 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...

  20. A conserved patch of hydrophobic amino acids modulates Myb activity by mediating protein-protein interactions.

    Science.gov (United States)

    Dukare, Sandeep; Klempnauer, Karl-Heinz

    2016-07-01

    The transcription factor c-Myb plays a key role in the control of proliferation and differentiation in hematopoietic progenitor cells and has been implicated in the development of leukemia and certain non-hematopoietic tumors. c-Myb activity is highly dependent on the interaction with the coactivator p300 which is mediated by the transactivation domain of c-Myb and the KIX domain of p300. We have previously observed that conservative valine-to-isoleucine amino acid substitutions in a conserved stretch of hydrophobic amino acids have a profound effect on Myb activity. Here, we have explored the function of the hydrophobic region as a mediator of protein-protein interactions. We show that the hydrophobic region facilitates Myb self-interaction and binding of the histone acetyl transferase Tip60, a previously identified Myb interacting protein. We show that these interactions are affected by the valine-to-isoleucine amino acid substitutions and suppress Myb activity by interfering with the interaction of Myb and the KIX domain of p300. Taken together, our work identifies the hydrophobic region in the Myb transactivation domain as a binding site for homo- and heteromeric protein interactions and leads to a picture of the c-Myb transactivation domain as a composite protein binding region that facilitates interdependent protein-protein interactions of Myb with regulatory proteins. PMID:27080133

  1. A conserved patch of hydrophobic amino acids modulates Myb activity by mediating protein-protein interactions.

    Science.gov (United States)

    Dukare, Sandeep; Klempnauer, Karl-Heinz

    2016-07-01

    The transcription factor c-Myb plays a key role in the control of proliferation and differentiation in hematopoietic progenitor cells and has been implicated in the development of leukemia and certain non-hematopoietic tumors. c-Myb activity is highly dependent on the interaction with the coactivator p300 which is mediated by the transactivation domain of c-Myb and the KIX domain of p300. We have previously observed that conservative valine-to-isoleucine amino acid substitutions in a conserved stretch of hydrophobic amino acids have a profound effect on Myb activity. Here, we have explored the function of the hydrophobic region as a mediator of protein-protein interactions. We show that the hydrophobic region facilitates Myb self-interaction and binding of the histone acetyl transferase Tip60, a previously identified Myb interacting protein. We show that these interactions are affected by the valine-to-isoleucine amino acid substitutions and suppress Myb activity by interfering with the interaction of Myb and the KIX domain of p300. Taken together, our work identifies the hydrophobic region in the Myb transactivation domain as a binding site for homo- and heteromeric protein interactions and leads to a picture of the c-Myb transactivation domain as a composite protein binding region that facilitates interdependent protein-protein interactions of Myb with regulatory proteins.

  2. Organization, Structure and Activity of Proteins in Monolayers

    Energy Technology Data Exchange (ETDEWEB)

    Boucher,J.; Trudel, E.; Methot, M.; Desmeules, P.; Salesse, C.

    2007-01-01

    Many different processes take place at the cell membrane interface. Indeed, for instance, ligands bind membrane proteins which in turn activate peripheral membrane proteins, some of which are enzymes whose action is also located at the membrane interface. Native cell membranes are difficult to use to gain information on the activity of individual proteins at the membrane interface because of the large number of different proteins involved in membranous processes. Model membrane systems, such as monolayers at the air-water interface, have thus been extensively used during the last 50 years to reconstitute proteins and to gain information on their organization, structure and activity in membranes. In the present paper, we review the recent work we have performed with membrane and peripheral proteins as well as enzymes in monolayers at the air-water interface. We show that the structure and orientation of gramicidin has been determined by combining different methods. Furthermore, we demonstrate that the secondary structure of rhodopsin and bacteriorhodopsin is indistinguishable from that in native membranes when appropriate conditions are used. We also show that the kinetics and extent of monolayer binding of myristoylated recoverin is much faster than that of the nonmyristoylated form and that this binding is highly favored by the presence polyunsaturated phospholipids. Moreover, we show that the use of fragments of RPE65 allow determine which region of this protein is most likely involved in membrane binding. Monomolecular films were also used to further understand the hydrolysis of organized phospholipids by phospholipases A2 and C.

  3. G protein activation stimulates phospholipase D signaling in plants

    NARCIS (Netherlands)

    Munnik, T.; Arisz, S.A.; Vrije, de T.; Musgrave, A.

    1995-01-01

    We provide direct evidence for phospholipase D (PLD) signaling in plants by showing that this enzyme is stimulated by the G protein activators mastoparan, ethanol, and cholera toxin. An in vivo assay for PLD activity in plant cells was developed based on the use of a "reporter alcohol" rather than w

  4. Factor H-related proteins determine complement-activating surfaces.

    Science.gov (United States)

    Józsi, Mihály; Tortajada, Agustin; Uzonyi, Barbara; Goicoechea de Jorge, Elena; Rodríguez de Córdoba, Santiago

    2015-06-01

    Complement factor H-related proteins (FHRs) are strongly associated with different diseases involving complement dysregulation, which suggests a major role for these proteins regulating complement activation. Because FHRs are evolutionarily and structurally related to complement inhibitor factor H (FH), the initial assumption was that the FHRs are also negative complement regulators. Whereas weak complement inhibiting activities were originally reported for these molecules, recent developments indicate that FHRs may enhance complement activation, with important implications for the role of these proteins in health and disease. We review these findings here, and propose that FHRs represent a complex set of surface recognition molecules that, by competing with FH, provide improved discrimination of self and non-self surfaces and play a central role in determining appropriate activation of the complement pathway.

  5. Mitogen-activated protein kinase signaling in plants

    DEFF Research Database (Denmark)

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

    2010-01-01

    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......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...... crossinhibition, feedback control, and scaffolding. Plant MAPK cascades regulate numerous processes, including stress and hormonal responses, innate immunity, and developmental programs. Genetic analyses have uncovered several predominant MAPK components shared by several of these processes including...

  6. Redox Regulation of the AMP-Activated Protein Kinase

    OpenAIRE

    Yingying Han; Qilong Wang; Ping Song; Yi Zhu; Ming-Hui Zou

    2010-01-01

    Redox state is a critical determinant of cell function, and any major imbalances can cause severe damage or death. Objectives 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). Methods Bovine aortic endothelial cells (BAEC) were exposed to Berberine. AMPK activity and reactive oxygen species were monitored after the incubation. Results In BAEC, Berberine caused a dos...

  7. Redox Regulation of Protein Tyrosine Phosphatase Activity by Hydroxyl Radical

    OpenAIRE

    Meng, Fan-Guo; Zhang, Zhong-Yin

    2012-01-01

    Substantial evidence suggests that transient production of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) is an important signaling event triggered by the activation of various cell surface receptors. Major targets of H2O2 include protein tyrosine phosphatases (PTPs). Oxidation of the active site Cys by H2O2 abrogates PTP catalytic activity, thereby potentially furnishing a mechanism to ensure optimal tyrosine phosphorylation in response to a variety of physiological stimuli. ...

  8. Raman spectroscopy of antifreeze glycoproteins and their interaction with various substrates

    Science.gov (United States)

    Cui, Y.; Turner, G.; Alexander, V.; Smith, I.; Sease, A.; Guo, M.; Burger, A.; Morgan, S.; Yeh, Yin

    2004-11-01

    Micro-Raman spectra of a mixture of antifreeze glycoproteins (AFGP) 6, 7 and 8 have been measured in the range of 100 - 4500 cm-1 with He-Ne laser excitation. The spectra were obtained for both bulk AFGP and films of AFGP deposited on various substrates. New vibrational peaks have been observed for films which are not present in the spectra of the bulk samples. The results will be presented and mechanisms of interaction between the AFGP molecule and substrates will be proposed. The assignment of new peaks and the effects of the water presence will also be discussed. Research supported by the NSF Center for Biophotonics, managed by U.C. Davis, CA No. PHY 0120999, NSF Research Experiences for Undergraduates DMR-0139180 and by the MBRS program through NIH/NIGMS grant 1S06-GM62813-01.

  9. 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....... in the human cervical carcinoma HeLa cells by up to 8-fold, and this could be blocked by the p38 MAP kinase inhibitor SB203580. We show that p38alpha MAP kinase, in a phosphorylation-dependent manner, can directly interact with the alpha and beta subunits of CK2 to activate the holoenzyme through what appears...

  10. Hydrodynamic collective effects of active proteins in biological membranes

    Science.gov (United States)

    Koyano, Yuki; Kitahata, Hiroyuki; Mikhailov, Alexander S.

    2016-08-01

    Lipid bilayers forming biological membranes are known to behave as viscous two-dimensional fluids on submicrometer scales; usually they contain a large number of active protein inclusions. Recently, it was shown [A. S. Mikhailov and R. Kapral, Proc. Natl. Acad. Sci. USA 112, E3639 (2015), 10.1073/pnas.1506825112] that such active proteins should induce nonthermal fluctuating lipid flows leading to diffusion enhancement and chemotaxislike drift for passive inclusions in biomembranes. Here, a detailed analytical and numerical investigation of such effects is performed. The attention is focused on the situations when proteins are concentrated within lipid rafts. We demonstrate that passive particles tend to become attracted by active rafts and are accumulated inside them.

  11. 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...

  12. Anti-tumor activities andapoptotic mechanism ofribosome-inactivating proteins

    Institute of Scientific and Technical Information of China (English)

    MeiqiZeng; ManyinZheng; DeshengLu; JunWang; WenqiJiang; OuSha

    2015-01-01

    Ribosome-inactivating proteins (RIPs) belong to a family of enzymes that attack eukaryotic ribosomes and potently inhibit cellular protein synthesis. RIPs possess several biomedical properties, including anti-viral and anti-tumor activi-ties. Multiple RIPs are known to inhibit tumor cell proliferation through inducing apoptosis in a variety of cancers, such as breast cancer, leukemia/lymphoma, and hepatoma. This review focuses on the anti-tumor activities of RIPs and their apoptotic effects through three closely related pathways: mitochondrial, death receptor, and endoplasmic reticulum pathways.

  13. Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis.

    Science.gov (United States)

    Kohansal-Nodehi, Mahdokht; Chua, John Je; Urlaub, Henning; Jahn, Reinhard; Czernik, Dominika

    2016-01-01

    Neurotransmitter release is mediated by the fast, calcium-triggered fusion of synaptic vesicles with the presynaptic plasma membrane, followed by endocytosis and recycling of the membrane of synaptic vesicles. While many of the proteins governing these processes are known, their regulation is only beginning to be understood. Here we have applied quantitative phosphoproteomics to identify changes in phosphorylation status of presynaptic proteins in resting and stimulated nerve terminals isolated from the brains of Wistar rats. Using rigorous quantification, we identified 252 phosphosites that are either up- or downregulated upon triggering calcium-dependent exocytosis. Particularly pronounced were regulated changes of phosphosites within protein constituents of the presynaptic active zone, including bassoon, piccolo, and RIM1. Additionally, we have mapped kinases and phosphatases that are activated upon stimulation. Overall, our study provides a snapshot of phosphorylation changes associated with presynaptic activity and provides a foundation for further functional analysis of key phosphosites involved in presynaptic plasticity. PMID:27115346

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

    International Nuclear Information System (INIS)

    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

  15. Controlling Protein Activity and Degradation Using Blue Light.

    Science.gov (United States)

    Lutz, Anne P; Renicke, Christian; Taxis, Christof

    2016-01-01

    Regulation of protein stability is a fundamental process in eukaryotic cells and pivotal to, e.g., cell cycle progression, faithful chromosome segregation, or protein quality control. Synthetic regulation of protein stability requires conditional degradation sequences (degrons) that induce a stability switch upon a specific signal. Fusion to a selected target protein permits to influence virtually every process in a cell. Light as signal is advantageous due to its precise applicability in time, space, quality, and quantity. Light control of protein stability was achieved by fusing the LOV2 photoreceptor domain of Arabidopsis thaliana phototropin1 with a synthetic degron (cODC1) derived from the carboxy-terminal degron of ornithine decarboxylase to obtain the photosensitive degron (psd) module. The psd module can be attached to the carboxy terminus of target proteins that are localized to the cytosol or nucleus to obtain light control over their stability. Blue light induces structural changes in the LOV2 domain, which in turn lead to activation of the degron and thus proteasomal degradation of the whole fusion protein. Variants of the psd module with diverse characteristics are useful to fine-tune the stability of a selected target at permissive (darkness) and restrictive conditions (blue light). PMID:26965116

  16. Installing hydrolytic activity into a completely de novo protein framework

    Science.gov (United States)

    Burton, Antony J.; Thomson, Andrew R.; Dawson, William M.; Brady, R. Leo; Woolfson, Derek N.

    2016-09-01

    The design of enzyme-like catalysts tests our understanding of sequence-to-structure/function relationships in proteins. Here we install hydrolytic activity predictably into a completely de novo and thermostable α-helical barrel, which comprises seven helices arranged around an accessible channel. We show that the lumen of the barrel accepts 21 mutations to functional polar residues. The resulting variant, which has cysteine-histidine-glutamic acid triads on each helix, hydrolyses p-nitrophenyl acetate with catalytic efficiencies that match the most-efficient redesigned hydrolases based on natural protein scaffolds. This is the first report of a functional catalytic triad engineered into a de novo protein framework. The flexibility of our system also allows the facile incorporation of unnatural side chains to improve activity and probe the catalytic mechanism. Such a predictable and robust construction of truly de novo biocatalysts holds promise for applications in chemical and biochemical synthesis.

  17. The role of activated protein C in cancer progression

    NARCIS (Netherlands)

    G.L. van Sluis; H.R. Büller; C.A. Spek

    2010-01-01

    Activated protein C (APC) is best known as a natural anticoagulant that also has direct cell signaling properties which (among others) enhance vascular barrier function. We recently established the relevance of APC-induced barrier enhancement by showing that endogenous APC limits cancer cell extrava

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

    Science.gov (United States)

    Khatib, Abdallah; Arhab, Yani; Bentebibel, Assia; Abousalham, Abdelkarim; Noiriel, Alexandre

    2016-01-01

    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. PMID:26745266

  19. 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.

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

    Science.gov (United States)

    Khatib, Abdallah; Arhab, Yani; Bentebibel, Assia; Abousalham, Abdelkarim; Noiriel, Alexandre

    2016-01-01

    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.

  1. 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. PMID:26920259

  2. 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.

  3. RNF4-Dependent Oncogene Activation by Protein Stabilization.

    Science.gov (United States)

    Thomas, Jane J; Abed, Mona; Heuberger, Julian; Novak, Rostislav; Zohar, Yaniv; Beltran Lopez, Angela P; Trausch-Azar, Julie S; Ilagan, Ma Xenia G; Benhamou, David; Dittmar, Gunnar; Kopan, Raphael; Birchmeier, Walter; Schwartz, Alan L; Orian, Amir

    2016-09-20

    Ubiquitylation regulates signaling pathways critical for cancer development and, in many cases, targets proteins for degradation. Here, we report that ubiquitylation by RNF4 stabilizes otherwise short-lived oncogenic transcription factors, including β-catenin, Myc, c-Jun, and the Notch intracellular-domain (N-ICD) protein. RNF4 enhances the transcriptional activity of these factors, as well as Wnt- and Notch-dependent gene expression. While RNF4 is a SUMO-targeted ubiquitin ligase, protein stabilization requires the substrate's phosphorylation, rather than SUMOylation, and binding to RNF4's arginine-rich motif domain. Stabilization also involves generation of unusual polyubiquitin chains and docking of RNF4 to chromatin. Biologically, RNF4 enhances the tumor phenotype and is essential for cancer cell survival. High levels of RNF4 mRNA correlate with poor survival of a subgroup of breast cancer patients, and RNF4 protein levels are elevated in 30% of human colon adenocarcinomas. Thus, RNF4-dependent ubiquitylation translates transient phosphorylation signal(s) into long-term protein stabilization, resulting in enhanced oncoprotein activation. PMID:27653698

  4. NRIP, a novel calmodulin binding protein, activates calcineurin to dephosphorylate human papillomavirus E2 protein.

    Science.gov (United States)

    Chang, Szu-Wei; Tsao, Yeou-Ping; Lin, Chia-Yi; Chen, Show-Li

    2011-07-01

    Previously, we found a gene named nuclear receptor interaction protein (NRIP) (or DCAF6 or IQWD1). We demonstrate that NRIP is a novel binding protein for human papillomavirus 16 (HPV-16) E2 protein. HPV-16 E2 and NRIP can directly associate into a complex in vivo and in vitro, and the N-terminal domain of NRIP interacts with the transactivation domain of HPV-16 E2. Only full-length NRIP can stabilize E2 protein and induce HPV gene expression, and NRIP silenced by two designed small interfering RNAs (siRNAs) decreases E2 protein levels and E2-driven gene expression. We found that NRIP can directly bind with calmodulin in the presence of calcium through its IQ domain, resulting in decreased E2 ubiquitination and increased E2 protein stability. Complex formation between NRIP and calcium/calmodulin activates the phosphatase calcineurin to dephosphorylate E2 and increase E2 protein stability. We present evidences for E2 phosphorylation in vivo and show that NRIP acts as a scaffold to recruit E2 and calcium/calmodulin to prevent polyubiquitination and degradation of E2, enhancing E2 stability and E2-driven gene expression. PMID:21543494

  5. Protein kinase domain of twitchin has protein kinase activity and an autoinhibitory region.

    Science.gov (United States)

    Lei, J; Tang, X; Chambers, T C; Pohl, J; Benian, G M

    1994-08-19

    Twitchin is a 753-kDa polypeptide located in the muscle A-bands of the nematode, Caenorhabditis elegans. It consists of multiple copies of both fibronectin III and immunoglobulin C2 domains and, near the C terminus, a protein kinase domain with greatest homology to the catalytic domains of myosin light chain kinases. We have expressed and purified from Escherichia coli twitchin's protein kinase catalytic core and flanking sequences that do not include fibronectin III and immunoglobulin C2 domains. The protein was shown to phosphorylate a model substrate and to undergo autophosphorylation. The autophosphorylation occurs at a slow rate, attaining a maximum at 3 h with a stoichiometry of about 1.0 mol of phosphate/mol of protein, probably through an intramolecular mechanism. Sequence analysis of proteolytically derived phosphopeptides revealed that autophosphorylation occurred N-terminal to the catalytic core, predominantly at Thr-5910, with possible minor sites at Ser5912 and/or Ser-5913. This portion of twitchin (residues 5890-6268) was also phosphorylated in vitro by protein kinase C in the absence of calcium and phosphotidylserine, but not by cAMP-dependent protein kinase. By comparing the activities of three twitchin segments, the enzyme appears to be inhibited by the 60-amino acid residues lying just C-terminal to the kinase catalytic core. Thus, like a number of other protein kinases including myosin light chain kinases, the twitchin kinase appears to be autoregulated. PMID:8063727

  6. Protein kinase C controls activation of the DNA integrity checkpoint

    Science.gov (United States)

    Soriano-Carot, María; Quilis, Inma; Bañó, M. Carmen; Igual, J. Carlos

    2014-01-01

    The protein kinase C (PKC) superfamily plays key regulatory roles in numerous cellular processes. Saccharomyces cerevisiae contains a single PKC, Pkc1, whose main function is cell wall integrity maintenance. In this work, we connect the Pkc1 protein to the maintenance of genome integrity in response to genotoxic stresses. Pkc1 and its kinase activity are necessary for the phosphorylation of checkpoint kinase Rad53, histone H2A and Xrs2 protein after deoxyribonucleic acid (DNA) damage, indicating that Pkc1 is required for activation of checkpoint kinases Mec1 and Tel1. Furthermore, Pkc1 electrophoretic mobility is delayed after inducing DNA damage, which reflects that Pkc1 is post-translationally modified. This modification is a phosphorylation event mediated by Tel1. The expression of different mammalian PKC isoforms at the endogenous level in yeast pkc1 mutant cells revealed that PKCδ is able to activate the DNA integrity checkpoint. Finally, downregulation of PKCδ activity in HeLa cells caused a defective activation of checkpoint kinase Chk2 when DNA damage was induced. Our results indicate that the control of the DNA integrity checkpoint by PKC is a mechanism conserved from yeast to humans. PMID:24792164

  7. Enhancement of rabbit protein S anticoagulant cofactor activity in vivo by modulation of the protein S C4B binding protein interaction.

    OpenAIRE

    Weinstein, R E; Walker, F. J.

    1990-01-01

    The carboxy-terminal region of protein S has been recently been observed to be involved in the interaction between protein S and C4b-binding protein (Walker, F. J. 1989. J. Biol. Chem. 264:17645-17658). A synthetic peptide, GVQLDLDEAI, corresponding to that region of protein S has been used to investigate the protein S/C4b-binding protein interaction in vitro and in vivo. Rabbit activated protein C possesses species-specific anticoagulant activity for which rabbit protein S functions as a cof...

  8. The pleiotropic activity of heat-shock proteins

    Directory of Open Access Journals (Sweden)

    Arleta Kaźmierczuk

    2009-10-01

    Full Text Available Stress or heat-shock proteins (HSPs are highly conserved proteins present in cells of both prokaryotes and eukaryotes, providing them with protection from cellular and environmental stress factors. Based on molecular-weight, HSPs can be divided into the large (HSP100: 100–110 kDa and HSP90: 75–96 kDa, intermediate (HSP70: 66–78 kDa, HSP60, and HSP40, and small (sHSP: 8.5–40 kDa subfamilies. These proteins play an essential role as molecular chaperones/co-chaperones by assisting the correct folding of nascent and stress-accumulated protein-substrate assembly, preventing the aggregation of these proteins, as well as transport across membranes and the degradation of other proteins. Members of HSP family display dual activity depending on their intra- or extracellular distribution. Intracellular HSPs mainly play a protective role. Extracellular or membrane-bound HSPs mediate immunological functions. Among the functions of HSPs is their participation in cell signaling. This review deals with the structure and properties of the main members of the HSPs and their role in a large number of cellular/extracellular processes.

  9. Heat Shock Protein 90 Indirectly Regulates ERK Activity by Affecting Raf Protein Metabolism

    Institute of Scientific and Technical Information of China (English)

    Fei DOU; Liu-Di YUAN; Jing-Jing ZHU

    2005-01-01

    Extracellular signal-regulated protein kinase (ERK) has been implicated in the pathogenesis of several nerve system diseases. As more and more kinases have been discovered to be the client proteins of the molecular chaperone Hsp90, the use of Hsp90 inhibitors to reduce abnormal kinase activity is a new treatment strategy for nerve system diseases. This study investigated the regulation of the ERK pathway by Hsp90. We showed that Hsp90 inhibitors reduce ERK phosphorylation without affecting the total ERK protein level. Further investigation showed that Raf, the upstream kinase in the Ras-Raf-MEK-ERK pathway,forms a complex with Hsp90 and Hsp70. Treating cells with Hsp90 inhibitors facilitates Raf degradation,thereby down-regulating the activity of ERK.

  10. Mechanism of antifreeze proteins action, based on Hierarchic theory of water and new ''clusterphilic'' interaction

    CERN Document Server

    Kaivarainen, A

    2001-01-01

    A basically new Hierarchic theory, general for solids and liquids (Kaivarainen, 2001, 2000, 1995, 1992), has been briefly described and illustrated by computer simulations on examples of water and ice. Full description of theory and its numerous applications are presented in series of articles at the arXiv of Los-Alamos (see http://arXiv.org/abs/physics/0102086). New clusterphilic interactions, intermediate between hydrophilic and hydrphobic, are introduced. They can be subdivided into: intramolecular - when water cluster is localized in the ''open'' states of big interdomain or intersubunit cavities and intermolecular clusterphilic interactions. Intermolecular clusterphilic interactions can be induced by very different macromolecules. The latter displays themselves in bordering of water cluster by macromolecules and forming so-called ''clustrons''. Clusterphilic interactions can play an important role in self-organization of biosystems, especially multiglobular allosteric enzymes, microtubules and the actin ...

  11. Pharmacological activities in thermal proteins: relationships in molecular evolution

    Science.gov (United States)

    Fox, S. W.; Hefti, F.; Hartikka, J.; Junard, E.; Przybylski, A. T.; Vaughan, G.

    1987-01-01

    The model of protobiological events that has been presented in these pages has increasing relevance to pharmacological research. The thermal proteins that function as key substances in the proteinoid theory have recently been found to prolong the survival of rat forebrain neurons in culture and to stimulate the growth of neurites. A search for such activity in thermal proteins added to cultures of modern neurons was suggested by the fact that some of the microspheres assembled from proteinoids rich in hydrophobic amino acids themselves generate fibrous outgrowths.

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

    Science.gov (United States)

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

    2015-07-01

    Sustamine™ (SUS) is a dipeptide composed of alanine and glutamine (AlaGln). Glutamine has been suggested to increase muscle protein accretion; however, the underlying molecular mechanisms of glutamine on muscle protein metabolism following resistance exercise have not been fully addressed. In the present study, 2-month-old rats climbed a ladder 10 times with a weight equal to 75 % of their body mass attached at the tail. Rats were then orally administered one of four solutions: placebo (PLA-glycine = 0.52 g/kg), whey protein (WP = 0.4 g/kg), low dose of SUS (LSUS = 0.1 g/kg), or high dose of SUS (HSUS = 0.5 g/kg). An additional group of sedentary (SED) rats was intubated with glycine (0.52 g/kg) at the same time as the ladder-climbing rats. Blood samples were collected immediately after exercise and at either 20 or 40 min after recovery. The flexor hallucis longus (FHL), a muscle used for climbing, was excised at 20 or 40 min post exercise and analyzed for proteins regulating protein synthesis and degradation. All supplements elevated the phosphorylation of FOXO3A above SED at 20 min post exercise, but only the SUS supplements significantly reduced the phosphorylation of AMPK and NF-kB p65. SUS supplements had no effect on mTOR signaling, but WP supplementation yielded a greater phosphorylation of mTOR, p70S6k, and rpS6 compared with PLA at 20 min post exercise. However, by 40 min post exercise, phosphorylation of mTOR and rpS6 in PLA had risen to levels not different than WP. These results suggest that SUS blocks the activation of intracellular signals for MPB, whereas WP accelerates mRNA translation. PMID:25837301

  13. Efficient expression and purification of biologically active human cystatin proteins.

    Science.gov (United States)

    Chauhan, Sakshi; Tomar, Raghuvir S

    2016-02-01

    Cystatins are reversible cysteine protease inhibitor proteins. They are known to play important roles in controlling cathepsins, neurodegenerative disease, and in immune system regulation. Production of recombinant cystatin proteins is important for biochemical and function characterization. In this study, we cloned and expressed human stefin A, stefin B and cystatin C in Escherichia coli. Human stefin A, stefin B and cystatin C were purified from soluble fraction. For cystatin C, we used various chaperone plasmids to make cystatin C soluble, as it is reported to localize in inclusion bodies. Trigger factor, GroES-GroEL, DnaK-DnaJ-GrpE chaperones lead to the presence of cystatin C in the soluble fraction. Immobilized metal affinity chromatography, glutathione sepharose and anion exchange chromatography techniques were employed for efficient purification of these proteins. Their biological activities were tested by inhibition assays against cathepsin L and H3 protease.

  14. Modulation of the protein kinase activity of mTOR.

    Science.gov (United States)

    Lawrence, J C; Lin, T A; McMahon, L P; Choi, K M

    2004-01-01

    mTOR is a founding member of a family of protein kinases having catalytic domains homologous to those in phosphatidylinositol 3-OH kinase. mTOR participates in the control by insulin of the phosphorylation of lipin, which is required for adipocyte differentiation, and the two translational regulators, p70S6K and PHAS-I. The phosphorylation of mTOR, itself, is stimulated by insulin in Ser2448, a site that is also phosphorylated by protein kinase B (PKB) in vitro and in response to activation of PKB activity in vivo. Ser2448 is located in a short stretch of amino acids not found in the two TOR proteins in yeast. A mutant mTOR lacking this stretch exhibited increased activity, and binding of the antibody, mTAb-1, to this region markedly increased mTOR activity. In contrast, rapamycin-FKBP12 inhibited mTOR activity towards both PHAS-I and p70S6K, although this complex inhibited the phosphorylation of some sites more than that of others. Mutating Ser2035 to Ile in the FKBP12-rapamycin binding domain rendered mTOR resistant to inhibition by rapamycin. Unexpectedly, this mutation markedly decreased the ability of mTOR to phosphorylate certain sites in both PHAS-I and p70S6K. The results support the hypotheses that rapamycin disrupts substrate recognition instead of directly inhibiting phosphotransferase activity and that mTOR activity in cells is controlled by the phosphorylation of an inhibitory regulatory domain containing the mTAb-1 epitope. PMID:14560959

  15. Antioxidant, Antibacterial, and Cytoprotective Activity of Agathi Leaf Protein

    Directory of Open Access Journals (Sweden)

    A. S. Zarena

    2014-01-01

    Full Text Available In the present study a protein termed agathi leaf protein (ALP from Sesbania grandiflora Linn. (agathi leaves was isolated after successive precipitation with 65% ammonium sulphate followed by purification on Sephadex G 75. The column chromatography of the crude protein resulted in four peaks of which Peak I (P I showed maximum inhibition activity against hydroxyl radical. SDS-PAGE analysis of P I indicated that the molecular weight of the protein is ≈29 kDa. The purity of the protein was 98.4% as determined by RP-HPLC and showed a single peak with a retention time of 19.9 min. ALP was able to reduce oxidative damage by scavenging lipid peroxidation against erythrocyte ghost (85.50 ± 6.25%, linolenic acid (87.67 ± 3.14% at 4.33 μM, ABTS anion (88 ± 3.22%, and DNA damage (83 ± 4.20% at 3.44 μM in a dose-dependent manner. The purified protein offered significant protection to lymphocyte (72% at 30 min induced damage by t-BOOH. In addition, ALP showed strong antibacterial activity against Pseudomonas aeruginosa (20 ± 3.64 mm and Staphylococcus aureus (19 ± 1.53 mm at 200 μg/mL. The safety assessment showed that ALP does not induce cytotoxicity towards human lymphocyte at the tested concentration of 0.8 mg/mL.

  16. Novel Biosensor of Membrane Protein Proximity Based on Fluorogen Activated Proteins.

    Science.gov (United States)

    Vasilev, Kalin V; Gallo, Eugenio; Shank, Nathaniel; Jarvik, Jonathan W

    2016-01-01

    We describe a novel biosensor system for reporting proximity between cell surface proteins in live cultured cells. The biosensor takes advantage of recently developed fluorogen-activating proteins (FAPs) that display fluorescence only when bound to otherwise-nonfluorescent fluorogen molecules. To demonstrate feasibility for the approach, two recombinant rapamycin-binding proteins were expressed as single-pass plasma membrane proteins in HeLa cells; one of the proteins (scAvd- FRB) carried an extracellular avidin tag; the other (HL1-TO1-FKBP) carried an extracellular FAP. Cells were incubated with a membrane-impermeable bivalent ligand (biotin-PEG2000-DIR) consisting of biotin joined to a dimethyl-indole red (DIR) fluorogen by a polyethylene glycol linker, thus tethering the fluorogen to the scAvd-FRB fusion protein. Addition of rapamycin, which promotes FKBP-FRB dimerization and thereby brings the FAP in close proximity to the tethered fluorogen, led to a significant increase in DIR fluorescence. We call the new proximity assay TEFLA, for tethered fluorogen assay. PMID:27055753

  17. Membrane lipids regulate ganglioside GM2 catabolism and GM2 activator protein activity[S

    Science.gov (United States)

    Anheuser, Susi; Breiden, Bernadette; Schwarzmann, Günter; Sandhoff, Konrad

    2015-01-01

    Ganglioside GM2 is the major lysosomal storage compound of Tay-Sachs disease. It also accumulates in Niemann-Pick disease types A and B with primary storage of SM and with cholesterol in type C. Reconstitution of GM2 catabolism with β-hexosaminidase A and GM2 activator protein (GM2AP) at uncharged liposomal surfaces carrying GM2 as substrate generated only a physiologically irrelevant catabolic rate, even at pH 4.2. However, incorporation of anionic phospholipids into the GM2 carrying liposomes stimulated GM2 hydrolysis more than 10-fold, while the incorporation of plasma membrane stabilizing lipids (SM and cholesterol) generated a strong inhibition of GM2 hydrolysis, even in the presence of anionic phospholipids. Mobilization of membrane lipids by GM2AP was also inhibited in the presence of cholesterol or SM, as revealed by surface plasmon resonance studies. These lipids also reduced the interliposomal transfer rate of 2-NBD-GM1 by GM2AP, as observed in assays using Förster resonance energy transfer. Our data raise major concerns about the usage of recombinant His-tagged GM2AP compared with untagged protein. The former binds more strongly to anionic GM2-carrying liposomal surfaces, increases GM2 hydrolysis, and accelerates intermembrane transfer of 2-NBD-GM1, but does not mobilize membrane lipids. PMID:26175473

  18. Protein Kinase Cδ mediates the activation of Protein Kinase D2 in Platelets

    OpenAIRE

    Bhavanasi, Dheeraj; Kim, Soochong; Goldfinger, Lawrence E.; Kunapuli, Satya P.

    2011-01-01

    Protein Kinase D (PKD) is a subfamily of serine/threonine specific family of kinases, comprised of PKD1, PKD2 and PKD3 (PKCμ, PKD2 and PKCν in humans). It is known that PKCs activate PKD, but the relative expression of isoforms of PKD or the specific PKC isoform/s responsible for its activation in platelets is not known. This study is aimed at investigating the pathway involved in activation of PKD in platelets. We show that PKD2 is the major isoform of PKD that is expressed in human as well ...

  19. 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.

  20. 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.

  1. 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.

  2. 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)

  3. Negative regulation of lymphocyte activation by the adaptor protein LAX.

    Science.gov (United States)

    Zhu, Minghua; Granillo, Olivia; Wen, Renren; Yang, Kaiyong; Dai, Xuezhi; Wang, Demin; Zhang, Weiguo

    2005-05-01

    The membrane-associated adaptor protein LAX is a linker for activation of T cells (LAT)-like molecule that is expressed in lymphoid tissues. Upon stimulation of T or B cells, it is phosphorylated and interacts with Grb2 and the p85 subunit of PI3K. LAX, however, is not capable of replacing LAT in the TCR signaling pathway. In this study we report that upon T or B cell activation, the LAX protein was up-regulated dramatically. Although disruption of the LAX gene by homologous recombination had no major impact on lymphocyte development, it caused a significant reduction in CD23 expression on mature B cells. Interestingly, naive LAX(-/-) mice had spontaneous germinal center formation. Compared with normal T and B cells, LAX(-/-) T and B cells were hyperresponsive and had enhanced calcium flux, protein tyrosine phosphorylation, MAPK and Akt activation, and cell survival upon engagement of the T or B AgRs. Our data demonstrate that LAX functions as a negative regulator in lymphocyte signaling.

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

    International Nuclear Information System (INIS)

    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 N2 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)

  5. Overinhibition of Mitogen-Activated Protein Kinase Inducing Tau Hyperphosphorylation

    Institute of Scientific and Technical Information of China (English)

    LI Hong-lian; CHEN Juan; LIU Shi-jie; ZHANG Jia-yu; WANG Qun; WANG Jian-zhi

    2005-01-01

    To reveal the relationship between mitogen-activated protein kinase (MAPK) and tau phosphorylation, we used different concentration of PD98059, an inhibitor of MEK (MAPK kinase), to treat mice neuroblastma (N2a) cell line for 6 h. It showed that the activity of MAPK decreased in a dose-dependent manner. But Western blot and immunofluorescence revealed that just when the cells were treated with 16 μmol/L PD98059, tau was hyperphosphorylated at Ser396/404 and Ser199/202 sites. We obtained the conclusion that overinhibited MAPK induced tau hyperphosphorylation at Ser396/404 and Ser199/202 sites.

  6. 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......Autocrine factors prevent cell death in the ciliate Tetrahymena thermophila, a unicellular eukaryote, in a chemically defined medium. At certain growth conditions these factors are released at a sufficient concentration by > 500 cells ml-1 to support cell survival and proliferation. The protein...... kinase C activators phorbol 12-myristate 13-acetate (PMA) or 1-oleyl 2-acetate glycerol (OAG) when added to 250 cells ml-1 supported cell survival and proliferation. In the presence of the serine and threonine kinase inhibitor staurosporine the cells died both at 250 cells ml-1 in cultures supplemented...

  7. 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. Paradoxicall...... and subsequent rise in cellular [G6P]....

  8. Computational Modeling for the Activation Cycle of G-proteins by G-protein-coupled Receptors

    CERN Document Server

    Bao, Yifei; Glavy, Joseph; White, Tommy; 10.4204/EPTCS.40.4

    2010-01-01

    In this paper, we survey five different computational modeling methods. For comparison, we use the activation cycle of G-proteins that regulate cellular signaling events downstream of G-protein-coupled receptors (GPCRs) as a driving example. Starting from an existing Ordinary Differential Equations (ODEs) model, we implement the G-protein cycle in the stochastic Pi-calculus using SPiM, as Petri-nets using Cell Illustrator, in the Kappa Language using Cellucidate, and in Bio-PEPA using the Bio-PEPA eclipse plug in. We also provide a high-level notation to abstract away from communication primitives that may be unfamiliar to the average biologist, and we show how to translate high-level programs into stochastic Pi-calculus processes and chemical reactions.

  9. 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

    2013-11-22

    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.

  10. 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.

  11. H pylori stimulates proliferation of gastric cancer cells through activating mitogen-activated protein kinase cascade

    Institute of Scientific and Technical Information of China (English)

    Yong-Chang Chen; Ying Wang; Jing-Yan Li; Wen-Rong Xu; You-Li Zhang

    2006-01-01

    AIM: To explore the mechanism by which H pylori causes activation of gastric epithelial cells.METHODS: A VacA (+) and CagA (+) standard Hpyloriline NCTC 11637 and a human gastric adenocarcinoma derived gastric epithelial cell line BGC-823 were applied in the study. MTT assay and 3H-TdR incorporation test were used to detect the proliferation of BGC-823 cells and Western blotting was used to detect the activity and existence of related proteins.RESULTS: Incubation with Hpylori extract increased the proliferation of gastric epithelial cells, reflected by both live cell number and DNA synthesis rate. The activity of extracellular signal-regulated protein kinase (ERK) signal transduction cascade increased within 20 min after incubation with Hpylori extract and appeared to be a sustained event. MAPK/ERK kinase (MEK) inhibitor PD98059abolished the action of H pylori extract on both ERK activity and cell proliferation. Incubation with H pyloriextract increased c-Fos expression and SRE-dependentgene expression. H pylori extract caused phosphorylation of several proteins including a protein with molecular size of 97.4 kDa and tyrosine kinase inhibitor genistein inhibited the activation of ERK and the proliferation of cells caused by H pylori extract.CONCLUSION: Biologically active elements in H pylori extract cause proliferation of gastric epithelial cells through activating tyrosine kinase and ERK signal transduction cascade.

  12. Berberine Promotes Glucose Consumption Independently of AMP-Activated Protein Kinase Activation

    OpenAIRE

    Miao Xu; Yuanyuan Xiao; Jun Yin; Wolin Hou; Xueying Yu; Li Shen; Fang Liu; Li Wei; Weiping Jia

    2014-01-01

    Berberine is a plant alkaloid with anti-diabetic action. Activation of AMP-activated protein kinase (AMPK) pathway has been proposed as mechanism for berberine's action. This study aimed to examine whether AMPK activation was necessary for berberine's glucose-lowering effect. We found that in HepG2 hepatocytes and C2C12 myotubes, berberine significantly increased glucose consumption and lactate release in a dose-dependent manner. AMPK and acetyl coenzyme A synthetase (ACC) phosphorylation wer...

  13. Involvement of Hypothalamic AMP-Activated Protein Kinase in Leptin-Induced Sympathetic Nerve Activation

    OpenAIRE

    Mamoru Tanida; Naoki Yamamoto; Toshishige Shibamoto; Kamal Rahmouni

    2013-01-01

    In mammals, leptin released from the white adipose tissue acts on the central nervous system to control feeding behavior, cardiovascular function, and energy metabolism. Central leptin activates sympathetic nerves that innervate the kidney, adipose tissue, and some abdominal organs in rats. AMP-activated protein kinase (AMPK) is essential in the intracellular signaling pathway involving the activation of leptin receptors (ObRb). We investigated the potential of AMPKα2 in the sympathetic effec...

  14. Cordycepin activates AMP-activated protein kinase (AMPK) via interaction with the γ1 subunit

    OpenAIRE

    Wu, Chongming; Guo, Yanshen; Su, Yan; Zhang, Xue; Luan, Hong; Zhang, Xiaopo; Zhu, Huixin; He, Huixia; Wang, Xiaoliang; Sun, Guibo; Sun, Xiaobo; Guo, Peng; Zhu, Ping

    2013-01-01

    Cordycepin is a bioactive component of the fungus Cordyceps militaris. Previously, we showed that cordycepin can alleviate hyperlipidemia through enhancing the phosphorylation of AMP-activated protein kinase (AMPK), but the mechanism of this stimulation is unknown. Here, we investigated the potential mechanisms of cordycepin-induced AMPK activation in HepG2 cells. Treatment with cordycepin largely reduced oleic acid (OA)-elicited intracellular lipid accumulation and increased AMPK activity in...

  15. Superoxide dismutase activity of Cu-bound prion protein

    Science.gov (United States)

    Hodak, Miroslav; Lu, Wenchang; Bernholc, Jerry

    2009-03-01

    Misfolding of the prion protein, PrP, has been linked to a group of neurodegenerative diseases, including the mad cow disease in cattle and the Creutzfeldt-Jakob disease in humans. The normal function of PrP is still unknown, but it was found that the PrP can efficiently bind Cu(II) ions. Early experiments suggested that Cu-PrP complex possesses significant superoxide dismutase (SOD) activity, but later experiments failed to confirm it and at present this issue remains unresolved. Using a recently developed hybrid DFT/DFT method, which combines Kohn-Sham DFT for the solute and its first solvation shells with orbital-free DFT for the remainder of the solvent, we have investigated SOD activity of PrP. The PrP is capable of incorporating Cu(II) ions in several binding modes and our calculations find that each mode has a different SOD activity. The highest activity found is comparable to those of well-known SOD proteins, suggesting that the conflicting experimental results may be due to different bindings of Cu(II) in those experiments.

  16. Activity of lactoperoxidase when adsorbed on protein layers.

    Science.gov (United States)

    Haberska, Karolina; Svensson, Olof; Shleev, Sergey; Lindh, Liselott; Arnebrant, Thomas; Ruzgas, Tautgirdas

    2008-09-15

    Lactoperoxidase (LPO) is an enzyme, which is used as an antimicrobial agent in a number of applications, e.g., food technology. In the majority of applications LPO is added to a homogeneous product phase or immobilised on product surface. In the latter case, however, the measurements of LPO activity are seldom reported. In this paper we have assessed LPO enzymatic activity on bare and protein modified gold surfaces by means of electrochemistry. It was found that LPO rapidly adsorbs to bare gold surfaces resulting in an amount of LPO adsorbed of 2.9mg/m(2). A lower amount of adsorbed LPO is obtained if the gold surface is exposed to bovine serum albumin, bovine or human mucin prior to LPO adsorption. The enzymatic activity of the adsorbed enzyme is in general preserved at the experimental conditions and varies only moderately when comparing bare gold and gold surface pretreated with the selected proteins. The measurement of LPO specific activity, however, indicate that it is about 1.5 times higher if LPO is adsorbed on gold surfaces containing a small amount of preadsorbed mucin in comparison to the LPO directly adsorbed on bare gold.

  17. Activated G Protein Gαs Samples Multiple Endomembrane Compartments.

    Science.gov (United States)

    Martin, Brent R; Lambert, Nevin A

    2016-09-23

    Heterotrimeric G proteins are localized to the plasma membrane where they transduce extracellular signals to intracellular effectors. G proteins also act at intracellular locations, and can translocate between cellular compartments. For example, Gαs can leave the plasma membrane and move to the cell interior after activation. However, the mechanism of Gαs translocation and its intracellular destination are not known. Here we use bioluminescence resonance energy transfer (BRET) to show that after activation, Gαs rapidly associates with the endoplasmic reticulum, mitochondria, and endosomes, consistent with indiscriminate sampling of intracellular membranes from the cytosol rather than transport via a specific vesicular pathway. The primary source of Gαs for endosomal compartments is constitutive endocytosis rather than activity-dependent internalization. Recycling of Gαs to the plasma membrane is complete 25 min after stimulation is discontinued. We also show that an acylation-deacylation cycle is important for the steady-state localization of Gαs at the plasma membrane, but our results do not support a role for deacylation in activity-dependent Gαs internalization. PMID:27528603

  18. Conservation, variability and the modeling of active protein kinases.

    Directory of Open Access Journals (Sweden)

    James D R Knight

    Full Text Available The human proteome is rich with protein kinases, and this richness has made the kinase of crucial importance in initiating and maintaining cell behavior. Elucidating cell signaling networks and manipulating their components to understand and alter behavior require well designed inhibitors. These inhibitors are needed in culture to cause and study network perturbations, and the same compounds can be used as drugs to treat disease. Understanding the structural biology of protein kinases in detail, including their commonalities, differences and modes of substrate interaction, is necessary for designing high quality inhibitors that will be of true use for cell biology and disease therapy. To this end, we here report on a structural analysis of all available active-conformation protein kinases, discussing residue conservation, the novel features of such conservation, unique properties of atypical kinases and variability in the context of substrate binding. We also demonstrate how this information can be used for structure prediction. Our findings will be of use not only in understanding protein kinase function and evolution, but they highlight the flaws inherent in kinase drug design as commonly practiced and dictate an appropriate strategy for the sophisticated design of specific inhibitors for use in the laboratory and disease therapy.

  19. Hepatitis B virus x protein induces autophagy via activating death-associated protein kinase.

    Science.gov (United States)

    Zhang, H-T; Chen, G G; Hu, B-G; Zhang, Z-Y; Yun, J-P; He, M-L; Lai, P B S

    2014-01-01

    Hepatitis B virus x protein (HBX), a product of hepatitis B virus (HBV), is a multifunctional protein that regulates viral replication and various cellular functions. Recently, HBX has been shown to induce autophagy; however, the responsible mechanism is not fully known. In this study, we established stable HBX-expressing epithelial Chang cells as the platform to study how HBX induced autophagy. The results showed that the overexpression of HBX resulted in starvation-induced autophagy. HBX-induced autophagy was related to its ability to dephosphorylate/activate death-associated protein kinase (DAPK). The block of DAPK by its siRNA significantly counteracted HBX-mediated autophagy, confirming the positive role of DAPK in this process. HBX also induced Beclin 1, which functions at the downstream of the DAPK-mediated autophagy pathway. Although HBX could activate JNK, a kinase known to participate in autophagy in certain conditions, the change in JNK failed to influence HBX-induced autophagy. In conclusion, HBX induces autophagy via activating DAPK in a pathway related to Beclin 1, but not JNK. This new finding should help us to understand the role of autophagy in HBX-mediated pathogenesis and thus may provide targets for intervening HBX-related disorders.

  20. TALE factors poise promoters for activation by Hox proteins.

    Science.gov (United States)

    Choe, Seong-Kyu; Ladam, Franck; Sagerström, Charles G

    2014-01-27

    Hox proteins form complexes with TALE cofactors from the Pbx and Prep/Meis families to control transcription, but it remains unclear how Hox:TALE complexes function. Examining a Hoxb1b:TALE complex that regulates zebrafish hoxb1a transcription, we find maternally deposited TALE proteins at the hoxb1a promoter already during blastula stages. These TALE factors recruit histone-modifying enzymes to promote an active chromatin profile at the hoxb1a promoter and also recruit RNA polymerase II (RNAPII) and P-TEFb. However, in the presence of TALE factors, RNAPII remains phosphorylated on serine 5 and hoxb1a transcription is inefficient. By gastrula stages, Hoxb1b binds together with TALE factors to the hoxb1a promoter. This triggers P-TEFb-mediated transitioning of RNAPII to the serine 2-phosphorylated form and efficient hoxb1a transcription. We conclude that TALE factors access promoters during early embryogenesis to poise them for activation but that Hox proteins are required to trigger efficient transcription.

  1. Fluctuation driven active molecular transport in passive channel proteins

    Science.gov (United States)

    Kosztin, Ioan

    2006-03-01

    Living cells interact with their extracellular environment through the cell membrane, which acts as a protective permeability barrier for preserving the internal integrity of the cell. However, cell metabolism requires controlled molecular transport across the cell membrane, a function that is fulfilled by a wide variety of transmembrane proteins, acting as either passive or active transporters. In this talk it is argued that, contrary to the general belief, in active cell membranes passive and spatially asymmetric channel proteins can act as active transporters by consuming energy from nonequilibrium fluctuations fueled by cell metabolism. This assertion is demonstrated in the case of the E. coli aquaglyceroporin GlpF channel protein, whose high resolution crystal structure is manifestly asymmetric. By calculating the glycerol flux through GlpF within the framework of a stochastic model, it is found that, as a result of channel asymmetry, glycerol uptake driven by a concentration gradient is enhanced significantly in the presence of non-equilibrium fluctuations. Furthermore, the enhancement caused by a ratchet-like mechanism is larger for the outward, i.e., from the cytoplasm to the periplasm, flux than for the inward one, suggesting that the same non-equilibrium fluctuations also play an important role in protecting the interior of the cell against poisoning by excess uptake of glycerol. Preliminary data on water and sugar transport through aquaporin and maltoporin channels, respectively, are indicative of the universality of the proposed nonequilibrium-fluctuation-driven active transport mechanism. This work was supported by grants from the Univ. of Missouri Research Board, the Institute for Theoretical Sciences and the Department of Energy (DOE Contract W-7405-ENG-36), and the National Science Foundation (FIBR-0526854).

  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. Mycobacteriophage putative GTPase-activating protein can potentiate antibiotics.

    Science.gov (United States)

    Yan, Shuangquan; Xu, Mengmeng; Duan, Xiangke; Yu, Zhaoxiao; Li, Qiming; Xie, Longxiang; Fan, Xiangyu; Xie, Jianping

    2016-09-01

    The soaring incidences of infection by antimicrobial resistant (AR) pathogens and shortage of effective antibiotics with new mechanisms of action have renewed interest in phage therapy. This scenario is exemplified by resistant tuberculosis (TB), caused by resistant Mycobacterium tuberculosis. Mycobacteriophage SWU1 A321_gp67 encodes a putative GTPase-activating protein. Mycobacterium smegmatis with gp67 overexpression showed changed colony formation and biofilm morphology and supports the efficacy of streptomycin and capreomycin against Mycobacterium. gp67 down-regulated the transcription of genes involved in cell wall and biofilm development. To our knowledge, this is the first report to show that phage protein in addition to lysin or recombination components can synergize with existing antibiotics. Phage components might represent a promising new clue for better antibiotic potentiators. PMID:27345061

  4. 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. PMID:25890482

  5. Amygdala kindling alters protein kinase C activity in dentate gyrus.

    Science.gov (United States)

    Chen, S J; Desai, M A; Klann, E; Winder, D G; Sweatt, J D; Conn, P J

    1992-11-01

    Kindling is a use-dependent form of synaptic plasticity and a widely used model of epilepsy. Although kindling has been widely studied, the molecular mechanisms underlying induction of this phenomenon are not well understood. We determined the effect of amygdala kindling on protein kinase C (PKC) activity in various regions of rat brain. Kindling stimulation markedly elevated basal (Ca(2+)-independent) and Ca(2+)-stimulated phosphorylation of an endogenous PKC substrate (which we have termed P17) in homogenates of dentate gyrus, assayed 2 h after kindling stimulation. The increase in P17 phosphorylation appeared to be due at least in part to persistent PKC activation, as basal PKC activity assayed in vitro using an exogenous peptide substrate was increased in kindled dentate gyrus 2 h after the last kindling stimulation. A similar increase in basal PKC activity was observed in dentate gyrus 2 h after the first kindling stimulation. These results document a kindling-associated persistent PKC activation and suggest that the increased activity of PKC could play a role in the induction of the kindling effect.

  6. Protein kinase D activity controls endothelial nitric oxide synthesis.

    Science.gov (United States)

    Aicart-Ramos, Clara; Sánchez-Ruiloba, Lucía; Gómez-Parrizas, Mónica; Zaragoza, Carlos; Iglesias, Teresa; Rodríguez-Crespo, Ignacio

    2014-08-01

    Vascular endothelial growth factor (VEGF) regulates key functions of the endothelium, such as angiogenesis or vessel repair in processes involving endothelial nitric oxide synthase (eNOS) activation. One of the effector kinases that become activated in endothelial cells upon VEGF treatment is protein kinase D (PKD). Here, we show that PKD phosphorylates eNOS, leading to its activation and a concomitant increase in NO synthesis. Using mass spectrometry, we show that the purified active kinase specifically phosphorylates recombinant eNOS on Ser1179. Treatment of endothelial cells with VEGF or phorbol 12,13-dibutyrate (PDBu) activates PKD and increases eNOS Ser1179 phosphorylation. In addition, pharmacological inhibition of PKD and gene silencing of both PKD1 and PKD2 abrogate VEGF signaling, resulting in a clear diminished migration of endothelial cells in a wound healing assay. Finally, inhibition of PKD in mice results in an almost complete disappearance of the VEGF-induced vasodilatation, as monitored through determination of the diameter of the carotid artery. Hence, our data indicate that PKD is a new regulatory kinase of eNOS in endothelial cells whose activity orchestrates mammalian vascular tone. PMID:24928905

  7. 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.

    2007-01-01

    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 a

  8. Egg Activation at Fertilization by a Soluble Sperm Protein.

    Science.gov (United States)

    Swann, Karl; Lai, F Anthony

    2016-01-01

    The most fundamental unresolved issue of fertilization is to define how the sperm activates the egg to begin embryo development. Egg activation at fertilization in all species thus far examined is caused by some form of transient increase in the cytoplasmic free Ca(2+) concentration. What has not been clear, however, is precisely how the sperm triggers the large changes in Ca(2+) observed within the egg cytoplasm. Here, we review the studies indicating that the fertilizing sperm stimulates a cytosolic Ca(2+) increase in the egg specifically by delivering a soluble factor that diffuses into the cytosolic space of the egg upon gamete membrane fusion. Evidence is primarily considered in species of eggs where the sperm has been shown to elicit a cytosolic Ca(2+) increase by initiating Ca(2+) release from intracellular Ca(2+) stores. We suggest that our best understanding of these signaling events is in mammals, where the sperm triggers a prolonged series of intracellular Ca(2+) oscillations. The strongest empirical studies to date suggest that mammalian sperm-triggered Ca(2+) oscillations are caused by the introduction of a sperm-specific protein, called phospholipase C-zeta (PLCζ) that generates inositol trisphosphate within the egg. We will discuss the role and mechanism of action of PLCζ in detail at a molecular and cellular level. We will also consider some of the evidence that a soluble sperm protein might be involved in egg activation in nonmammalian species.

  9. Genome activation by raspberry bushy dwarf virus coat protein.

    Science.gov (United States)

    Macfarlane, Stuart A; McGavin, Wendy J

    2009-03-01

    Two sets of infectious cDNA clones of raspberry bushy dwarf virus (RBDV) have been constructed, enabling either the synthesis of infectious RNA transcripts or the delivery of infectious binary plasmid DNA by infiltration of Agrobacterium tumefaciens. In whole plants and in protoplasts, inoculation of RBDV RNA1 and RNA2 transcripts led to a low level of infection, which was greatly increased by the addition of RNA3, a subgenomic RNA coding for the RBDV coat protein (CP). Agroinfiltration of RNA1 and RNA2 constructs did not produce a detectable infection but, again, inclusion of a construct encoding the CP led to high levels of infection. Thus, RBDV replication is greatly stimulated by the presence of the CP, a mechanism that also operates with ilarviruses and alfalfa mosaic virus, where it is referred to as genome activation. Mutation to remove amino acids from the N terminus of the CP showed that the first 15 RBDV CP residues are not required for genome activation. Other experiments, in which overlapping regions at the CP N terminus were fused to the monomeric red fluorescent protein, showed that sequences downstream of the first 48 aa are not absolutely required for genome activation. PMID:19218221

  10. AccaDueO - Solar heating system without antifreeze; AccaDueO - Solaranlage ohne Frostschutzmittel - Schlussbericht

    Energy Technology Data Exchange (ETDEWEB)

    Engeler, L.; Salerno, B.

    2003-12-15

    This illustrated final report for the Swiss Federal Office of Energy (SFOE) describes a solar collector system that uses a heat transfer fluid without antifreeze additives. The so-called 'drain-back' system supplies heat for heating and hot water preparation in a three-family house in Waldenburg, Switzerland, together with a wood-fired boiler. The results of measurements made on the collectors and the storage tank are presented in tabular and graphical form and discussed. The opinions of experts, inhabitants and the general public are noted.

  11. A protein tyrosine phosphatase-like protein from baculovirus has RNA 5'-triphosphatase and diphosphatase activities.

    Science.gov (United States)

    Takagi, T; Taylor, G S; Kusakabe, T; Charbonneau, H; Buratowski, S

    1998-08-18

    The superfamily of protein tyrosine phosphatases (PTPs) includes at least one enzyme with an RNA substrate. We recently showed that the RNA triphosphatase domain of the Caenorhabditis elegans mRNA capping enzyme is related to the PTP enzyme family by sequence similarity and mechanism. The PTP most similar in sequence to the capping enzyme triphosphatase is BVP, a dual-specificity PTP encoded by the Autographa californica nuclear polyhedrosis virus. Although BVP previously has been shown to have modest tyrosine and serine/threonine phosphatase activity, we find that it is much more potent as an RNA 5'-phosphatase. BVP sequentially removes gamma and beta phosphates from the 5' end of triphosphate-terminated RNA, leaving a 5'-monophosphate end. The activity was specific for polynucleotides; nucleotide triphosphates were not hydrolyzed. A mutant protein in which the active site cysteine was replaced with serine was inactive. Three other dual-specificity PTPs (VH1, VHR, and Cdc14) did not exhibit detectable RNA phosphatase activity. Therefore, capping enzyme and BVP are members of a distinct PTP-like subfamily that can remove phosphates from RNA. PMID:9707557

  12. Immersion freezing of ice nucleation active protein complexes

    Science.gov (United States)

    Hartmann, S.; Augustin, S.; Clauss, T.; Wex, H.; Šantl-Temkiv, T.; Voigtländer, J.; Niedermeier, D.; Stratmann, F.

    2013-06-01

    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 nucleation are attached

  13. 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

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

    Directory of Open Access Journals (Sweden)

    Yingying Han

    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.

  15. Protein composition of catalytically active human telomerase from immortal cells

    DEFF Research Database (Denmark)

    Cohen, Scott B; Graham, Mark E; Lovrecz, George O;

    2007-01-01

    Telomerase is a ribonucleoprotein enzyme complex that adds 5'-TTAGGG-3' repeats onto the ends of human chromosomes, providing a telomere maintenance mechanism for approximately 90% of human cancers. We have purified human telomerase approximately 10(8)-fold, with the final elution dependent on the...... enzyme's ability to catalyze nucleotide addition onto a DNA oligonucleotide of telomeric sequence, thereby providing specificity for catalytically active telomerase. Mass spectrometric sequencing of the protein components and molecular size determination indicated an enzyme composition of two molecules...... each of telomerase reverse transcriptase, telomerase RNA, and dyskerin....

  16. The Metastasis-associated Proteins 1 and 2 Form Distinct Protein Complexes with Histone Deacetylase Activity

    Institute of Scientific and Technical Information of China (English)

    Ya-LiYao; Wcn-MingYang

    2005-01-01

    The metastasis-associated protein MTA1 has been shown to express differentially to high levels in metastatic cells. MTA2, which is homologous to MTA1, is a component of the NURD ATP-dependcnt chromatin remodeling and histone deacetylase complex. Here we report evidence that although both human MTA1 and MTA2 repress transcription specifically, are located in the nucleus, and contain associated histone deacetylase activity, they exist in two biochemically distinct protein complexes and may perform different functions pertaining to tumor metastasis. Specifically, both MTA1 and MTA2 complexes exert histone deacetylase activity. However, the MTA1 complex contained HDAC1/2, RbAp46/48, and MBD3, but not Sin3 or Mi2, two important components of the MTA2 complex. Moreover, the MTA2 complex is similar to the HDAC1 complex, suggesting a housekeeping role of the MTA2 complex. The MTA1 complex could be further separated, resulting in acore MTA1-HDAC complex, showing that the histone deacetylase activity and transcriptional repression activity were integral properties of the MTA1 complex. Finally, MTA1, unlike MTA2, did not interact with the pleotropic transcription factor YY1 or the immunophilin FKBP25. We suggest that MTA1 associates with adifferent set of transcription factors from MTA2 and that this property may contribute to the metastatic potential of cells overexpressing MTA1. We also report the finding of human MTA3, which is highly homologous toboth MTA1 and MTA2. However, MTA3 does not repress transcription to a significant level and appears to have a diffused pattern of subcellular localization, suggesting a biological role distinct from that of the other two MTA proteins.

  17. Monomeric G protein-coupled receptor rhodopsin in solution activates its G protein transducin at the diffusion limit

    OpenAIRE

    Ernst, Oliver P.; Gramse, Verena; Kolbe, Michael; Hofmann, Klaus Peter; Heck, Martin

    2007-01-01

    G protein-coupled receptors mediate biological signals by stimulating nucleotide exchange in heterotrimeric G proteins (Gαβγ). Receptor dimers have been proposed as the functional unit responsible for catalytic interaction with Gαβγ. To investigate whether a G protein-coupled receptor monomer can activate Gαβγ, we used the retinal photoreceptor rhodopsin and its cognate G protein transducin (Gt) to determine the stoichiometry of rhodopsin/Gt binding and the rate of catalyzed nucleotide exchan...

  18. Thermally activated charge transport in microbial protein nanowires.

    Science.gov (United States)

    Lampa-Pastirk, Sanela; Veazey, Joshua P; Walsh, Kathleen A; Feliciano, Gustavo T; Steidl, Rebecca J; Tessmer, Stuart H; Reguera, Gemma

    2016-01-01

    The bacterium Geobacter sulfurreducens requires the expression of conductive protein filaments or pili to respire extracellular electron acceptors such as iron oxides and uranium and to wire electroactive biofilms, but the contribution of the protein fiber to charge transport has remained elusive. Here we demonstrate efficient long-range charge transport along individual pili purified free of metal and redox organic cofactors at rates high enough to satisfy the respiratory rates of the cell. Carrier characteristics were within the orders reported for organic semiconductors (mobility) and inorganic nanowires (concentration), and resistivity was within the lower ranges reported for moderately doped silicon nanowires. However, the pilus conductance and the carrier mobility decreased when one of the tyrosines of the predicted axial multistep hopping path was replaced with an alanine. Furthermore, low temperature scanning tunneling microscopy demonstrated the thermal dependence of the differential conductance at the low voltages that operate in biological systems. The results thus provide evidence for thermally activated multistep hopping as the mechanism that allows Geobacter pili to function as protein nanowires between the cell and extracellular electron acceptors. PMID:27009596

  19. Physical activity and high-sensitivity C-reactive protein.

    Science.gov (United States)

    Plaisance, Eric P; Grandjean, Peter W

    2006-01-01

    Cardiovascular disease (CVD) remains one of the leading causes of death and disability in developed countries around the world despite the documented success of lifestyle and pharmacological interventions. This illustrates the multifactorial nature of atherosclerosis and the use of novel inflammatory markers as an adjunct to risk factor reduction strategies. As evidence continues to accumulate that inflammation is involved in all stages of the development and progression of atherosclerosis, markers of inflammation such as high-sensitivity C-reactive protein (CRP) may provide additional information regarding the biological status of the atherosclerotic lesion. Recent investigations suggest that physical activity reduces CRP levels. Higher levels of physical activity and cardiorespiratory fitness are consistently associated with 6-35% lower CRP levels. Longitudinal training studies that have demonstrated reductions in CRP concentrations range from 16% to 41%, an effect that may be independent of baseline levels of CRP, body composition or weight loss. The average change in CRP associated with physical activity appears to be at least as good, if not better, than currently prescribed pharmacological interventions in similar populations. The primary purpose of this review will be to present evidence from both cross-sectional and longitudinal investigations that physical activity lowers CRP levels in a dose-response manner. Finally, this review will examine factors such as body composition, sex, blood sample timing, diet and smoking, which may influence the CRP response to physical activity. PMID:16646631

  20. TALE proteins bind to both active and inactive chromatin.

    Science.gov (United States)

    Scott, James N F; Kupinski, Adam P; Kirkham, Christopher M; Tuma, Roman; Boyes, Joan

    2014-02-15

    TALE (transcription activator-like effector) proteins can be tailored to bind to any DNA sequence of choice and thus are of immense utility for genome editing and the specific delivery of transcription activators. However, to perform these functions, they need to occupy their sites in chromatin. In the present study, we have systematically assessed TALE binding to chromatin substrates and find that in vitro TALEs bind to their target site on nucleosomes at the more accessible entry/exit sites, but not at the nucleosome dyad. We show further that in vivo TALEs bind to transcriptionally repressed chromatin and that transcription increases binding by only 2-fold. These data therefore imply that TALEs are likely to bind to their target in vivo even at inactive loci.

  1. Gene activation by triplex-forming oligonucleotide coupled to the activating domain of protein VP16.

    OpenAIRE

    Kuznetsova, S.; Ait-Si-Ali, S; Nagibneva, I; Troalen, F; Le Villain, J P; Harel-Bellan, A; Svinarchuk, F

    1999-01-01

    Triplex-forming oligonucleotides (TFOs) are generally designed to inhibit transcription or DNA replication but can be used for more diverse purposes. Here we have designed a chimera peptide-TFO able to activate transcription from a target gene. The designed hybrid molecule contains a triplex-forming sequence, linked through a phosphoroamidate bond to several minimal transcriptional activation domains derived from Herpes simplex virus protein 16 (VP16). We show here that this TFO-peptide chime...

  2. 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

  3. “Fuzzy oil drop” model applied to individual small proteins built of 70 amino acids

    OpenAIRE

    Prymula, Katarzyna; Sałapa, Kinga; Roterman, Irena

    2010-01-01

    Abstract The proteins composed of short polypeptides (about 70 amino acid residues) representing the following functional groups (according to PDB notation): growth hormones, serine protease inhibitors, antifreeze proteins, chaperones and proteins of unknown function, were selected for structural and functional analysis. Classification based on the distribution of hydrophobicity in terms of deficiency/excess as the measure of structural and functional specificity is presented. The ...

  4. Protein phosphatase-1 activates CDK9 by dephosphorylating Ser175.

    Directory of Open Access Journals (Sweden)

    Tatiana Ammosova

    Full Text Available The cyclin-dependent kinase CDK9/cyclin T1 induces HIV-1 transcription by phosphorylating the carboxyterminal domain (CTD of RNA polymerase II (RNAPII. CDK9 activity is regulated by protein phosphatase-1 (PP1 which was previously shown to dephosphorylate CDK9 Thr186. Here, we analyzed the effect of PP1 on RNAPII phosphorylation and CDK9 activity. The selective inhibition of PP1 by okadaic acid and by NIPP1 inhibited phosphorylation of RNAPII CTD in vitro and in vivo. Expression of the central domain of NIPP1 in cultured cells inhibited the enzymatic activity of CDK9 suggesting its activation by PP1. Comparison of dephosphorylation of CDK9 phosphorylated by ((32P in vivo and dephosphorylation of CDK9's Thr186 analyzed by Thr186 phospho-specific antibodies, indicated that a residue other than Thr186 might be dephosphorylated by PP1. Analysis of dephosphorylation of phosphorylated peptides derived from CDK9's T-loop suggested that PP1 dephosphorylates CDK9 Ser175. In cultured cells, CDK9 was found to be phosphorylated on Ser175 as determined by combination of Hunter 2D peptide mapping and LC-MS analysis. CDK9 S175A mutant was active and S175D--inactive, and dephosphorylation of CDK9's Ser175 upregulated HIV-1 transcription in PP1-dependent manner. Collectively, our results point to CDK9 Ser175 as novel PP1-regulatory site which dephosphorylation upregulates CDK9 activity and contribute to the activation of HIV-1 transcription.

  5. 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...

  6. Osmotic cell shrinkage activates ezrin/radixin/moesin (ERM) proteins : activation mechanisms and physiological implications

    NARCIS (Netherlands)

    Rasmussen, M.; Alexander, R.T.; Darborg, B.V.; Mobjerg, N.; Hoffmann, E.K.; Kapus, A.; Pedersen, S.F.

    2008-01-01

    Hyperosmotic shrinkage induces multiple cellular responses, including activation of volume-regulatory ion transport, cytoskeletal reorganization, and cell death. Here we investigated the possible roles of ezrin/radixin/moesin (ERM) proteins in these events. Osmotic shrinkage of Ehrlich Lettre ascite

  7. Protein kinase C-dependent activation of P44/42 mitogen-activated protein kinase and heat shock protein 70 in signal transduction during hepatocyte ischemic preconditioning

    Institute of Scientific and Technical Information of China (English)

    Yi Gao; Yu-Qiang Shan; Ming-Xin Pan; Yu Wang; Li-Jun Tang; Hao Li; Zhi Zhang

    2004-01-01

    AIM: To investigate the significance of protein kinase C (PKC), P44/42 mitogen-activated protein kinase (MAPKs) and heat shock protein (HSP)70 signal transduction during hepatocyte ischemic preconditioning.METHODS: In this study we used an in vitro ischemic preconditioning (IP) model for hepatocytes and an in vivo model for rat liver to investigate the significance of protein kinase C (PKC), P44/42 mitogen-activated protein kinase (P44/42 MAPKs) and heat shock protein 70 (HSP70) signal transduction in IP. Through a normal liver cell hypoxic preconditioning (HP) model in which cultured normal liver cells were subjected to 3 cycles of 5 min of incubation under hypoxic conditions followed by 5 min of reoxygenation and subsequently exposed to hypoxia and reoxygenation for 6 h and 9 h respectively. PKC inhibitor, activator and MEK inhibitor were utilized to analyze the phosphorylation of PKC, the expression of P44/42 MAPKs and HSP70.Viability and cellular ultrastructure were also observed. By using rat liver as an in vivo model of liver preconditioning (3 cycles of 10-min occlusion and 10-min reperfusion),in vivo phosphorylation of PKC and P44/42MAPKs, HSP70 expression were further analyzed. AST/ALT concentration,cellular structure and ultrastruture were also observed.All the data were statistically analyzed.RESULTS: Similar results were obtained in both in vivo and in vitro IP models. Compared with the control without IP (or HP), the phosphorylation of PKC and P44/42 MAPKs and the expression of HSP70 were obviously increased in IP (or HP) treated model in which cytoprotection could be found. The effects of preconditioning were mimicked by stimulating PKC with 4β phorobol-12-myristate13-acetate (PMA). Conversely, inhibiting PKC with chelerythrine abolished the protection given by preconditioning. PD98059,inhibitor of MEK (the upstream kinase of P44/42MAPKs),also reverted the cytoprotection exerted by preconditioning.CONCLUSION: The results demonstrate that

  8. Advanced oxidation protein products induce monocyte chemoattractant protein-1 expression via p38 mitogen-activated protein kinase activation in rat vascular smooth muscle cells

    Institute of Scientific and Technical Information of China (English)

    PENG Kan-fu; WU Xiong-fei; ZHAO Hong-wen; SUN Yan

    2006-01-01

    Background Advanced oxidation protein products (AOPPs) are new uremic toxins reported by Witko-Sarsat in 1996, which are associated with the pathogenesis of atherosclerosis. However, the mechanisms by which AOPPs enhance atherosclerosis have not been fully understood. Monocyte chemoattractant protein-1 (MCP-1) is a chemokine which stimulates migration of monocytes and plays a critical role in the development of atherosclerosis. In this study, we investigated the effect of AOPPs on MCP-1 expression in cultured vascular smooth muscle cells (VSMCs).Methods VSMCs were cultured and then co-incubated with AOPP (200 μ mol/L, 400 μ mol/L) for different times with or without pretreatment with specific p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580. RT-PCR and Western blott were used to detect MCP-1 mRNA and protein expression at different time points after AOPP stimulation in rat smooth muscle cells. Western blot was used to detect the expression of phosphorylated p38 MAPK.Results Treatment of VSMC with AOPPs resulted in a significant increase of the expression of MCP- 1 mRNA and protein in time- and dose-dependent manner, and could activated p38 MAPK. Pretreatment of VSMCs with SB203580 resulted in a dose-dependent inhibition of AOPPs-induced MCP-1 mRNA and protein expression.Conclusions AOPPs can stimulate MCP-1 expression via p38 MAPK in VSMCs. This suggests that AOPPs might contribute to the formation of atherosclerosis through this proinflammatory effect.

  9. Protein kinase A binds and activates heat shock factor 1.

    Directory of Open Access Journals (Sweden)

    Ayesha Murshid

    Full Text Available BACKGROUND: Many inducible transcription factors are regulated through batteries of posttranslational modifications that couple their activity to inducing stimuli. We have studied such regulation of Heat Shock Factor 1 (HSF1, a key protein in control of the heat shock response, and a participant in carcinogenisis, neurological health and aging. As the mechanisms involved in the intracellular regulation of HSF1 in good health and its dysregulation in disease are still incomplete we are investigating the role of posttranslational modifications in such regulation. METHODOLOGY/PRINCIPAL FINDINGS: In a proteomic study of HSF1 binding partners, we have discovered its association with the pleiotropic protein kinase A (PKA. HSF1 binds avidly to the catalytic subunit of PKA, (PKAcα and becomes phosphorylated on a novel serine phosphorylation site within its central regulatory domain (serine 320 or S320, both in vitro and in vivo. Intracellular PKAcα levels and phosphorylation of HSF1 at S320 were both required for HSF1 to be localized to the nucleus, bind to response elements in the promoter of an HSF1 target gene (hsp70.1 and activate hsp70.1 after stress. Reduction in PKAcα levels by small hairpin RNA led to HSF1 exclusion from the nucleus, its exodus from the hsp70.1 promoter and decreased hsp70.1 transcription. Likewise, null mutation of HSF1 at S320 by alanine substitution for serine led to an HSF1 species excluded from the nucleus and deficient in hsp70.1 activation. CONCLUSIONS: These findings of PKA regulation of HSF1 through S320 phosphorylation add to our knowledge of the signaling networks converging on this factor and may contribute to elucidating its complex roles in the stress response and understanding HSF1 dysregulation in disease.

  10. 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.

  11. A Variable Light Domain Fluorogen Activating Protein Homodimerizes To Activate Dimethylindole Red

    Energy Technology Data Exchange (ETDEWEB)

    Senutovitch, Nina; Stanfield, Robyn L.; Bhattacharyya, Shantanu; Rule, Gordon S.; Wilson, Ian A.; Armitage, Bruce A.; Waggoner, Alan S.; Berget, Peter B. (Scripps); (CM)

    2012-07-11

    Novel fluorescent tools such as green fluorescent protein analogues and fluorogen activating proteins (FAPs) are useful in biological imaging for tracking protein dynamics in real time with a low fluorescence background. FAPs are single-chain variable fragments (scFvs) selected from a yeast surface display library that produce fluorescence upon binding a specific dye or fluorogen that is normally not fluorescent when present in solution. FAPs generally consist of human immunoglobulin variable heavy (V{sub H}) and variable light (V{sub L}) domains covalently attached via a glycine- and serine-rich linker. Previously, we determined that the yeast surface clone, V{sub H}-V{sub L} M8, could bind and activate the fluorogen dimethylindole red (DIR) but that the fluorogen activation properties were localized to the M8V{sub L} domain. We report here that both nuclear magnetic resonance and X-ray diffraction methods indicate the M8V{sub L} forms noncovalent, antiparallel homodimers that are the fluorogen activating species. The M8V{sub L} homodimers activate DIR by restriction of internal rotation of the bound dye. These structural results, together with directed evolution experiments with both V{sub H}-V{sub L} M8 and M8V{sub L}, led us to rationally design tandem, covalent homodimers of M8V{sub L} domains joined by a flexible linker that have a high affinity for DIR and good quantum yields.

  12. Danthron activates AMP-activated protein kinase and regulates lipid and glucose metabolism in vitro

    Institute of Scientific and Technical Information of China (English)

    Rong ZHOU; Ling WANG; Xing XU; Jing CHEN; Li-hong HU; Li-li CHEN; Xu SHEN

    2013-01-01

    Aim:To discover the active compound on AMP-activated protein kinase (AMPK) activation and investigate the effects of the active compound 1,8-dihydroxyanthraquinone (danthron) from the traditional Chinese medicine rhubarb on AMPK-mediated lipid and glucose metabolism in vitro.Methods:HepG2 and C2C12 cells were used.Cell viability was determined using MTT assay.Real-time PCR was performed to measure the gene expression.Western blotting assay was applied to investigate the protein phosphorylation level.Enzymatic assay kits were used to detect the total cholesterol (TC),triglyceride (TG) and glucose contents.Results:Danthron (0.1,1,and 10 μmol/L) dose-dependently promoted the phosphorylation of AMPK and acetyl-CoA carboxylase (ACC)in both HepG2 and C2C12 cells.Meanwhile,danthron treatment significantly reduced the lipid synthesis related sterol regulatory element-binding protein 1c (SREBP1c) and fatty acid synthetase (FAS) gene expressions,and the TC and TG levels.In addition,danthron treatment efficiently increased glucose consumption.The actions of danthron on lipid and glucose metabolism were abolished or reversed by co-treatment with the AMPK inhibitor compound C.Conclusion:Danthron effectively reduces intracellular lipid contents and enhanced glucose consumption in vitro via activation of AMPK signaling pathway.

  13. Leishmania amazonensis: PKC-like protein kinase modulates the (Na++K+)ATPase activity.

    Science.gov (United States)

    Almeida-Amaral, Elmo Eduardo de; Caruso-Neves, Celso; Lara, Lucienne Silva; Pinheiro, Carla Mônica; Meyer-Fernandes, José Roberto

    2007-08-01

    The present study aimed to identify the presence of protein kinase C-like (PKC-like) in Leishmania amazonensis and to elucidate its possible role in the modulation of the (Na(+)+K(+))ATPase activity. Immunoblotting experiments using antibody against a consensus sequence (Ac 543-549) of rabbit protein kinase C (PKC) revealed the presence of a protein kinase of 80 kDa in L. amazonensis. Measurements of protein kinase activity showed the presence of both (Ca(2+)-dependent) and (Ca(2+)-independent) protein kinase activity in plasma membrane and cytosol. Phorbol ester (PMA) activation of the Ca(2+)-dependent protein kinase stimulated the (Na(+)+K(+))ATPase activity, while activation of the Ca(2+)-independent protein kinase was inhibitory. Both effects of protein kinase on the (Na(+)+K(+))ATPase of the plasma membrane were lower than that observed in intact cells. PMA induced the translocation of protein kinase from cytosol to plasma membrane, indicating that the maximal effect of protein kinase on the (Na(+)+K(+))ATPase activity depends on the synergistic action of protein kinases from both plasma membrane and cytosol. This is the first demonstration of a protein kinase activated by PMA in L. amazonensis and the first evidence for a possible role in the regulation of the (Na(+)+K(+))ATPase activity in this trypanosomatid. Modulation of the (Na(+)+K(+))ATPase by protein kinase in a trypanosomatid opens up new possibilities to understand the regulation of ion homeostasis in this parasite. PMID:17475255

  14. AMP-activated protein kinase (AMPK) activation regulates in vitro bone formation and bone mass

    OpenAIRE

    Shah, M; Kola, B; Bataveljic, A.; Arnett, T. R.; Viollet, B.; Saxon, L.; Korbonits, M.; C. Chenu

    2010-01-01

    Adenosine 5′-monophosphate-activated protein kinase (AMPK), a regulator of energy homeostasis, has a central role in mediating the appetite-modulating and metabolic effects of many hormones and antidiabetic drugs metformin and glitazones. The objective of this study was to determine if AMPK can be activated in osteoblasts by known AMPK modulators and if AMPK activity is involved in osteoblast function in vitro and regulation of bone mass in vivo. ROS 17/2.8 rat osteoblast-like cells were cult...

  15. 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.

  16. 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.

  17. Stimulation of IGF-binding protein-1 secretion by AMP-activated protein kinase.

    Science.gov (United States)

    Lewitt, M S

    2001-04-20

    Insulin-like growth factor-binding protein-1 (IGFBP-1) is stimulated during intensive exercise and in catabolic conditions to very high concentrations, which are not completely explained by known regulators such as insulin and glucocorticoids. The role of AMP-activated protein kinase (AMPK), an important signaling system in lipid and carbohydrate metabolism, in regulating IGFBP-1 was studied in H4-II-E rat hepatoma cells. Arsenic(III) oxide and 5-aminoimidazole-4-carboxamide-riboside (AICAR) were used as activators. AICAR (150 microM) stimulated IGFBP-1 secretion twofold during a 5-h incubation (P = 0.002). Insulin (100 ng/ml) inhibited IGFBP-1 by 80% (P < 0.001), but this was completely abolished in the presence of 150 microM AICAR. The effect of dexamethasone in stimulating IGFBP-1 threefold was additive to the effect of AICAR (P < 0.001) and, in the presence of AICAR, was incompletely inhibited by insulin. In conclusion AMPK is identified as a novel regulatory pathway for IGFBP-1, stimulating secretion and blocking the inhibitory effect of insulin. PMID:11302732

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

    Science.gov (United States)

    Zółtowska, Krystyna; Lipiński, Zbigniew; Dmitryjuk, Małgorzata

    2005-01-01

    The proteins level and activities of acid and alkaline proteases in whole body extracts of drone prepupae of Apis mellifera naturally infested with Varroa destructor were studied. The infested and a non-infested group did not differ significantly in their total protein content. However, some differences in protein profiles were found. A lack of three protein fractions of moderate and lower molecular weight in infested prepupae was noted. Moreover, some differences in the quantity of protein in most of the fractions were observed. The activity of acid proteases from infested prepupae was lower (p < 0.05) compared with the activity of these proteases from the non-infested one group. The infested drone had higher activity of alkaline proteases than non-infested but this difference was not statisticaly significant. PMID:16841690

  19. Activation of mitogen-activated protein kinase pathway by extremely low-dose ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Keiji; Kodama, Seiji; Watanabe, Masami [Nagasaki Univ., Graduate School of Biomedical Sciences, Nagasaki (Japan)

    2003-07-01

    We demonstrated here that X-ray irradiation at very low doses of between 2 and 5 cGy stimulated activity of a member of mitogen-activated protein (MAP) kinase, the extracellular signal-regulated kinase (ERK) 1/2, in normal human diploid cells. Higher doses of irradiation at more than 1 Gy induced phosphorylation of ERK1/2 and accumulated p53 protein. Phosphorylation of ERK1/2 decreased with dose down to 50 cGy, however, doses of between 5 cGy and 2 cGy phosphorylated ERK1/2 as efficiently as higher doses of X-rays, while the p53 protein level was no longer changed by doses below 50 cGy. ATM-dependent phosphorylation of p53 protein at Ser15 and histone H2AX at Ser139 was only observed at higher doses at more than 10 cGy of X-rays. We found that MEK1 was phosphorylated with both 2 cGy and 6 Gy of X-rays, and that the MEK1 inhibitor, PD98059 decreased phosphorylation of the ERK1/2 proteins induced by 2 cGy or 6 Gy of X-rays. Similar suppressive effect was observed with the specific epidermal growth factor (EGF) receptor tyrosine kinase inhibitor, AG1478. These results indicate that a limited range of low dose ionizing radiation differentially activate ERK1/2 kinases via activation of EGF receptor and MEK, which mediates various effects of cells receiving very low doses of ionizing radiation. (author)

  20. Cordycepin activates AMP-activated protein kinase (AMPK) via interaction with the γ1 subunit

    Science.gov (United States)

    Wu, Chongming; Guo, Yanshen; Su, Yan; Zhang, Xue; Luan, Hong; Zhang, Xiaopo; Zhu, Huixin; He, Huixia; Wang, Xiaoliang; Sun, Guibo; Sun, Xiaobo; Guo, Peng; Zhu, Ping

    2014-01-01

    Cordycepin is a bioactive component of the fungus Cordyceps militaris. Previously, we showed that cordycepin can alleviate hyperlipidemia through enhancing the phosphorylation of AMP-activated protein kinase (AMPK), but the mechanism of this stimulation is unknown. Here, we investigated the potential mechanisms of cordycepin-induced AMPK activation in HepG2 cells. Treatment with cordycepin largely reduced oleic acid (OA)-elicited intracellular lipid accumulation and increased AMPK activity in a dose-dependent manner. Cordycepin-induced AMPK activation was not accompanied by changes in either the intracellular levels of AMP or the AMP/ATP ratio, nor was it influenced by calmodulin-dependent protein kinase kinase (CaMKK) inhibition; however, this activation was significantly suppressed by liver kinase B1 (LKB1) knockdown. Molecular docking, fluorescent and circular dichroism measurements showed that cordycepin interacted with the γ1 subunit of AMPK. Knockdown of AMPKγ1 by siRNA substantially abolished the effects of cordycepin on AMPK activation and lipid regulation. The modulating effects of cordycepin on the mRNA levels of key lipid regulatory genes were also largely reversed when AMPKγ1 expression was inhibited. Together, these data suggest that cordycepin may inhibit intracellular lipid accumulation through activation of AMPK via interaction with the γ1 subunit. PMID:24286368

  1. Activation of purified calcium channels by stoichiometric protein phosphorylation

    International Nuclear Information System (INIS)

    Purified dihydropyridine-sensitive calcium channels from rabbit skeletal muscle were reconstituted into phosphatidylcholine vesicles to evaluate the effect of phosphorylation by cyclic AMP-dependent protein kinase (PK-A) on their function. Both the rate and extent of 45Ca2+ uptake into vesicles containing reconstituted calcium channels were increased severalfold after incubation with ATP and PK-A. The degree of stimulation of 45Ca2+ uptake was linearly proportional to the extent of phosphorylation of the alpha 1 and beta subunits of the calcium channel up to a stoichiometry of approximately 1 mol of phosphate incorporated into each subunit. The calcium channels activated by phosphorylation were determined to be incorporated into the reconstituted vesicles in the inside-out orientation and were completely inhibited by low concentrations of dihydropyridines, phenylalkylamines, Cd2+, Ni2+, and Mg2+. The results demonstrate a direct relationship between PK-A-catalyzed phosphorylation of the alpha 1 and beta subunits of the purified calcium channel and activation of the ion conductance activity of the dihydropyridine-sensitive calcium channels

  2. Multifunctional antimicrobial proteins and peptides: natural activators of immune systems.

    Science.gov (United States)

    Niyonsaba, François; Nagaoka, Isao; Ogawa, Hideoki; Okumura, Ko

    2009-01-01

    In addition to the physical barrier of the stratum corneum, cutaneous innate immunity also includes the release of various humoral mediators, such as cytokines and chemokines, recruitment and activation of phagocytes, and the production of antimicrobial proteins/peptides (AMPs). AMPs form an innate epithelial chemical shield, which provides a front-line component in innate immunity to inhibit microbial invasion; however, this might be an oversimplification of the diverse functions of these molecules. In fact, apart from exhibiting a broad spectrum of microbicidal properties, it is increasingly evident that AMPs display additional activities that are related to the stimulation and modulation of the cutaneous immune system. These diverse functions include chemoattraction and activation of immune and/or inflammatory cells, the production and release of cytokines and chemokines, acceleration of angiogenesis, promotion of wound healing, neutralization of harmful microbial products, and bridging of both innate and adaptive immunity. Thus, better understanding of the functions of AMPs in skin and identification of their signaling mechanisms may offer new strategies for the development of potential therapeutics for the treatment of infection- and/or inflammation-related skin diseases. Here, we briefly outline the structure, regulation of expression, and multifunctional roles of principal skin-derived AMPs.

  3. Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 Deficiency Reduces Insulin Sensitivity in High-Fat Diet-Fed Mice

    NARCIS (Netherlands)

    de Boer, Jan Freark; Dikkers, Arne; Jurdzinski, Angelika; von Felden, Johann; Gaestel, Matthias; Bavendiek, Udo; Tietge, Uwe J. F.

    2014-01-01

    Adipose tissue inflammation is considered an important contributor to insulin resistance. Mitogen-activated protein kinase-activated protein kinase 2 (MK2) is a major downstream target of p38 MAPK and enhances inflammatory processes. In line with the role of MK2 as contributor to inflammation, MK2(-

  4. Mitogen-activated protein kinase activator with WD40 repeats (MAWD) and MAWD-binding protein induce cell differentiation in gastric cancer

    OpenAIRE

    Li, Dongmei; Zhang, Jun; Xi, Yu; Zhang, Lei; Li, Wenmei; Cui, Jiantao; Xing, Rui; Pan, Yuanmin; Pan, Zemin; Li, Feng; Lu, Youyong

    2015-01-01

    Background Our previous proteomic analysis revealed that mitogen-activated protein kinase activator with WD40 repeats (MAWD) and MAWD-binding protein (MAWBP) were downregulated in gastric cancer (GC) tissues. These proteins interacted and formed complexes in GC cells. To investigate the role of MAWD and MAWBP in GC differentiation, we analyzed the relationship between MAWD/MAWBP and clinicopathologic characteristics of GC tissues and examined the expression of E-cadherin and pepsinogen C (PGC...

  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. Tribomechanical micronization and activation of whey protein concentrate and zeolite

    Indian Academy of Sciences (India)

    Z Herceg; V Lelas; M Brnčić; B Tripalo; D Ježek

    2004-02-01

    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 treated with the laboratory TMA equipment. The treatment was carried out at two various rotor speeds: 16,000 and 22,000 r.p.m. at ambient temperature. Analyses of the particle size and distribution as well as the specific area and scanning electron microscopy were carried out on the powdered WPC and zeolite, before and after the TMA treatment. Suspensions of the WPC and zeolite were treated with ultrasound, just before determining the particle size distribution, at 50 kHz. The results showed that tribomechanical treatment causes significant decrease in particle size, change in particle size distribution and increase in specific area of WPC and zeolite. These changes of the treated materials depend on the type of the material, the level of inserting particles, the planned angle of the impact, internal rubbing and the planned number of impacts. The effects found became stronger as the rotor speed of the TMA equipment increased (16,000 to 22,000 rpm). Ultrasonic treatment of suspension of tribomechanically treated WPC resulted infurther breakdown of partly damaged protein globules as proved with the statistic analyses. No further changes in their granulometric composition were caused by ultrasonic treatment of a suspension of tribomechanically treated zeolite.

  7. Involvement of protein kinase C activation in L-leucine-induced stimulation of protein synthesis in l6 myotubes.

    Science.gov (United States)

    Yagasaki, Kazumi; Morisaki, Naoko; Kitahara, Yoshiro; Miura, Atsuhito; Funabiki, Ryuhei

    2003-11-01

    Effects of leucine and related compounds on protein synthesis were studied in L6 myotubes. The incorporation of [(3)H]tyrosine into cellular protein was measured as an index of protein synthesis. In leucine-depleted L6 myotubes, leucine and its keto acid, alpha-ketoisocaproic acid (KIC), stimulated protein synthesis, while D-leucine did not. Mepacrine, an inhibitor of both phospholipases A(2) and C, canceled stimulatory actions of L-leucine and KIC on protein synthesis. Neither indomethacin, an inhibitor of cyclooxygenase, nor caffeic acid, an inhibitor of lipoxygenase, diminished their stimulatory actions, suggesting no involvement of arachidonic acid metabolism. Conversely, 1-O-hexadecyl-2-O-methylglycerol, an inhibitor of proteinkinase C, significantly canceled the stimulatory actions of L-leucine and KIC on protein synthesis, suggesting an involvement of phosphatidylinositol degradation and activation of protein kinase C. L-Leucine caused a rapid activation of protein kinase C in both cytosol and membrane fractions of the cells. These results strongly suggest that both L-leucine and KIC stimulate protein synthesis in L6 myotubes through activation of phospholipase C and protein kinase C. PMID:19003213

  8. 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.

  9. Protein engineering,expression,and activity of a novel fusion protein possessing keratinocyte growth factor 2 and fibronectin

    Institute of Scientific and Technical Information of China (English)

    Wonmo Kang; Junhyeog Jang

    2009-01-01

    Growth factor-induced proliferation and differentiation often require adhesion of cells to the extracellular matrix proteins such as fibronectin(FN).In this study,we aimed to investigate the effect of protein engineering of the keratinocyte growth factor 2(KGF2)fused to the FN on the mitogenic activity of KGF2.The fusion protein(KGF2-FN10),which was expressed in Escherichia coli,showed significantly enhanced mitogenic activity of KGF2 on human keratinocytes.Moreover,KGF2-FN10 fusion protein showed significantly increased activity to differentiate keratinocytes from native KGF2.In conclusion,these results suggest that KGF2-FN10 fusion protein has certain advantages over native KGF2 and may offer a novel strategy to potentiate the therapeutic effect of KGF2.

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

    Science.gov (United States)

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

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

    KAUST Repository

    Al-Tawashi, 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.

  12. Enhanced biocontrol activity of Trichoderma through inactivation of a mitogen-activated protein kinase

    OpenAIRE

    Mendoza-Mendoza, Artemio; Pozo, María J.; Grzegorski, Darlene; Martínez, Pedro; García, Juan M.; Olmedo-Monfil, Vianey; Cortés, Carlos; Kenerley, Charles; Herrera-Estrella, Alfredo

    2003-01-01

    The production of lytic enzymes in Trichoderma is considered determinant in its parasitic response against fungal species. A mitogen-activated protein kinase encoding gene, tvk1, from Trichoderma virens was cloned, and its role during the mycoparasitism, conidiation, and biocontrol was examined in tvk1 null mutants. These mutants showed a clear increase in the level of the expression of mycoparasitism-related genes under simulated mycoparasitism and during direct confrontation with the plant ...

  13. Mitogen-Activated Protein Kinases and Hypoxic/Ischemic Nephropathy

    Directory of Open Access Journals (Sweden)

    Fengbao Luo

    2016-08-01

    Full Text Available Tissue hypoxia/ischemia is a pathological feature of many human disorders including stroke, myocardial infarction, hypoxic/ischemic nephropathy, as well as cancer. In the kidney, the combination of limited oxygen supply to the tissues and high oxygen demand is considered the main reason for the susceptibility of the kidney to hypoxic/ischemic injury. In recent years, increasing evidence has indicated that a reduction in renal oxygen tension/blood supply plays an important role in acute kidney injury, chronic kidney disease, and renal tumorigenesis. However, the underlying signaling mechanisms, whereby hypoxia alters cellular behaviors, remain poorly understood. Mitogen-activated protein kinases (MAPKs are key signal-transducing enzymes activated by a wide range of extracellular stimuli, including hypoxia/ischemia. There are four major family members of MAPKs: the extracellular signal-regulated kinases-1 and -2 (ERK1/2, the c-Jun N-terminal kinases (JNK, p38 MAPKs, and extracellular signal-regulated kinase-5 (ERK5/BMK1. Recent studies, including ours, suggest that these MAPKs are differentially involved in renal responses to hypoxic/ischemic stress. This review will discuss their changes in hypoxic/ischemic pathophysiology with acute kidney injury, chronic kidney diseases and renal carcinoma.

  14. Activation of tracheal smooth muscle contraction: synergism between Ca2+ and activators of protein kinase C.

    OpenAIRE

    Park, S.; Rasmussen, H

    1985-01-01

    The effects of divalent ionophores (A23187 and ionomycin), Ca2+ channel agonist (BAY K 8644), and protein kinase C (C-kinase) activators [phorbol 12-myristate 13-acetate (PMA), mezerein] on bovine tracheal smooth muscle contraction were investigated. A23187 (5 microM) and ionomycin (0.5 microM) produced a prompt but transient contraction. C-kinase activators either produced no effect--e.g., PMA at 200 nM--or produced a rise in tension that was slow in onset but then gradually increased--e.g.,...

  15. Activation of multiple mitogen-activated protein kinases by recombinant calcitonin gene-related peptide receptor.

    Science.gov (United States)

    Parameswaran, N; Disa, J; Spielman, W S; Brooks, D P; Nambi, P; Aiyar, N

    2000-02-18

    Calcitonin gene-related peptide is a 37-amino-acid neuropeptide and a potent vasodilator. Although calcitonin gene-related peptide has been shown to have a number of effects in a variety of systems, the mechanisms of action and the intracellular signaling pathways, especially the regulation of mitogen-activated protien kinase (MAPK) pathway, is not known. In the present study we investigated the role of calcitonin gene-related peptide in the regulation of MAPKs in human embryonic kidney (HEK) 293 cells stably transfected with a recombinant porcine calcitonin gene-related peptide-1 receptor. Calcitonin gene-related peptide caused a significant dose-dependent increase in cAMP response and the effect was inhibited by calcitonin gene-related peptide(8-37), the calcitonin gene-related peptide-receptor antagonist. Calcitonin gene-related peptide also caused a time- and concentration-dependent increase in extracellular signal-regulated kinase (ERK) and P38 mitogen-activated protein kinase (P38 MAPK) activities, with apparently no significant change in cjun-N-terminal kinase (JNK) activity. Forskolin, a direct activator of adenylyl cyclase also stimulated ERK and P38 activities in these cells suggesting the invovement of cAMP in this process. Calcitonin gene-related peptide-stimulated ERK and P38 MAPK activities were inhibited significantly by calcitonin gene-related peptide receptor antagonist, calcitonin gene-related peptide-(8-37) suggesting the involvement of calcitonin gene-related peptide-1 receptor. Preincubation of the cells with the cAMP-dependent protein kinase inhibitor, H89 [¿N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride¿] inhibited calcitonin gene-related peptide-mediated activation of ERK and p38 kinases. On the other hand, preincubation of the cells with wortmannin ¿[1S-(1alpha,6balpha,9abeta,11alpha, 11bbeta)]-11-(acetyloxy)-1,6b,7,8,9a,10,11, 11b-octahydro-1-(methoxymethyl)-9a,11b-dimethyl-3H-furo[4,3, 2-de]indeno[4,5-h]-2

  16. Regulation of orange carotenoid protein activity in cyanobacterial photoprotection

    NARCIS (Netherlands)

    Thurotte, A.; Lopez Igual, R.; Wilson, A.; Comolet, L.; Bourcier de Carbon, C.; Xiao, F.; Kirilovsky, D.

    2015-01-01

    Plants, algae, and cyanobacteria have developed mechanisms to decrease the energy arriving at reaction centers to protect themselves from high irradiance. In cyanobacteria, the photoactive Orange Carotenoid Protein (OCP) and the Fluorescence Recovery Protein are essential elements in this mechanism.

  17. The Identification of Novel Protein-Protein Interactions in Liver that Affect Glucagon Receptor Activity.

    Directory of Open Access Journals (Sweden)

    Junfeng Han

    Full Text Available Glucagon regulates glucose homeostasis by controlling glycogenolysis and gluconeogenesis in the liver. Exaggerated and dysregulated glucagon secretion can exacerbate hyperglycemia contributing to type 2 diabetes (T2D. Thus, it is important to understand how glucagon receptor (GCGR activity and signaling is controlled in hepatocytes. To better understand this, we sought to identify proteins that interact with the GCGR to affect ligand-dependent receptor activation. A Flag-tagged human GCGR was recombinantly expressed in Chinese hamster ovary (CHO cells, and GCGR complexes were isolated by affinity purification (AP. Complexes were then analyzed by mass spectrometry (MS, and protein-GCGR interactions were validated by co-immunoprecipitation (Co-IP and Western blot. This was followed by studies in primary hepatocytes to assess the effects of each interactor on glucagon-dependent glucose production and intracellular cAMP accumulation, and then in immortalized CHO and liver cell lines to further examine cell signaling. Thirty-three unique interactors were identified from the AP-MS screening of GCGR expressing CHO cells in both glucagon liganded and unliganded states. These studies revealed a particularly robust interaction between GCGR and 5 proteins, further validated by Co-IP, Western blot and qPCR. Overexpression of selected interactors in mouse hepatocytes indicated that two interactors, LDLR and TMED2, significantly enhanced glucagon-stimulated glucose production, while YWHAB inhibited glucose production. This was mirrored with glucagon-stimulated cAMP production, with LDLR and TMED2 enhancing and YWHAB inhibiting cAMP accumulation. To further link these interactors to glucose production, key gluconeogenic genes were assessed. Both LDLR and TMED2 stimulated while YWHAB inhibited PEPCK and G6Pase gene expression. In the present study, we have probed the GCGR interactome and found three novel GCGR interactors that control glucagon

  18. Curcumin attenuates diet-induced hepatic steatosis by activating AMP-activated protein kinase.

    Science.gov (United States)

    Um, Min Young; Hwang, Kwang Hyun; Ahn, Jiyun; Ha, Tae Youl

    2013-09-01

    Curcumin is a well-known component of traditional turmeric (Curcuma longa), which has been reported to prevent obesity and diabetes. However, the effect of curcumin on hepatic lipid metabolism remains unclear. The aim of this study was to examine the effects of curcumin on hepatic steatosis in high-fat/cholesterol diet (HFD)-induced obese mice. Male C57BL/6J mice were fed a normal diet (ND), HFD or HFD with 0.15% curcumin (HFD+C) for 11 weeks. We found that curcumin significantly lowered the body-weight and adipose tissue weight of mice in the HFD+C group compared with the findings for the HFD group (p cholesterol, fasting glucose and insulin in serum were decreased, and HFD-induced impairment of insulin sensitivity was improved by curcumin supplementation (p Curcumin protected against the development of hepatic steatosis by reducing hepatic fat accumulation. Moreover, curcumin activated AMP-activated protein kinase (AMPK) and elevated the gene expression of peroxisome proliferator-activated receptor alpha. By contrast, curcumin suppressed the HFD-mediated increases in sterol regulatory element-binding protein-1, acetyl-CoA carboxylase 1, fatty acid synthase and cluster of differentiation 36 expression. Taken together, these findings indicate that curcumin attenuates HFD-induced hepatic steatosis by regulating hepatic lipid metabolism via AMPK activation, suggesting its use as a therapeutic for hepatic steatosis.

  19. New Tricks for Old Proteins: Single Mutations in a Nonenzymatic Protein Give Rise to Various Enzymatic Activities.

    Science.gov (United States)

    Moroz, Yurii S; Dunston, Tiffany T; Makhlynets, Olga V; Moroz, Olesia V; Wu, Yibing; Yoon, Jennifer H; Olsen, Alissa B; McLaughlin, Jaclyn M; Mack, Korrie L; Gosavi, Pallavi M; van Nuland, Nico A J; Korendovych, Ivan V

    2015-12-01

    Design of a new catalytic function in proteins, apart from its inherent practical value, is important for fundamental understanding of enzymatic activity. Using a computationally inexpensive, minimalistic approach that focuses on introducing a single highly reactive residue into proteins to achieve catalysis we converted a 74-residue-long C-terminal domain of calmodulin into an efficient esterase. The catalytic efficiency of the resulting stereoselective, allosterically regulated catalyst, nicknamed AlleyCatE, is higher than that of any previously reported de novo designed esterases. The simplicity of our design protocol should complement and expand the capabilities of current state-of-art approaches to protein design. These results show that even a small nonenzymatic protein can efficiently attain catalytic activities in various reactions (Kemp elimination, ester hydrolysis, retroaldol reaction) as a result of a single mutation. In other words, proteins can be just one mutation away from becoming entry points for subsequent evolution.

  20. 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.

  1. Enhanced transcriptional activation by E2 proteins from the oncogenic human papillomaviruses.

    OpenAIRE

    Kovelman, R; Bilter, G K; Glezer, E; Tsou, A Y; Barbosa, M S

    1996-01-01

    A systematic comparison of transcriptional activation by papillomavirus E2 proteins revealed that the E2 proteins from high-risk human papillomaviruses (human papillomavirus type 16 [HPV-16] and HPV-18) are much more active than are the E2 proteins from low-risk HPVs (HPV-6b and HPV-11). Despite the tropism of HPVs for particular epithelial cell types, this difference in transcriptional activation was observed in a number of different epithelial and nonepithelial cells. The enhanced activitie...

  2. AMP-activated protein kinase (AMPK) activation regulates in vitro bone formation and bone mass.

    Science.gov (United States)

    Shah, M; Kola, B; Bataveljic, A; Arnett, T R; Viollet, B; Saxon, L; Korbonits, M; Chenu, C

    2010-08-01

    Adenosine 5'-monophosphate-activated protein kinase (AMPK), a regulator of energy homeostasis, has a central role in mediating the appetite-modulating and metabolic effects of many hormones and antidiabetic drugs metformin and glitazones. The objective of this study was to determine if AMPK can be activated in osteoblasts by known AMPK modulators and if AMPK activity is involved in osteoblast function in vitro and regulation of bone mass in vivo. ROS 17/2.8 rat osteoblast-like cells were cultured in the presence of AMPK activators (AICAR and metformin), AMPK inhibitor (compound C), the gastric peptide hormone ghrelin and the beta-adrenergic blocker propranolol. AMPK activity was measured in cell lysates by a functional kinase assay and AMPK protein phosphorylation was studied by Western Blotting using an antibody recognizing AMPK Thr-172 residue. We demonstrated that treatment of ROS 17/2.8 cells with AICAR and metformin stimulates Thr-172 phosphorylation of AMPK and dose-dependently increases its activity. In contrast, treatment of ROS 17/2.8 cells with compound C inhibited AMPK phosphorylation. Ghrelin and propranolol dose-dependently increased AMPK phosphorylation and activity. Cell proliferation and alkaline phosphatase activity were not affected by metformin treatment while AICAR significantly inhibited ROS 17/2.8 cell proliferation and alkaline phosphatase activity at high concentrations. To study the effect of AMPK activation on bone formation in vitro, primary osteoblasts obtained from rat calvaria were cultured for 14-17days in the presence of AICAR, metformin and compound C. Formation of 'trabecular-shaped' bone nodules was evaluated following alizarin red staining. We demonstrated that both AICAR and metformin dose-dependently increase trabecular bone nodule formation, while compound C inhibits bone formation. When primary osteoblasts were co-treated with AICAR and compound C, compound C suppressed the stimulatory effect of AICAR on bone nodule formation

  3. A specific, transmembrane interface regulates fibroblast activation protein (FAP) homodimerization, trafficking and exopeptidase activity.

    Science.gov (United States)

    Wonganu, Benjamaporn; Berger, Bryan W

    2016-08-01

    Fibroblast activation protein (FAP) is a cell-surface serine protease which promotes invasiveness of certain epithelial cancers and is therefore a potential target for cancer drug development and delivery. Unlike dipeptidyl peptidase IV (DPPIV), FAP exhibits prolyl endopeptidase activity and is active as a homodimer with specificity for type I collagen. The mechanism that regulates FAP homodimerization and its relation to prolyl endopeptidase activity is not completely understood. Here, we investigate key residues in the FAP TM domain that may be significant for FAP homodimerization. Mutations to predicted TM interfacial residues (G10L, S14L, and A18L) comprising a small-X3-small motif reduced FAP TM-CYTO dimerization relative to wild type as measured using the AraTM assay, whereas predicted off-interface residues showed no significant change from wild type. The results implied that the predicted small-X3-small dimer interface affect stabilization of FAP TM-CYTO homodimerization. Compared with FAPwild-type, the interfacial TM residue G10L significantly decreased FAP endopeptidase activity more than 25%, and also reduced cell-surface versus intracellular expression relative to other interfacial residues S14L and A18L. Thus, our results suggest FAP dimerization is important for both trafficking and protease activity, and is dependent on a specific TM interface. PMID:27155568

  4. 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

    activity), anticoagulant proteins (protein C, antithrombin) and fibrinolytic factors (tissue plasminogen activator, plasminogen activator inhibitor-1) were performed for 5 h. RESULTS: ANP was confirmed by elevated serum amylase, development of ascites, and histological changes of the pancreas. A moderate...... 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....

  5. The biphasic virulence activities of gingipains: activation and inactivation of host proteins.

    Science.gov (United States)

    Imamura, Takahisa; Travis, James; Potempa, Jan

    2003-12-01

    Gingipains are trypsin-like cysteine proteinases produced by Porphyromonas gingivalis, a major causative bacterium of adult periodontitis. Rgps (HRgpA and RgpB) and Kgp are specific for -Arg-Xaa- and -Lys-Xaa- peptide bonds, respectively. HRgpA and Kgp are non-covalent complexes containing separate catalytic and adhesion/hemagglutinin domains, while RgpB has only a catalytic domain with a primary structure essentially identical to that of the cata-lytic subunit of HRgpA. The multiple virulence activities of gingipains are reviewed in view of the biphasic mechanisms: activation and inactivation of host proteins. Rgps enhanced vascular permeability through prekallikrein activation or direct bradykinin release in combination with Kgp. This Rgp action is potentially associated with gingival edema and crevicular fluid production. Rgps activate the blood coagulation system, leading to progression of inflammation and consequent alveolar bone loss in the periodontitis site. Rgps also activate protease-activated receptors and induce platelet aggregation, which, together with the coagulation-inducing activity, may explain an emerging link between periodontitis and cardiovascular disease. Kgp is the most potent fibrinogen/fibrin degrading enzyme of the three gingipains in human plasma, being involved in the bleeding tendency at the diseased gingiva. Gingipains stimulate expression of matrix metalloproteinases (MMPs) in fibroblasts and activate secreted latent MMPs that can destroy periodontal tissues. Gingipains degrade cytokines, components of the complement system and several receptors, including macrophage CD14, T cell CD4 and CD8, thus perturbing the host-defense systems and thereby facilitating sustained colonization of P. gingivalis. Gingipains are potent virulence factors of P. gingivalis, and in many regards their pathogenic activities constitute new mechanisms of bacterial virulence. PMID:14683429

  6. Evolutionary Conservation of a GPCR-Independent Mechanism of Trimeric G Protein Activation.

    Science.gov (United States)

    Coleman, Brantley D; Marivin, Arthur; Parag-Sharma, Kshitij; DiGiacomo, Vincent; Kim, Seongseop; Pepper, Judy S; Casler, Jason; Nguyen, Lien T; Koelle, Michael R; Garcia-Marcos, Mikel

    2016-03-01

    Trimeric G protein signaling is a fundamental mechanism of cellular communication in eukaryotes. The core of this mechanism consists of activation of G proteins by the guanine-nucleotide exchange factor (GEF) activity of G protein coupled receptors. However, the duration and amplitude of G protein-mediated signaling are controlled by a complex network of accessory proteins that appeared and diversified during evolution. Among them, nonreceptor proteins with GEF activity are the least characterized. We recently found that proteins of the ccdc88 family possess a Gα-binding and activating (GBA) motif that confers GEF activity and regulates mammalian cell behavior. A sequence similarity-based search revealed that ccdc88 genes are highly conserved across metazoa but the GBA motif is absent in most invertebrates. This prompted us to investigate whether the GBA motif is present in other nonreceptor proteins in invertebrates. An unbiased bioinformatics search in Caenorhabditis elegans identified GBAS-1 (GBA and SPK domain containing-1) as a GBA motif-containing protein with homologs only in closely related worm species. We demonstrate that GBAS-1 has GEF activity for the nematode G protein GOA-1 and that the two proteins are coexpressed in many cells of living worms. Furthermore, we show that GBAS-1 can activate mammalian Gα-subunits and provide structural insights into the evolutionarily conserved determinants of the GBA-G protein interface. These results demonstrate that the GBA motif is a functional GEF module conserved among highly divergent proteins across evolution, indicating that the GBA-Gα binding mode is strongly constrained under selective pressure to mediate receptor-independent G protein activation in metazoans. PMID:26659249

  7. Anticoagulant synergism of heparin and activated protein C in vitro. Role of a novel anticoagulant mechanism of heparin, enhancement of inactivation of factor V by activated protein C.

    OpenAIRE

    Petäjä, J; Fernández, J. A.; Gruber, A.; Griffin, J H

    1997-01-01

    Interactions between standard heparin and the physiological anticoagulant plasma protein, activated protein C (APC) were studied. The ability of heparin to prolong the activated partial thromboplastin time and the factor Xa- one-stage clotting time of normal plasma was markedly enhanced by addition of purified APC to the assays. Experiments using purified clotting factors showed that heparin enhanced by fourfold the phospholipid-dependent inactivation of factor V by APC. In contrast to factor...

  8. Cheese from Ultrafiltered Milk : whey proteins and chymosin activity

    NARCIS (Netherlands)

    Buijsse, C.

    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 and the

  9. Furin is involved in baculovirus envelope fusion protein activation

    NARCIS (Netherlands)

    Westenberg, M.; Wang, H.; IJkel, W.F.J.; Goldbach, R.W.; Vlak, J.M.; Zuidema, D.

    2002-01-01

    The Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) Se8 gene was recently shown to encode the viral envelope fusion (F) protein. A 60-kDa C-terminal subunit (F1) of the 76-kDa primary translation product of this gene was found to be the major envelope protein of SeMNPV budded virus (BV)

  10. Astragaloside Ⅱ triggers T cell activation through regulation of CD45 protein tyrosine phosphatase activity

    Institute of Scientific and Technical Information of China (English)

    Chun-ping WAN; Li-xin GAO; Li-fei HOU; Xiao-qian YANG; Pei-lan HE; Yi-fu YANG; Wei TANG

    2013-01-01

    Aim:To investigate the immunomodulating activity of astragalosides,the active compounds from a traditional tonic herb Astragalus membranaceus Bge,and to explore the molecular mechanisms underlying the actions,focusing on CD45 protein tyrosine phosphatase (CD45 PTPase),which plays a critical role in T lymphocyte activation.Methods:Primary splenocytes and T cells were prepared from mice.CD45 PTPase activity was assessed using a colorimetric assay.Cell proliferation was measured using a [3H]-thymidine incorporation assay.Cytokine proteins and mRNAs were examined with ELISA and RT-PCR,respectively.Activation markers,including CD25 and CD69,were analyzed using flow cytometry.Activation of LCK (Tyr505) was detected using Western blot analysis.Mice were injected with the immunosuppressant cyclophosphamide (CTX,80 mg/kg),and administered astragaloside Ⅱ (50 mg/kg).Results:Astragaloside Ⅰ,Ⅱ,Ⅲ,and Ⅳ concentration-dependently increased the CD45-mediated of pNPP/OMFP hydrolysis with the EC50 values ranged from 3.33 to 10.42 μg/mL.Astragaloside Ⅱ (10 and 30 μg/mL) significantly enhanced the proliferation of primary splenocytes induced by ConA,alloantigen or anti-CD3.Astragaloside Ⅱ (30 μg/mL) significantly increased IL-2 and IFN-y secretion,upregulated the mRNA levels of IFN-y and T-bet in primary splenocytes,and promoted CD25 and CD69 expression on primary CD4+T cells upon TCR stimulation.Furthermore,astragaloside Ⅱ (100 ng/mL) promoted CD45-mediated dephosphorylation of LCK (Tyr505) in primary T cells,which could be blocked by a specific CD45 PTPase inhibitor.In CTX-induced immunosuppressed mice,oral administration of astragaloside Ⅱ restored the proliferation of splenic T cells and the production of IFN-Y and IL-2.However,astragaloside Ⅱ had no apparent effects on B cell proliferation.Conclusion:Astragaloside Ⅱ enhances T cell activation by regulating the activity of CD45 PTPase,which may explain why Astragalus membranaceus Bge is used as a tonic

  11. Refolding Techniques for Recovering Biologically Active Recombinant Proteins from Inclusion Bodies

    Directory of Open Access Journals (Sweden)

    Hiroshi Yamaguchi

    2014-02-01

    Full Text Available 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.

  12. Activated Alveolar Epithelial Cells Initiate Fibrosis through Secretion of Mesenchymal Proteins

    OpenAIRE

    Yang, Jibing; Wheeler, Sarah E.; Velikoff, Miranda; Kleaveland, Kathryn R.; LaFemina, Michael J.; Frank, James A.; Chapman, Harold A.; Christensen, Paul J; Kim, Kevin K.

    2013-01-01

    Fibrosis is characterized by accumulation of activated fibroblasts and pathological deposition of fibrillar collagens. Activated fibroblasts overexpress matrix proteins and release factors that promote further recruitment of activated fibroblasts, leading to progressive fibrosis. The contribution of epithelial cells to this process remains unknown. Epithelium-directed injury may lead to activation of epithelial cells with phenotypes and functions similar to activated fibroblasts. Prior report...

  13. Protein Synthesis Inhibition Activity by Strawberry Tissue Protein Extracts during Plant Life Cycle and under Biotic and Abiotic Stresses

    Directory of Open Access Journals (Sweden)

    Walther Faedi

    2013-07-01

    Full Text Available Ribosome-inactivating proteins (RIPs, enzymes that are widely distributed in the plant kingdom, inhibit protein synthesis by depurinating rRNA and many other polynucleotidic substrates. Although RIPs show antiviral, antifungal, and insecticidal activities, their biological and physiological roles are not completely understood. Additionally, it has been described that RIP expression is augmented under stressful conditions. In this study, we evaluated protein synthesis inhibition activity in partially purified basic proteins (hereafter referred to as RIP activity from tissue extracts of Fragaria × ananassa (strawberry cultivars with low (Dora and high (Record tolerance to root pathogens and fructification stress. Association between the presence of RIP activity and the crop management (organic or integrated soil, growth stage (quiescence, flowering, and fructification, and exogenous stress (drought were investigated. RIP activity was found in every tissue tested (roots, rhizomes, leaves, buds, flowers, and fruits and under each tested condition. However, significant differences in RIP distribution were observed depending on the soil and growth stage, and an increase in RIP activity was found in the leaves of drought-stressed plants. These results suggest that RIP expression and activity could represent a response mechanism against biotic and abiotic stresses and could be a useful tool in selecting stress-resistant strawberry genotypes.

  14. Mechanistic pathways and biological roles for receptor-independent activators of G-protein signaling.

    Science.gov (United States)

    Blumer, Joe B; Smrcka, Alan V; Lanier, Stephen M

    2007-03-01

    Signal processing via heterotrimeric G-proteins in response to cell surface receptors is a central and much investigated aspect of how cells integrate cellular stimuli to produce coordinated biological responses. The system is a target of numerous therapeutic agents and plays an important role in adaptive processes of organs; aberrant processing of signals through these transducing systems is a component of various disease states. In addition to G-protein coupled receptor (GPCR)-mediated activation of G-protein signaling, nature has evolved creative ways to manipulate and utilize the Galphabetagamma heterotrimer or Galpha and Gbetagamma subunits independent of the cell surface receptor stimuli. In such situations, the G-protein subunits (Galpha and Gbetagamma) may actually be complexed with alternative binding partners independent of the typical heterotrimeric Galphabetagamma. Such regulatory accessory proteins include the family of regulator of G-protein signaling (RGS) proteins that accelerate the GTPase activity of Galpha and various entities that influence nucleotide binding properties and/or subunit interaction. The latter group of proteins includes receptor-independent activators of G-protein signaling (AGS) proteins that play surprising roles in signal processing. This review provides an overview of our current knowledge regarding AGS proteins. AGS proteins are indicative of a growing number of accessory proteins that influence signal propagation, facilitate cross talk between various types of signaling pathways, and provide a platform for diverse functions of both the heterotrimeric Galphabetagamma and the individual Galpha and Gbetagamma subunits.

  15. Vegetative Storage Protein with Trypsin Inhibitor Activity Occurs in Sapindus mukorassi,a Sapindaceae Deciduous Tree

    Institute of Scientific and Technical Information of China (English)

    Shi-Biao Liu; Xu-Chu Wang; Min-Jing Shi; Yue-Yi Chen; Zheng-Hai Hu; Wei-Min Tian

    2009-01-01

    A vegetative storage protein (VSP) with trypsin inhibitor activity in a deciduous tree,Sapindus mukorassi,was characterized by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis,Western-blot,immuno-histochemical localization,light- and electro-microscopy,together with analysis of proteinase inhibitor activity of the purified VSP in vitro.There were two proteins with molecular masses of about 23 and 27 kDa in a relatively high content in the bark tissues of terminal branches of S.mukorassi in leafless periods.The proteins decreased markedly during young shoot development,indicating their role in seasonal nitrogen storage.Immuno-histochemical localization with the polyclonal antibodies raised against the 23 kDa protein demonstrated that the 23 kDa protein was the major component of protein inclusions in protein-storing cells.The protein inclusions were identified by protein-specific staining and should correspond to the electron-dense materials in different forms in the vacuoles of phloem parenchyma cells and phloem ray parenchyma cells under an electron microscope.So,the 23 kDa protein was a typical VSP in S.mukorassi.The 23 and 27 kDa proteins shared no immuno-relatedness,whereas the 23 kDa protein was immuno-related with the 22 kDa VSP in lychee and possessed trypsin inhibitor activity.The 23 kDa protein may confer dual functions:nitrogen storage and defense.

  16. 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

    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......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....... Moreover, MK2 activity was required for damage response, accumulation of ssDNA, and decreased survival when cells were treated with the nucleoside analogue gemcitabine or when the checkpoint kinase Chk1 was antagonized. By using DNA fiber assays, we found that MK2 inhibition or knockdown rescued DNA...

  17. Influence of physical activity of racehorses on lactate dehydrogenase and creatine kinase activities, and protein synthesis

    Directory of Open Access Journals (Sweden)

    Jović Slavoljub

    2013-01-01

    Full Text Available The aim of the research was to assess the effects of physical activity of various intensity on the degree of damage to certain organs resulting from increased free radical production, as well as the adaptability of the organism to physical exercise. Two groups of healthy 3-5-year-old full-blooded racehorses were assessed. The first one ran a 2400-meter gallop race, which is considered a short-lasting, intense physical activity; lipid status was assessed prior to, and 48 and 72 h after the race. The second group ran a forty-kilometer endurance ride, which is a long-lasting moderate physical activity; the lipid status was assessed immediately before, on finishing and 48, 72, 96, 120 and 144 h after the race. The total activity of LDH changed 72 h and 96 h following the gallop race (p>0.05, whilst the maximum activity was measured immediately after the endurance ride. By means of electrophoresis LDH in all the horses 5 isoforms were detected. The activity of LDH1 72 h after the gallop race significantly rose in comparison to the one before the race (p0.05. Following the endurance ride LDH1 activity rose at all sampling times, reaching the maximum at 96 h and 144 h in comparison to the values both before and on finishing the ride (p<0.01. The increase in the activity of LDH2 was significantly higher 48 h, 72 h, 96 h and 120 h (p<0.05 after the race in comparison to that before the race, and at 48 h, 72 h, 96 h, 120 h and 144 h (p<0.05 in comparison to the values on finishing the race. LDH3 activity significantly decreased and LDH5 rose immediately after the endurance ride (p<0.01, whilst LDH4 significantly rose at all times following the endurance ride (p<0.01. The CK activity pointed to high, medium and low degree of adaptation of horses to physical activity. The concentrations of total proteins, albumins and globulins remained within the physiological range at all sampling times, with the exception of 96 h after the endurance ride, when the fall

  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

    2015-01-01

    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. PMID:26516115

  19. 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...

  20. 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. PMID:24297163

  1. 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.

  2. 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...

  3. Exchange Protein Activated by cAMP Enhances Long-Term Memory Formation Independent of Protein Kinase A

    Science.gov (United States)

    Ma, Nan; Abel, Ted; Hernandez, Pepe J.

    2009-01-01

    It is well established that cAMP signaling within neurons plays a major role in the formation of long-term memories--signaling thought to proceed through protein kinase A (PKA). However, here we show that exchange protein activated by cAMP (Epac) is able to enhance the formation of long-term memory in the hippocampus and appears to do so…

  4. 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. PMID:24691882

  5. Extracellular protein analysis of activated sludge and their functions in wastewater treatment plant by shotgun proteomics

    OpenAIRE

    Peng Zhang; Yu Shen; Jin-Song Guo; Chun Li; Han Wang; You-Peng Chen; Peng Yan; Ji-Xiang Yang; Fang Fang

    2015-01-01

    In this work, proteins in extracellular polymeric substances extracted from anaerobic, anoxic and aerobic sludges of wastewater treatment plant (WWTP) were analyzed to probe their origins and functions. Extracellular proteins in WWTP sludges were identified using shotgun proteomics, and 130, 108 and 114 proteins in anaerobic, anoxic and aerobic samples were classified, respectively. Most proteins originated from cell and cell part, and their most major molecular functions were catalytic activ...

  6. Effect of Protein Binding on the Pharmacological Activity of Highly Bound Antibiotics▿

    OpenAIRE

    Schmidt, Stephan; Röck, Katharina; Sahre, Martina; Burkhardt, Olaf; Brunner, Martin; Lobmeyer, Maximilian T.; Derendorf, Hartmut

    2008-01-01

    During antibiotic drug development, media are frequently spiked with either serum/plasma or protein supplements to evaluate the effect of protein binding. Usually, previously reported serum or plasma protein binding values are applied in the analysis. The aim of this study was to evaluate this approach by experimentally measuring free, unbound concentrations for antibiotics with reportedly high protein binding and their corresponding antimicrobial activities in media containing commonly used ...

  7. Alteration and modulation of protein activity by varying post-translational modification

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, David N; Reed, David W; Thompson, Vicki S; Lacey, Jeffrey A; Apel, William A

    2015-03-03

    Embodiments of the invention include methods of altering the enzymatic activity or solubility of an extremophilic enzyme or post-translationally modifying a protein of interest via using isolated or partially purified glycosyltransferases and/or post-translational modification proteins, extracts of cells comprising glycosyltransferases and/or post-translational modification proteins, and/or in cells comprising one or more glycosyltransferases and/or post-translational modification proteins.

  8. Alteration and modulation of protein activity by varying post-translational modification

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, David N.; Reed, David W.; Thompson, Vicki S.; Lacey, Jeffrey A.; Apel, William A.

    2016-07-12

    Embodiments of the invention include methods of altering the enzymatic activity or solubility of an extremophilic enzyme or post-translationally modifying a protein of interest via using isolated or partially purified glycosyltransferases and/or post-translational modification proteins, extracts of cells comprising glycosyltransferases and/or post-translational modification proteins, and/or in cells comprising one or more glycosyltransferases and/or post-translational modification proteins.

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

    Institute of Scientific and Technical Information of China (English)

    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.

  10. Coagulation factor Va binds to human umbilical vein endothelial cells and accelerates protein C activation.

    OpenAIRE

    Maruyama, I.; Salem, H H; Majerus, P W

    1984-01-01

    In vitro the rate of protein C activation by thrombin is significantly accelerated by two distinct cofactors (a) the endothelial cell surface protein, thrombomodulin, and (b) human coagulation Factor Va. We have recently reported that the activity of Factor Va is contained in the 78,000-D light chain. In this study we have investigated the effects of Factor Va and its light chain on the activation of protein C in the presence of cultured endothelial cells. Thrombin-catalyzed protein C activat...

  11. The AU-rich element mRNA decay-promoting activity of BRF1 is regulated by mitogen-activated protein kinase-activated protein kinase 2

    OpenAIRE

    Maitra, Sushmit; Chou, Chu-Fang; Luber, Christian A.; Lee, Kyung-Yeol; Mann, Matthias; Chen, Ching-Yi

    2008-01-01

    Regulated mRNA decay is a highly important process for the tight control of gene expression. Inherently unstable mRNAs contain AU-rich elements (AREs) in the 3′ untranslated regions that direct rapid mRNA decay by interaction with decay-promoting ARE-binding proteins (ARE-BPs). The decay of ARE-containing mRNAs is regulated by signaling pathways that are believed to directly target ARE-BPs. Here, we show that BRF1 involved in ARE-mediated mRNA decay (AMD) is phosphorylated by MAPK-activated p...

  12. Antibacterial activity of a lectin-like Burkholderia cenocepacia protein

    OpenAIRE

    Ghequire, Maarten; De Canck, Evelien; Wattiau, Pierre; Van Winge, Iris; Loris, Remy; Coenye, Tom; De Mot, René

    2013-01-01

    Abstract Bacteriocins of the LlpA family have previously been characterized in the γ-proteobacteria Pseudomonas and Xanthomonas. These proteins are composed of two MMBL (monocot mannose-binding lectin) domains, a module predominantly and abundantly found in lectins from monocot plants. Genes encoding four different types of LlpA-like proteins were identified in genomes from strains belonging to the Burkholderia cepacia complex (Bcc) and the Burkholderia pseudomallei group. A selected recombin...

  13. Activation of mitogen activated protein kinases via complement receptor type 2

    Institute of Scientific and Technical Information of China (English)

    LUO Min-hua 罗敏华; CHEN Ming-liang 陈明亮; Heribert Stoiber; Manfred P Dierich

    2004-01-01

    Background Complement receptor type 2 (CR2) is the receptor for C3d and C3dg and for Epstein-Barr virus. The aim of our study was to explore whether CR2 can independently mediate the activation of mitogen-activated protein kinases (MAPKs, including ERK, JNK, and p38MAPK), and to highlight the molecular mechanism of CD4+ cell deletion in AIDS.Results FACS results showed that the positive rates of HOS-CR2 and HOS-CD4CR2 cells were greater than 96%, and Western blot showed that the CR2 expression levels on HOS-CR2 and HOS-CD4CR2 cells were high. Activation and blocking tests of MAPKs (ERK, JNK, and p38MAPK) were carried out in HOS-CR2, HOS-CD4, and HOS-CD4CR2 cells. The activation of MAPKs in HOS-CR2 cells stimulated with PMA (100 ng/ml) and NHS (10%) was identical. The activation of MAPKs increased at 5 minutes, reached a peak at 10 minutes, and decreased to baseline within 30 minutes, all in a time-dependent manner; the activation of MAPKs was blocked by anti-CR2 McAb, PD98059 (inhibitor of ERK), and Wortmanin (inhibitor of PI-3K), respectively. In HOS-CD4 cells, MAPKs were activated by HIV-gp160. In HOS-CD4CR2 cells, MAPK activation was induced by HIV-gp160, 10% NHS, and HIV-gp160+10%NHS; phosphorylation of p38MAPK was dramatically induced by HIV-gp160+NHS, and lasted for 1 hour. The cell proliferation results showed that HIV-gp160 inhibited the proliferation of HOS-CD4 and HOS-CD4CR2 cells (P<0.01) and that NHS enhanced the effect of HIV-gp160 (P<0.01).Conclusions The activation of MAPKs is independently mediated by CR2 and that anti-CR2 McAb, PD98059, and Wortmanin block the activation of MAPKs, respectively. The results of the signal transduction and cell proliferation assays of HOS-CD4CR2 cells show that CR2 plays a role in the pathogenesis of HIV infection, especially in the inhibition of CD4+ cell proliferation.

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

    International Nuclear Information System (INIS)

    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

  15. Osthole enhances glucose uptake through activation of AMP-activated protein kinase in skeletal muscle cells.

    Science.gov (United States)

    Lee, Wei-Hwa; Lin, Ren-Jye; Lin, Shyr-Yi; Chen, Yu-Chien; Lin, Hsiu-Ming; Liang, Yu-Chih

    2011-12-28

    AMP-activated protein kinase (AMPK) is an energy sensor that regulates cellular metabolism. Activation of AMPK in skeletal muscles, the liver, and adipose tissues results in a favorable metabolic milieu for preventing and treating type 2 diabetes, i.e., decreased levels of circulating glucose, plasma lipids, and ectopic fat accumulation and enhanced insulin sensitivity. Osthole was extracted from a Chinese herbal medicine, and we found that it had glucose lowering activity in our previous study. However, the detailed glucose lowering mechanisms of osthole are still unclear. In this study, we used skeletal muscle cells to examine the underlying molecular mechanisms of osthole's glucose lowering activity. A Western blot analysis revealed that osthole significantly induced phosphorylation of AMPK and acetyl-CoA carboxylase (ACC). Next, we found that osthole significantly increased the level of translocation of glucose transporter 4 (GLUT4) to plasma membranes and glucose uptake in a dose-dependent manner. Osthole-induced glucose uptake was reversed by treatment with Compound C, an AMPK inhibitor, suggesting that osthole-induced glucose uptake was mediated in an AMPK-dependent manner. The increase in the AMP:ATP ratio was involved in osthole's activation of AMPK. Finally, we found that osthole counteracted hyperglycemia in mice with streptozotocin-induced diabetes. These results suggest that the increase in the AMP:ATP ratio by osthole triggered activation of the AMPK signaling pathway and led to increases in plasma membrane GLUT4 content and glucose uptake level. Therefore, osthole might have potential as an antidiabetic agent for treating diabetes. PMID:22098542

  16. Activation of brain B-Raf protein kinase by Rap1B small GTP-binding protein.

    Science.gov (United States)

    Ohtsuka, T; Shimizu, K; Yamamori, B; Kuroda, S; Takai, Y

    1996-01-19

    Rap1 small GTP-binding protein has the same amino acid sequence at its effector domain as that of Ras. Rap1 has been shown to antagonize the Ras functions, such as the Ras-induced transformation of NIH 3T3 cells and the Ras-induced activation of the c-Raf-1 protein kinase-dependent mitogen-activated protein (MAP) kinase cascade in Rat-1 cells, whereas we have shown that Rap1 as well as Ras stimulates DNA synthesis in Swiss 3T3 cells. We have established a cell-free assay system in which Ras activates bovine brain B-Raf protein kinase. Here we have used this assay system and examined the effect of Rap1 on the B-Raf activity to phosphorylate recombinant MAP kinase kinase (MEK). Recombinant Rap1B stimulated the activity of B-Raf, which was partially purified from bovine brain and immunoprecipitated by an anti-B-Raf antibody. The GTP-bound form was active, but the GDP-bound form was inactive. The fully post-translationally lipid-modified form was active, but the unmodified form was nearly inactive. The maximum B-Raf activity stimulated by Rap1B was nearly the same as that stimulated by Ki-Ras. Rap1B enhanced the Ki-Ras-stimulated B-Raf activity in an additive manner. These results indicate that not only Ras but also Rap1 is involved in the activation of the B-Raf-dependent MAP kinase cascade.

  17. 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.

  18. 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.

  19. Protective effects of inhibition of adenosine monophosphate activated protein kinase activity against cerebral ischemia-reperfusion injury in mice

    Institute of Scientific and Technical Information of China (English)

    补娟

    2013-01-01

    Objective To observe the effect of inhibition of adenosine monophosphate activated protein kinase (AMPK) on shape,function and inflammatory factor of microglia for mice after cerebral ischemia-reperfusion

  20. AMP-activated protein kinase regulates nicotinamide phosphoribosyl transferase expression in skeletal muscle

    DEFF Research Database (Denmark)

    Brandauer, Josef; Vienberg, Sara Gry; Andersen, Marianne Agerholm;

    2013-01-01

    -activated protein kinase (AMPK) increases sirtuin activity by elevating NAD levels. As NAM directly inhibits sirtuins, increased Nampt activation or expression could be a metabolic stress response. Evidence suggests that AMPK regulates Nampt mRNA content, but whether repeated AMPK activation is necessary for...... increasing Nampt protein levels is unknown. To this end, we assessed whether exercise training- or 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR)-mediated increases in skeletal muscle Nampt abundance are AMPK dependant. One-legged knee-extensor exercise training in humans increased Nampt protein...

  1. Helicobacter pylori neutrophil activating protein as target for new drugs against H.pylori inflammation

    Institute of Scientific and Technical Information of China (English)

    Theodora Choli-Papadopoulou; Filippos Kottakis; Georgios Papadopoulos; Stefanos Pendas

    2011-01-01

    Helicobacter pylori (H. pylori ) infection is among the most common human infections and the major risk factor for peptic ulcer disease and gastric cancer. Within this work we present the implication of C-terminal region of H. pylori neutrophil activating protein in the stimulation of neutrophil activation as well as the evidence that the C-terminal region of H. pylori activating protein is indispensable for neutrophil adhesion to endothelial cells, a step necessary to H. pylori inflammation. In addition we show that arabino galactan proteins derived from chios mastic gum, the natural resin of the plant Pistacia lentiscus var. Chia inhibit neutrophil activation in vitro .

  2. 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.

  3. Study on the Anti-freezing Technology to Mountain Tea Plantations%山地茶园防冻技术研究

    Institute of Scientific and Technical Information of China (English)

    王烨军; 黄建琴; 罗仲兴; 张必桦; 徐奕鼎

    2009-01-01

    Six kinds of technical measures (pruning, covering couch grass, mulching plastic film, sprinkling irri-gation antifreeze etc.) were used to mountain tea plantations to improve the capacity of frost resistance and study the relationship between the enzyme activity of SOD, POD and freezing rates. The results showed that: (1) with freezing rates increase, the enzyme activity of SOD also increases accordingly, in a certain range to various technical measures. This is because tea plants have the self-repairing ability; (2) to mountain tea plantations; the technical measures used directly to crown of tea plant have better effect than used to environment and soil because of wind-chill effect. The result of six kinds of the technical measures is: mulching plastic film > cover-ing couch grass > CK> no pruning>irrigating roots> spraying crown; (3) It's beneficial to tea plant to reduce freezing injury with suitable pruning in autumn.%研究了修剪、铺草、覆膜、喷灌防冻剂等6项技术措施对山地茶园茶树的抗寒性影响及SOD、POD酶活性与受冻率之间的关系,结果表明:①各处理随着受冻率的增加,在一定范围内SOD酶活性也相应增加,这与茶树自身的修复功能有关;②对于山地茶园,受风冻影响,直接应用于茶树树冠的技术措施要比应用于环境及土壤的技术措施效果好.6种处理效果为:覆膜>铺草>对照>未修剪>灌根>喷冠;③秋天进行适当的修剪,对降低茶树冻害有利.

  4. Mapping the homodimer interface of an optimized, artificial, transmembrane protein activator of the human erythropoietin receptor.

    Directory of Open Access Journals (Sweden)

    Emily B Cohen

    Full Text Available Transmembrane proteins constitute a large fraction of cellular proteins, and specific interactions involving membrane-spanning protein segments play an important role in protein oligomerization, folding, and function. We previously isolated an artificial, dimeric, 44-amino acid transmembrane protein that activates the human erythropoietin receptor (hEPOR in trans. This artificial protein supports limited erythroid differentiation of primary human hematopoietic progenitor cells in vitro, even though it does not resemble erythropoietin, the natural ligand of this receptor. Here, we used a directed-evolution approach to explore the structural basis for the ability of transmembrane proteins to activate the hEPOR. A library that expresses thousands of mutants of the transmembrane activator was screened for variants that were more active than the original isolate at inducing growth factor independence in mouse cells expressing the hEPOR. The most active mutant, EBC5-16, supports erythroid differentiation in human cells with activity approaching that of EPO, as assessed by cell-surface expression of glycophorin A, a late-stage marker of erythroid differentiation. EBC5-16 contains a single isoleucine to serine substitution at position 25, which increases its ability to form dimers. Genetic studies confirmed the importance of dimerization for activity and identified the residues constituting the homodimer interface of EBC5-16. The interface requires a GxxxG dimer packing motif and a small amino acid at position 25 for maximal activity, implying that tight packing of the EBC5-16 dimer is a crucial determinant of activity. These experiments identified an artificial protein that causes robust activation of its target in a natural host cell, demonstrated the importance of dimerization of this protein for engagement of the hEPOR, and provided the framework for future structure-function studies of this novel mechanism of receptor activation.

  5. Membrane lipids regulate ganglioside GM2 catabolism and GM2 activator protein activity.

    Science.gov (United States)

    Anheuser, Susi; Breiden, Bernadette; Schwarzmann, Günter; Sandhoff, Konrad

    2015-09-01

    Ganglioside GM2 is the major lysosomal storage compound of Tay-Sachs disease. It also accumulates in Niemann-Pick disease types A and B with primary storage of SM and with cholesterol in type C. Reconstitution of GM2 catabolism with β-hexosaminidase A and GM2 activator protein (GM2AP) at uncharged liposomal surfaces carrying GM2 as substrate generated only a physiologically irrelevant catabolic rate, even at pH 4.2. However, incorporation of anionic phospholipids into the GM2 carrying liposomes stimulated GM2 hydrolysis more than 10-fold, while the incorporation of plasma membrane stabilizing lipids (SM and cholesterol) generated a strong inhibition of GM2 hydrolysis, even in the presence of anionic phospholipids. Mobilization of membrane lipids by GM2AP was also inhibited in the presence of cholesterol or SM, as revealed by surface plasmon resonance studies. These lipids also reduced the interliposomal transfer rate of 2-NBD-GM1 by GM2AP, as observed in assays using Förster resonance energy transfer. Our data raise major concerns about the usage of recombinant His-tagged GM2AP compared with untagged protein. The former binds more strongly to anionic GM2-carrying liposomal surfaces, increases GM2 hydrolysis, and accelerates intermembrane transfer of 2-NBD-GM1, but does not mobilize membrane lipids.

  6. Enhanced biocontrol activity of Trichoderma through inactivation of a mitogen-activated protein kinase.

    Science.gov (United States)

    Mendoza-Mendoza, Artemio; Pozo, María J; Grzegorski, Darlene; Martínez, Pedro; García, Juan M; Olmedo-Monfil, Vianey; Cortés, Carlos; Kenerley, Charles; Herrera-Estrella, Alfredo

    2003-12-23

    The production of lytic enzymes in Trichoderma is considered determinant in its parasitic response against fungal species. A mitogen-activated protein kinase encoding gene, tvk1, from Trichoderma virens was cloned, and its role during the mycoparasitism, conidiation, and biocontrol was examined in tvk1 null mutants. These mutants showed a clear increase in the level of the expression of mycoparasitism-related genes under simulated mycoparasitism and during direct confrontation with the plant pathogen Rhizoctonia solani. The null mutants displayed an increased protein secretion phenotype as measured by the production of lytic enzymes in culture supernatant compared to the wild type. Consistently, biocontrol assays demonstrated that the null mutants were considerably more effective in disease control than the wild-type strain or a chemical fungicide. In addition, tvk1 gene disruptant strains sporulated abundantly in submerged cultures, a condition that is not conducive to sporulation in the wild type. These data suggest that Tvk1 acts as a negative modulator during host sensing and sporulation in T. virens. PMID:14673101

  7. Membrane lipids regulate ganglioside GM2 catabolism and GM2 activator protein activity.

    Science.gov (United States)

    Anheuser, Susi; Breiden, Bernadette; Schwarzmann, Günter; Sandhoff, Konrad

    2015-09-01

    Ganglioside GM2 is the major lysosomal storage compound of Tay-Sachs disease. It also accumulates in Niemann-Pick disease types A and B with primary storage of SM and with cholesterol in type C. Reconstitution of GM2 catabolism with β-hexosaminidase A and GM2 activator protein (GM2AP) at uncharged liposomal surfaces carrying GM2 as substrate generated only a physiologically irrelevant catabolic rate, even at pH 4.2. However, incorporation of anionic phospholipids into the GM2 carrying liposomes stimulated GM2 hydrolysis more than 10-fold, while the incorporation of plasma membrane stabilizing lipids (SM and cholesterol) generated a strong inhibition of GM2 hydrolysis, even in the presence of anionic phospholipids. Mobilization of membrane lipids by GM2AP was also inhibited in the presence of cholesterol or SM, as revealed by surface plasmon resonance studies. These lipids also reduced the interliposomal transfer rate of 2-NBD-GM1 by GM2AP, as observed in assays using Förster resonance energy transfer. Our data raise major concerns about the usage of recombinant His-tagged GM2AP compared with untagged protein. The former binds more strongly to anionic GM2-carrying liposomal surfaces, increases GM2 hydrolysis, and accelerates intermembrane transfer of 2-NBD-GM1, but does not mobilize membrane lipids. PMID:26175473

  8. C4b-binding protein protects coagulation factor Va from inactivation by activated protein C

    NARCIS (Netherlands)

    van de Poel, RHL; Meijers, JCM; Rosing, J; Tans, G; Bouma, Bonno N.

    2000-01-01

    We investigated the effect of C4BP on APC-mediated inactivation of factor Va (FVa) in the absence and presence of protein S. FVa inactivation was biphasic (k(506) = 4.4 x 10(8) M-1 s(-1), k(306) = 2.7 x 10(7) M-1 s(-1)), and protein S accelerated Arg(306) cleavage approximately 10-fold. Preincubatio

  9. 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.

  10. The WW-HECT protein Smurf2 interacts with the Docking Protein NEDD9/HEF1 for Aurora A activation

    Directory of Open Access Journals (Sweden)

    Moore Finola E

    2010-09-01

    Full Text Available Abstract The multi-functional adaptor protein NEDD9/HEF1/Cas-L regulates cell motility, invasion and cell cycle progression, and plays key roles in cancer progression and metastasis. NEDD9 is localized to the centrosome and is required for activation of Aurora A kinase in mitosis. Here we demonstrate that the HECT-WW protein Smurf2 physically associates with NEDD9 and is required for the stability of NEDD9 protein. Smurf2 depletion results in a marked decrease in NEDD9 protein levels, by facilitating polyubiquitination and proteasomal degradation of NEDD9. Conversely, forced overexpression of Smurf2 results in upregulation of endogenous NEDD9 protein, confirming the role for Smurf2 in NEDD9 stability. Cells with Smurf2 depletion fail to activate Aurora A at the G2/M boundary, leading to a marked delay in mitotic entry. These observations suggest that the stable complex of Smurf2 and NEDD9 is required for timely entry into mitosis via Aurora A activation.

  11. Mitogen-activated protein kinases mediate Mycobacterium tuberculosis–induced CD44 surface expression in monocytes

    Indian Academy of Sciences (India)

    Natarajan Palaniappan; S Anbalagan; Sujatha Narayanan

    2012-03-01

    CD44, an adhesion molecule, has been reported to be a binding site for Mycobacterium tuberculosis (M. tuberculosis) in macrophages and it also mediates mycobacterial phagocytosis, macrophage recruitment and protective immunity against pulmonary tuberculosis in vivo. However, the signalling pathways that are involved in M. tuberculosis–induced CD44 surface expression in monocytic cells are currently unknown. Exposure of THP-1 human monocytes to M. tuberculosis H37Rv and H37Ra induced distinct, time-dependent, phosphorylation of mitogen-activated protein kinase kinase-1, extracellular signal regulated kinase 1/2, mitogen-activated protein kinase kinase 3/6, p38 mitogen-activated protein kinase and c-jun N-terminal kinases. The strains also differed in their usage of CD14 and human leukocyte antigen-DR (HLA-DR) receptors in mediating mitogen-activated protein kinase activation. M. tuberculosis H37Rv strain induced lower CD44 surface expression and tumour necrosis factor-alpha levels, whereas H37Ra the reverse. Using highly specific inhibitors of mitogen-activated protein kinase kinase-1, p38 mitogen-activated protein kinase and c-jun N-terminal kinase, we report that inhibition of extracellular signal regulated kinase 1/2 and c-jun N-terminal kinases increases, but that inhibition of p38 mitogen-activated protein kinase decreases M. tuberculosis–induced CD44 surface expression in THP-1 human monocytes.

  12. Phosphorylation of synaptosomal cytoplasmic proteins: Inhibition of calcium-activated, phospholipid-dependent protein kinase (protein kinase c) by bay k 8644.

    Science.gov (United States)

    Robinson, P J; Lovenberg, W

    1988-01-01

    The phosphorylation of specific substrates of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) was examined in striatal synaptosomal cytoplasm. The phosphoprotein substrata were termed group C phosphoprotems and were divided into two subgroups: group C(1) phosphoproteins (P83, P45A, P21 and P18) were found in both cytoplasm and synaptosomal membranes and, although stimulated by phosphatidylserine, only required exogamous calcium for their labeling; group C(2) phosphoproteins (P120, P96, P21.5, P18.5 and P16) were found predominantly in the cytoplasm and were absolutely dependent upon exogenous calcium and phosphatidylserme for their labeling. Several criteria were used to identify these proteins as specific protein kinase C substrates: (a) their phosphorylation was stimulated to a greater extent by Ca(2+) /phosphatidylserine/diolein than by Ca(2+) alone or Cal(2+) /calmodulin (group C(1)) or was completely dependent upon Ca(2+) /phosphatdylserine/diolein (group C(2)); (b) supermaximal concentrations of the cAMP-dependent protein kinase inhibitor were without effect; (c) their phosphorylation was stimulated by oleic acid, which selectively activates protein kinase C in the absence of Ca(2+); (d) NaCl, which inhibited cAMP- and Ca(2+)/calmodulindependent phosphorylation, slightly increased phosphorylation of group C(1) and slightly decreased phosphorylation of group C(2) phosphoproteins. Maximal phosphorylation of P96 and other group C phosphoproteins occurred within 60 s and was followed by a slow decay rate while substrata of calmodulin-dependent protein kinase were maximally labeled within 20-30 s and rapidly dephosphorylated. The phosphorylation of all group C phosphoproteins was inhibited by the calcium channel agomst BAY K 8644, however, group C(2) phosphoproteins were considerably more sensitive. The IC(50) for inhibition of P96 labeling was 19 ?M. but for P83 was 190 ?M. Group B phosphoproteins were also slightly inhibited, and the

  13. Interactive protein network of FXIII-A1 in lipid rafts of activated and non-activated platelets.

    Science.gov (United States)

    Rabani, Vahideh; Montange, Damien; Davani, Siamak

    2016-09-01

    Lipid-rafts are defined as membrane microdomains enriched in cholesterol and glycosphingolipids within platelet plasma membrane. Lipid raft-mediated clot retraction requires factor XIII and other interacting proteins. The aim of this study was to investigate the proteins that interact with factor XIII in raft and non-raft domains of activated and non-activated platelet plasma membrane. By lipidomics analysis, we identified cholesterol- and sphingomyelin-enriched areas as lipid rafts. Platelets were activated by thrombin. Proteomics analysis provided an overview of the pathways in which proteins of rafts and non-rafts participated in the interaction network of FXIII-A1, a catalytic subunit of FXIII. "Platelet activation" was the principal pathway among KEGG pathways for proteins of rafts, both before and after activation. Network analysis showed four types of interactions (activation, binding, reaction, and catalysis) in raft and non-raft domains in interactive network of FXIII-A1. FXIII-A1 interactions with other proteins in raft domains and their role in homeostasis highlight the specialization of the raft domain in clot retraction via the Factor XIII protein network.

  14. Stabilizing effects of G protein on the active conformation of adenosine A1 receptor differ depending on G protein type.

    Science.gov (United States)

    Tateyama, Michihiro; Kubo, Yoshihiro

    2016-10-01

    G protein coupled receptors (GPCRs) trigger various cellular and physiological responses upon the ligand binding. The ligand binding induces conformational change in GPCRs which allows G protein to interact with the receptor. The interaction of G protein also affects the active conformation of GPCRs. In this study, we have investigated the effects of Gαi1, Gαo and chimeric Gαqi5 on the active conformation of the adenosine A1 receptor, as each Gα showed difference in the interaction with adenosine A1 receptor. The conformational changes in the adenosine A1 receptor were detected as the agonist-induced decreases in efficiency of Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) fused at the two intracellular domains of the adenosine A1 receptor. Amplitudes of the agonist-induced FRET decreases were subtle when the FP-tagged adenosine A1 receptor was expressed alone, whereas they were significantly enhanced when co-expressed with Gαi1Gβ1Gγ22 (Gi1) or Gαqi5Gβ1Gγ22 (Gqi5) but not with GαοGβ1Gγ22 (Go). The enhancement of the agonist-induced FRET decrease in the presence of Gqi5 was significantly larger than that of Gi1. Furthermore, the FRET recovery upon the agonist removal in the presence of Gqi5 was significantly slower than that of Gi1. From these results it was revealed that the agonist-bound active conformation of adenosine A1 receptor is unstable without the binding of G protein and that the stabilizing effects of G protein differ depending on the types of G protein.

  15. Characterization of Adapter Protein NRBP as a Negative Regulator of T Cell Activation

    Institute of Scientific and Technical Information of China (English)

    WANG Hui; LIN Zhi-xin; WU Jun

    2008-01-01

    Adapter proteins can regulate the gene transcriptions in disparate signaling pathway by interacting with multiple signaling molecules, including T cell activation signaling. Nuclear receptor binding protein (NRBP), a novel adapter protein, represents a small family of evolutionarily conserved proteins with homologs in Caenorhabditis elegans (C. elegans), Drosophila melanogaster (D.melanogaster), mouse and human. Here, we demonstrated that overexpression of NRBP in Jurkat TAg cells specifically impairs T cell receptor (TCR) or phorbol myristate acetate (PMA)/ionomycin-mediated signaling leading to nuclear factor of activated T cells (NFAT) promoter activation. Furthermore, the N-terminal of NRBP is necessary for its regulation of NFAT activation. Finally, we showed that NRBP has minimal effect on both TCR- and PMA-induced CD69 up-regulation in Jurkat TAg cells, which suggests that NRBP may function downstream of protein kinase C (PKC)/Ras pathway.

  16. Oscillatory change of SR-protein kinase activities during oocyte maturation meiosis in fish

    Institute of Scientific and Technical Information of China (English)

    杨仲安; 曹丹; 桂建芳

    2000-01-01

    The SR-protein kinase activity was analyzed and the cytological changes were observed during oocyte maturation in bisexual transparent color crucian carp ( Carassius auratus color variety). The results revealed that the SR-protein kinase activity was sensitive to the artificially induced spawning hormones, and the change of oscillatory activity was similar to that of the maturation-promoting factor (MPF) kinase that regulates meiotic cell cycle in fish.

  17. Probing the Biomimetic Ice Nucleation Inhibition Activity of Poly(vinyl alcohol) and Comparison to Synthetic and Biological Polymers

    OpenAIRE

    Congdon, Thomas; Dean, Bethany T.; Kasperczak-Wright, James; Biggs, Caroline I.; Notman, Rebecca; Gibson, Matthew I.

    2015-01-01

    Nature has evolved many elegant solutions to enable life to flourish at low temperatures by either allowing (tolerance) or preventing (avoidance) ice formation. These processes are typically controlled by ice nucleating proteins or antifreeze proteins, which act to either promote nucleation, prevent nucleation or inhibit ice growth depending on the specific need, respectively. These proteins can be expensive and their mechanisms of action are not understood, limiting their translation, especi...

  18. Competition between members of the tribbles pseudokinase protein family shapes their interactions with mitogen activated protein kinase pathways.

    Science.gov (United States)

    Guan, Hongtao; Shuaib, Aban; Leon, David Davila De; Angyal, Adrienn; Salazar, Maria; Velasco, Guillermo; Holcombe, Mike; Dower, Steven K; Kiss-Toth, Endre

    2016-01-01

    Spatio-temporal regulation of intracellular signalling networks is key to normal cellular physiology; dysregulation of which leads to disease. The family of three mammalian tribbles proteins has emerged as an important controller of signalling via regulating the activity of mitogen activated protein kinases (MAPK), the PI3-kinase induced signalling network and E3 ubiquitin ligases. However, the importance of potential redundancy in the action of tribbles and how the differences in affinities for the various binding partners may influence signalling control is currently unclear. We report that tribbles proteins can bind to an overlapping set of MAPK-kinases (MAPKK) in live cells and dictate the localisation of the complexes. Binding studies in transfected cells reveal common regulatory mechanisms and suggest that tribbles and MAPKs may interact with MAPKKs in a competitive manner. Computational modelling of the impact of tribbles on MAPK activation suggests a high sensitivity of this system to changes in tribbles levels, highlighting that these proteins are ideally placed to control the dynamics and balance of activation of concurrent signalling pathways. PMID:27600771

  19. Targeted reengineering of protein geranylgeranyltransferase type I selectivity functionally implicates active-site residues in protein-substrate recognition.

    Science.gov (United States)

    Gangopadhyay, Soumyashree A; Losito, Erica L; Hougland, James L

    2014-01-21

    Posttranslational modifications are vital for the function of many proteins. Prenylation is one such modification, wherein protein geranylgeranyltransferase type I (GGTase-I) or protein farnesyltransferase (FTase) modify proteins by attaching a 20- or 15-carbon isoprenoid group, respectively, to a cysteine residue near the C-terminus of a target protein. These enzymes require a C-terminal Ca1a2X sequence on their substrates, with the a1, a2, and X residues serving as substrate-recognition elements for FTase and/or GGTase-I. While crystallographic structures of rat GGTase-I show a tightly packed and hydrophobic a2 residue binding pocket, consistent with a preference for moderately sized a2 residues in GGTase-I substrates, the functional impact of enzyme-substrate contacts within this active site remains to be determined. Using site-directed mutagenesis and peptide substrate structure-activity studies, we have identified specific active-site residues within rat GGTase-I involved in substrate recognition and developed novel GGTase-I variants with expanded/altered substrate selectivity. The ability to drastically alter GGTase-I selectivity mirrors similar behavior observed in FTase but employs mutation of a distinct set of structurally homologous active-site residues. Our work demonstrates that tunable selectivity may be a general phenomenon among multispecific enzymes involved in posttranslational modification and raises the possibility of variable substrate selectivity among GGTase-I orthologues from different organisms. Furthermore, the GGTase-I variants developed herein can serve as tools for studying GGTase-I substrate selectivity and the effects of prenylation pathway modifications on specific proteins. PMID:24344934

  20. Separation of the transcriptional activation and replication functions of the bovine papillomavirus-1 E2 protein.

    OpenAIRE

    Winokur, P. L.; McBride, A A

    1992-01-01

    Replication of bovine papillomavirus-1 (BPV-1) DNA requires two viral gene products, the E1 protein and the full-length E2 protein. The 48 kDa E2 protein is a site-specific DNA-binding protein that binds to several sites which lie adjacent to the BPV-1 origin of replication. The 85 amino acid C-terminal domain contains the specific DNA binding and dimerization properties of the protein. The approximately 200 amino acid N-terminal domain is crucial for transcriptional activation. Both of these...

  1. Involvement of hypothalamic AMP-activated protein kinase in leptin-induced sympathetic nerve activation.

    Directory of Open Access Journals (Sweden)

    Mamoru Tanida

    Full Text Available In mammals, leptin released from the white adipose tissue acts on the central nervous system to control feeding behavior, cardiovascular function, and energy metabolism. Central leptin activates sympathetic nerves that innervate the kidney, adipose tissue, and some abdominal organs in rats. AMP-activated protein kinase (AMPK is essential in the intracellular signaling pathway involving the activation of leptin receptors (ObRb. We investigated the potential of AMPKα2 in the sympathetic effects of leptin using in vivo siRNA injection to knockdown AMPKα2 in rats, to produce reduced hypothalamic AMPKα2 expression. Leptin effects on body weight, food intake, and blood FFA levels were eliminated in AMPKα2 siRNA-treated rats. Leptin-evoked enhancements of the sympathetic nerve outflows to the kidney, brown and white adipose tissues were attenuated in AMPKα2 siRNA-treated rats. To check whether AMPKα2 was specific to sympathetic changes induced by leptin, we examined the effects of injecting MT-II, a melanocortin-3 and -4 receptor agonist, on the sympathetic nerve outflows to the kidney and adipose tissue. MT-II-induced sympatho-excitation in the kidney was unchanged in AMPKα2 siRNA-treated rats. However, responses of neural activities involving adipose tissue to MT-II were attenuated in AMPKα2 siRNA-treated rats. These results suggest that hypothalamic AMPKα2 is involved not only in appetite and body weight regulation but also in the regulation of sympathetic nerve discharges to the kidney and adipose tissue. Thus, AMPK might function not only as an energy sensor, but as a key molecule in the cardiovascular, thermogenic, and lipolytic effects of leptin through the sympathetic nervous system.

  2. Diverse activation pathways in class A GPCRs converge near the G-protein-coupling region.

    Science.gov (United States)

    Venkatakrishnan, A J; Deupi, Xavier; Lebon, Guillaume; Heydenreich, Franziska M; Flock, Tilman; Miljus, Tamara; Balaji, Santhanam; Bouvier, Michel; Veprintsev, Dmitry B; Tate, Christopher G; Schertler, Gebhard F X; Babu, M Madan

    2016-08-25

    Class A G-protein-coupled receptors (GPCRs) are a large family of membrane proteins that mediate a wide variety of physiological functions, including vision, neurotransmission and immune responses. They are the targets of nearly one-third of all prescribed medicinal drugs such as beta blockers and antipsychotics. GPCR activation is facilitated by extracellular ligands and leads to the recruitment of intracellular G proteins. Structural rearrangements of residue contacts in the transmembrane domain serve as 'activation pathways' that connect the ligand-binding pocket to the G-protein-coupling region within the receptor. In order to investigate the similarities in activation pathways across class A GPCRs, we analysed 27 GPCRs from diverse subgroups for which structures of active, inactive or both states were available. Here we show that, despite the diversity in activation pathways between receptors, the pathways converge near the G-protein-coupling region. This convergence is mediated by a highly conserved structural rearrangement of residue contacts between transmembrane helices 3, 6 and 7 that releases G-protein-contacting residues. The convergence of activation pathways may explain how the activation steps initiated by diverse ligands enable GPCRs to bind a common repertoire of G proteins. PMID:27525504

  3. Separation of the transcriptional activation and replication functions of the bovine papillomavirus-1 E2 protein.

    Science.gov (United States)

    Winokur, P L; McBride, A A

    1992-11-01

    Replication of bovine papillomavirus-1 (BPV-1) DNA requires two viral gene products, the E1 protein and the full-length E2 protein. The 48 kDa E2 protein is a site-specific DNA-binding protein that binds to several sites which lie adjacent to the BPV-1 origin of replication. The 85 amino acid C-terminal domain contains the specific DNA binding and dimerization properties of the protein. The approximately 200 amino acid N-terminal domain is crucial for transcriptional activation. Both of these domains are highly conserved among different papillomaviruses. An internal hinge region separates the two functional domains. The region varies in amino acid sequence and length among the E2 proteins of different papillomaviruses. A series of mutations were constructed within the E2 open reading frame which delete various regions of the conserved DNA binding and transactivation domains as well as the internal hinge region. Two mutated E2 proteins that lack portions of the conserved DNA-binding domain but which support DNA replication were identified using transient replication assays. These mutated E2 proteins were unable to function as transcriptional activators. Conversely, two E2 proteins containing large deletions of the hinge region were able to activate transcription, but were defective for replication. Thus, the replication and transactivation functions of the E2 protein are separable. PMID:1327758

  4. Cannabinoid receptor 2 expression modulates Gβ(1)γ(2) protein interaction with the activator of G protein signalling 2/dynein light chain protein Tctex-1.

    Science.gov (United States)

    Nagler, Marina; Palkowitsch, Lysann; Rading, Sebastian; Moepps, Barbara; Karsak, Meliha

    2016-01-01

    The activator of G protein signalling AGS2 (Tctex-1) forms protein complexes with Gβγ, and controls cell proliferation by regulating cell cycle progression. A direct interaction of Tctex-1 with various G protein-coupled receptors has been reported. Since the carboxyl terminal portion of CB2 carries a putative Tctex-1 binding motif, we investigated the potential interplay of CB2 and Tctex-1 in the absence and presence of Gβγ. The supposed interaction of cannabinoid receptor CB2 with Tctex-1 and the influence of CB2 on the formation of Tctex-1-Gβγ-complexes were studied by co- and/or immunoprecipitation experiments in transiently transfected HEK293 cells. The analysis on Tctex-1 protein was performed in the absence and presence of the ligands JWH 133, 2-AG, and AM 630, the protein biosynthesis inhibitor cycloheximide or the protein degradation blockers MG132, NH4Cl/leupeptin or bafilomycin. Our results show that CB2 neither directly nor indirectly via Gβγ interacts with Tctex-1, but competes with Tctex-1 in binding to Gβγ. The Tctex-1-Gβγ protein interaction was disrupted by CB2 receptor expression resulting in a release of Tctex-1 from the complex, and its degradation by the proteasome and partly by lysosomes. The decrease in Tctex-1 protein levels is induced by CB2 expression "dose-dependently" and is independent of stimulation by agonist or blocking by an inverse agonist treatment. The results suggest that CB2 receptor expression independent of its activation by agonists is sufficient to competitively disrupt Gβγ-Tctex-1 complexes, and to initiate Tctex-1 degradation. These findings implicate that CB2 receptor expression modifies the stability of intracellular protein complexes by a non-canonical pathway.

  5. The Ginkgo biloba Extract EGb 761 Modulates Proteasome Activity and Polyglutamine Protein Aggregation

    Directory of Open Access Journals (Sweden)

    Marcel Stark

    2014-01-01

    Full Text Available The standardized Ginkgo biloba extract EGb 761 has well-described antioxidative activities and effects on different cytoprotective signaling pathways. Consequently, a potential use of EGb 761 in neurodegenerative diseases has been proposed. A common characteristic feature of a variety of such disorders is the pathologic formation of protein aggregates, suggesting a crucial role for protein homeostasis. In this study, we show that EGb 761 increased the catalytic activity of the proteasome and enhanced protein degradation in cultured cells. We further investigated this effect in a cellular model of Huntington’s disease (HD by employing cells expressing pathologic variants of a polyglutamine protein (polyQ protein. We show that EGb 761 affected these cells by (i increasing proteasome activity and (ii inducing a more efficient degradation of aggregation-prone proteins. These results demonstrate a novel activity of EGb 761 on protein aggregates by enhancing proteasomal protein degradation, suggesting a therapeutic use in neurodegenerative disorders with a disturbed protein homeostasis.

  6. Coexistence of dopamine-beta-hydroxylase and activated protein-2 alpha in rat cerebellar Purkinje cells

    Institute of Scientific and Technical Information of China (English)

    Kejian Wang; Wei Li; Shanquan Sun; Zhongqin Ren; Guiqiong He

    2009-01-01

    BACKGROUND:Tyrosine hydroxylase and phenylethanolamine-n-methyl transferase expression coexist in Purkinje cells of the rat cerebellum.Numerous reports have also been published addressing whether dopamine-beta-hydroxylase (DBH) expression exists in cerebellar Purkinje cells.OBJECTIVE:To investigate the coexistence of DBH and activator protein-2α expression in rat cerebellar Purkinje cells.DESIGN,TIME AND SETTING:A cell morphological study was performed at the Institute of Neuroscience,Chongqing Medical University,China in May 2007.MATERIALS:Ten healthy Wistar rats,of either gender,aged 14 weeks,served as experimental animals.Rabbit anti-mouse DBH,goat anti-mouse activator protein-2α and rabbit anti-mouse β-actin (Santa Cruz Biotechnology,Inc.,USA),horseradish peroxidase-labeled goat anti-rabbit IgG,FITC-labeled mouse anti-rabbit IgG,and Cy3-labeled mouse anti-goat IgG (Boster,Wuhan,China),were used in this study.METHODS:Immunohistochemical staining was used to measure the expression of DBH or activator protein-2α,with double-label immunofluorescence being employed to determine coexpression of both,in the cerebellum of 5 randomly selected rats.Western blot assay was utilized to determine the expression of DBH and activator protein-2α in the cerebellum of the remaining 5 rats.MAIN OUTCOME MEASURES:Expression,localization and coexistence of DBH and activator protein-2α in the cerebellum were measured separately.RESULTS:Immunohistochemical staining demonstrated that cerebellar Purkinje cells stained positive for DBH and activator protein-2α.Western blot assay also demonstrated DBH and activator protein-2α expression in the cerebellum.Double-labeling immunofluorescence showed the coexistence of DBH and activator protein-2α in cerebellar Purkinje cells.CONCLUSION:Norepinephrine and activator protein-2α coexist in rat cerebellar Purkinje cells.

  7. 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

    2016-10-01

    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.

  8. Coat protein activation of alfalfa mosaic virus replication is concentration dependent.

    Science.gov (United States)

    Guogas, Laura M; Laforest, Siana M; Gehrke, Lee

    2005-05-01

    Alfalfa mosaic virus (AMV) and ilarvirus RNAs are infectious only in the presence of the viral coat protein; therefore, an understanding of coat protein's function is important for defining viral replication mechanisms. Based on in vitro replication experiments, the conformational switch model states that AMV coat protein blocks minus-strand RNA synthesis (R. C. Olsthoorn, S. Mertens, F. T. Brederode, and J. F. Bol, EMBO J. 18:4856-4864, 1999), while another report states that coat protein present in an inoculum is required to permit minus-strand synthesis (L. Neeleman and J. F. Bol, Virology 254:324-333, 1999). Here, we report on experiments that address these contrasting results with a goal of defining coat protein's function in the earliest stages of AMV replication. To detect coat-protein-activated AMV RNA replication, we designed and characterized a subgenomic luciferase reporter construct. We demonstrate that activation of viral RNA replication by coat protein is concentration dependent; that is, replication was strongly stimulated at low coat protein concentrations but decreased progressively at higher concentrations. Genomic RNA3 mutations preventing coat protein mRNA translation or disrupting coat protein's RNA binding domain diminished replication. The data indicate that RNA binding and an ongoing supply of coat protein are required to initiate replication on progeny genomic RNA transcripts. The data do not support the conformational switch model's claim that coat protein inhibits the initial stages of viral RNA replication. Replication activation may correlate with low local coat protein concentrations and low coat protein occupancy on the multiple binding sites present in the 3' untranslated regions of the viral RNAs. PMID:15827190

  9. Covalent binding and hemolytic activity of complement proteins.

    OpenAIRE

    Law, S K; Lichtenberg, N A; Levine, R P

    1980-01-01

    We report the inactivation of the third component of complement (C3) by hydroxylamine. C3 hemolytic and covalent binding activities decline with identical kinetics, demonstrating a direct correlation between the two activities. We conclude that covalent, surface-bound C3b is hemolytically active. The inactivation of C3 is first order with respect to hydroxylamine. We also studied C3 inactivation with [14C]methylamine. The inactivation corresponds quantitatively with the labeling of C3 in the ...

  10. 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.

  11. Beneficial effects of metformin on primary cardiomyocytes via activation of adenosine monophosphate-activated protein kinase

    Institute of Scientific and Technical Information of China (English)

    WANG Xiao-fang; ZHANG Jin-ying; LI Ling; ZHAO Xiao-yan

    2011-01-01

    Background Metformin has become a cornerstone in the treatment of patients with type-2 diabetes. Accumulated evidence suggests that metformin supports direct cardiovascular effects. The present study aimed to investigate if metformin has beneficial effects on primary cardiomyocytes damaged by H2O2, and reveal the potential mechanism of action of metformin.Methods Cardiomyocytes were incubated in the presence of 100 umol/L. H2O2 for 12 hours. Cardiomyocytes were pretreated with metformin at different concentrations and time and with aminoimidazole carboxamide ribonucleotide (AICAR) (500 umol/L), an adenosine monophophate (AMP)-activated protein kinase (AMPK) agonist for 60 minutes before the addition of H2O2. Other cells were preincubated with compound C (an AMPK antagonist, 20 umol/L) for 4 hours. The viability and apoptosis of cells were analyzed. AMPK, endothelial nitric oxide synthase (eNOS), and transforming growth factor (TGF)-β1 were analyzed using immunblotting.Results Metformin had antagonistic effects on the influences of H2O2 on cell viability and attenuated oxidative stress-induced apoptosis. Metformin also increased phosphorylation of AMPK and eNOS, and reduced the expression of TGF-β1, basic fibroblast growth factor (bFGF), and tumor necrosis factor (TNF)-α.Conclusions Metformin has beneficial effects on cardiomyocytes, and this effect involves activation of the AMPK-eNOS pathway. Metformin may be potentially beneficial for the treatment of heart disease.

  12. Resveratrol improves non-alcoholic fatty liver disease by activating AMP-activated protein kinase

    Institute of Scientific and Technical Information of China (English)

    Jing SHANG; Lu-lu CHEN; Eang-xi XIAO; Hui SUN; Hong-cheng DING; Hu XIAO

    2008-01-01

    Aim: To investigate whether resveratrol (RSV) can improve non-alcoholic fatty liver disease (NAFLD) and to find the possible mechanism. Methods: Rats fed a high-fat diet were treated with RSV. The liver histology was observed. Hyperinsulinemic euglycemic clamp was performed to assess insulin sensitivity. Fat accumulation was induced in HepG2 cells, and the cells were treated with RSV. AMP-activated protein kinase (AMPK) phosphorylation levels were de-termined both in the animal study and cell study. Results: Rats fed a high-fat diet developed abdominal obesity, NAFLD, and insulin resistance (IR), which were markedly improved by 10 weeks of RSV administration. RSV treatment prevented triacylglycerol (TG) accumulation in HepG2 cells that were incubated with high concentration of glucose and insulin. Both in vivo and in vitro studies showed that RSV treatment could promote the phosphorylation of AMPK, which in this study, suppressed 2 lipogenesis gene expressions, contributing to the improvement of NAFLD and IR. Conclusion: The results indicated that by re-ducing TG accumulation and improving IR, RSV could protect the liver from NAFLD. The activation of AMPK was involved in the mechanism. RSV has the therapeutic potential for preventing or treating NAFLD and IR-related metabolic disorders.

  13. 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.

  14. Computer-aided design of modular protein devices: Boolean AND gene activation

    Science.gov (United States)

    Salis, H.; Kaznessis, Y. N.

    2006-12-01

    Many potentially useful synthetic gene networks require the expression of an engineered gene if and only if two different DNA-binding proteins exist in sufficient concentration. While some natural and engineered systems activate gene expression according to a logical AND-like behavior, they often utilize allosteric or cooperative protein-protein interactions, rendering their components unsuitable for a toolbox of modular parts for use in multiple applications. Here, we develop a quantitative model to demonstrate that a small system of interacting fusion proteins, called a protein device, can activate an engineered gene according to the Boolean AND behavior while using only modular protein domains and DNA sites. The fusion proteins are created from transactivating, DNA-binding, non-DNA binding, and protein-protein interaction domains along with the corresponding peptide ligands. Using a combined kinetic and thermodynamic model, we identify the characteristics of the molecular components and their rates of constitutive production that maximize the fidelity of AND behavior. These AND protein devices facilitate the creation of complex genetic programs and may be used to create gene therapies, biosensors and other biomedical and biotechnological applications that turn on gene expression only when multiple DNA-binding proteins are simultaneously present.

  15. Development of orally active inhibitors of protein and cellular fucosylation

    OpenAIRE

    Okeley, Nicole M.; Alley, Stephen C.; Anderson, Martha E.; Boursalian, Tamar E.; Burke, Patrick J.; Emmerton, Kim M.; Jeffrey, Scott C.; Klussman, Kerry; Law, Che-Leung; Sussman, Django; Toki, Brian E.; Westendorf, Lori; Zeng, Weiping; Zhang, XinQun; Benjamin, Dennis R.

    2013-01-01

    The key role played by fucose in glycoprotein and cellular function has prompted significant research toward identifying recombinant and biochemical strategies for blocking its incorporation into proteins and membrane structures. Technologies surrounding engineered cell lines have evolved for the inhibition of in vitro fucosylation, but they are not applicable for in vivo use and drug development. To address this, we screened a panel of fucose analogues and identified 2-fluorofucose and 5-alk...

  16. Thioredoxin interacting protein inhibits hypoxia-inducible factor transcriptional activity

    OpenAIRE

    Farrell, Michael R; Rogers, Lynette K.; Liu, Yusen; Welty, Stephen E.; Tipple, Trent E.

    2010-01-01

    Vascular endothelial growth factor (VEGF) is required for proper lung development and is transcriptionally regulated in alveolar epithelial cells by hypoxia inducible factor (HIF). Previous findings in a newborn mouse model of bronchopulmonary dysplasia (BPD) suggest that thioredoxin interacting protein (Txnip) is a novel regulator of VEGF expression. The present studies were designed to test the hypothesis that Txnip negatively regulates VEGF through effects on HIF-mediated gene expression. ...

  17. Probing heterotrimeric G protein activation: applications to biased ligands.

    OpenAIRE

    Denis, Colette; Saulière, Aude; Galandrin, Ségolène; Sénard, Jean-Michel; Galés, Céline

    2012-01-01

    Cell surface G protein-coupled receptors (GPCRs) drive numerous signaling pathways involved in the regulation of a broad range of physiologic processes. Today, they represent the largest target for modern drugs development with potential application in all clinical fields. Recently, the concept of “ligand-directed trafficking” has led to a conceptual revolution in pharmacological theory, thus opening new avenues for drug discovery. Accordingly, GPCRs do not function as simple on-off switch bu...

  18. Structural basis for activation of G-protein-coupled receptors

    DEFF Research Database (Denmark)

    Gether, Ulrik; Asmar, Fazila; Meinild, Anne Kristine;

    2002-01-01

    Our understanding of how G-protein-coupled receptors (GPCRs) operate at the molecular level has been considerably improved over the last few years. The application of advanced biophysical techniques as well as the availability of high-resolution structural information has allowed insight both int......-expression with the cAMP sensitive Cl- channel CFTR (cystic fibrosis transmembrane conductance regulator) and electrophysiological measurements....

  19. INHIBITION OF IL-6-INDUCED STAT3 ACTIVATION IN MYELOMA CELLS BY PROTEIN KINASE A

    Institute of Scientific and Technical Information of China (English)

    宋伦; 黎燕; 沈倍奋

    2001-01-01

    To investigate the regulation effect of protein kinase A on IL-6-induced STAT3 activation in myeloma cells. Methods: Two human myeloma cell lines-Sko-007 and U266 were pretreated with Forskolin, a protein kinase A antagonist, and then stimulated by IL-6. The activation state of STAT3 in these two cells were examined by electrophoretic mobility shift assay (EMSA). Results: Although PKA pathway itself doesn't participate in IL-6 signal transduction in Sko-007 and U266 cells, activation of protein kinase A can inhibit IL-6-induced STAT3 activation in these two cell lines. Conclusion: There exists an inhibitory effect of protein kinase A on STAT3 activation in human myeloma cells treated by IL-6.

  20. Monitoring and validating active site redox states in protein crystals.

    Science.gov (United States)

    Antonyuk, Svetlana V; Hough, Michael A

    2011-06-01

    High resolution protein crystallography using synchrotron radiation is one of the most powerful tools in modern biology. Improvements in resolution have arisen from the use of X-ray beamlines with higher brightness and flux and the development of advanced detectors. However, it is increasingly recognised that the benefits brought by these advances have an associated cost, namely deleterious effects of X-ray radiation on the sample (radiation damage). In particular, X-ray induced reduction and damage to redox centres has been shown to occur much more rapidly than other radiation damage effects, such as loss of resolution or damage to disulphide bridges. Selection of an appropriate combination of in-situ single crystal spectroscopies during crystallographic experiments, such as UV-visible absorption and X-ray absorption spectroscopy (XAFS), allows for effective monitoring of redox states in protein crystals in parallel with structure determination. Such approaches are also essential in cases where catalytic intermediate species are generated by exposure to the X-ray beam. In this article, we provide a number of examples in which multiple single crystal spectroscopies have been key to understanding the redox status of Fe and Cu centres in crystal structures. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.

  1. Identification of a protein kinase activity in purified foot- and-mouth disease virus.

    OpenAIRE

    Grubman, M J; Baxt, B; La Torre, J L; Bachrach, H L

    1981-01-01

    Purified preparations of foot-and-mouth disease virus types A, O, and C contain a protein kinase activity which can transfer the gamma phosphate of [32P]ATP to virion structural proteins VP2 and VP3 and exogenous acceptor proteins. Utilizing protamine sulfate as an acceptor, the kinase activity can be demonstrated in disrupted virus but not in intact virus. The enzyme is heat labile with optimal activity at pH 7 or greater. Serine residues of protamine sulfate were identified as the amino aci...

  2. Protein palmitoylation activate zygotic gene expression during the maternal-to-zygotic transition.

    Science.gov (United States)

    Du, Zhaoxia; Chen, Xueran; Li, Xian; He, Kun; Ji, Shufang; Shi, Wei; Hao, Aijun

    2016-06-24

    Upon fertilization, maternal factors direct development and trigger zygotic genome activation at the maternal-to-zygotic transition (MZT). However, the factors that activate the zygotic program in vertebrates are not well defined. Here, we found that protein palmitoylation played an important role in acquiring transcriptional competency and orchestrating the clearance of the maternal program in zebrafish. After inhibition of protein palmitoylation, zebrafish embryos developed normally before the Mid-Blastula Transition (MBT); however, they did not initiate epiboly. Moreover, our results showed that protein palmitoylation is required to initiate the zygotic developmental program and induce clearance of the maternal program by activating miR-430 expression. PMID:27235108

  3. Immunotherapy for Prostate Cancer with Gc Protein-Derived Macrophage-Activating Factor, GcMAF.

    Science.gov (United States)

    Yamamoto, Nobuto; Suyama, Hirofumi; Yamamoto, Nobuyuki

    2008-07-01

    Serum Gc protein (known as vitamin D(3)-binding protein) is the precursor for the principal macrophage-activating factor (MAF). The MAF precursor activity of serum Gc protein of prostate cancer patients was lost or reduced because Gc protein was deglycosylated by serum alpha-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Therefore, macrophages of prostate cancer patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Stepwise treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generated the most potent MAF (termed GcMAF) ever discovered, which produces no adverse effect in humans. Macrophages activated by GcMAF develop a considerable variation of receptors that recognize the abnormality in malignant cell surface and are highly tumoricidal. Sixteen nonanemic prostate cancer patients received weekly administration of 100 ng of GcMAF. As the MAF precursor activity increased, their serum Nagalase activity decreased. Because serum Nagalase activity is proportional to tumor burden, the entire time course analysis for GcMAF therapy was monitored by measuring the serum Nagalase activity. After 14 to 25 weekly administrations of GcMAF (100 ng/week), all 16 patients had very low serum Nagalase levels equivalent to those of healthy control values, indicating that these patients are tumor-free. No recurrence occurred for 7 years. PMID:18633461

  4. Differential AMP-activated Protein Kinase (AMPK) Recognition Mechanism of Ca2+/Calmodulin-dependent Protein Kinase Kinase Isoforms.

    Science.gov (United States)

    Fujiwara, Yuya; Kawaguchi, Yoshinori; Fujimoto, Tomohito; Kanayama, Naoki; Magari, Masaki; Tokumitsu, Hiroshi

    2016-06-24

    Ca(2+)/calmodulin-dependent protein kinase kinase β (CaMKKβ) is a known activating kinase for AMP-activated protein kinase (AMPK). In vitro, CaMKKβ phosphorylates Thr(172) in the AMPKα subunit more efficiently than CaMKKα, with a lower Km (∼2 μm) for AMPK, whereas the CaMKIα phosphorylation efficiencies by both CaMKKs are indistinguishable. Here we found that subdomain VIII of CaMKK is involved in the discrimination of AMPK as a native substrate by measuring the activities of various CaMKKα/CaMKKβ chimera mutants. Site-directed mutagenesis analysis revealed that Leu(358) in CaMKKβ/Ile(322) in CaMKKα confer, at least in part, a distinct recognition of AMPK but not of CaMKIα. PMID:27151216

  5. Influenza B virus non-structural protein 1 counteracts ISG15 antiviral activity by sequestering ISGylated viral proteins.

    Science.gov (United States)

    Zhao, Chen; Sridharan, Haripriya; Chen, Ran; Baker, Darren P; Wang, Shanshan; Krug, Robert M

    2016-01-01

    The ubiquitin-like protein ISG15 and its conjugation to proteins (ISGylation) are strongly induced by type I interferon. Influenza B virus encodes non-structural protein 1 (NS1B) that binds human ISG15 and provides an appropriate model for determining how ISGylation affects virus replication in human cells. Here using a recombinant virus encoding a NS1B protein defective in ISG15 binding, we show that NS1B counteracts ISGylation-mediated antiviral activity by binding and sequestering ISGylated viral proteins, primarily ISGylated viral nucleoprotein (NP), in infected cells. ISGylated NP that is not sequestered by mutant NS1B acts as a dominant-negative inhibitor of oligomerization of the more abundant unconjugated NP. Consequently formation of viral ribonucleoproteins that catalyse viral RNA synthesis is inhibited, causing decreased viral protein synthesis and virus replication. We verify that ISGylated NP is largely responsible for inhibition of viral RNA synthesis by generating recombinant viruses that lack known ISGylation sites in NP. PMID:27587337

  6. Metallocarbene Artificial Enzymes : Extending Transition Metal Selectivity and Protein Activity

    NARCIS (Netherlands)

    Basauri Molina, M.

    2015-01-01

    A series of new semi-synthetic metalloprotein hybrids were created via the covalent binding of organometallic species in the active site of lipases, accordingly resulting in the first active site-directed (ASD) homogeneous artificial metalloenzymes. The use of this method promises the generation of

  7. 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.

  8. Cellular fatty acid composition, protein profile and antimicrobial activity of Bacillus sp., isolated from fish gut

    Institute of Scientific and Technical Information of China (English)

    Pushparaj Sujith; Baskaran Rohini; Singaram Jayalakshmi

    2014-01-01

    Objective: To purify and partially characterize the antimicrobial compounds from bacteriaBacillus sp., isolated from fish gut. Methods: Protein and fatty acids were isolated from the bacteria and checked for the presence of antibacterial activity. Protein has been purified to apparent homogeneity from the supernatants of culture by means of ammonium sulphate precipitation followed by dialysis. Fourier transform infrared spectroscopy analyses were performed for proteins to identify the functional groups.Results:sulfate polyacrylamide gel electrophoresis. Fatty acids were extracted and subjected to gas chromatographic analysis.Conclusions:Protein showed an apparent molecular mass 56, 47 and 39 kDa on sodium dodecyl acids and proteins which holds promise for the development of new drugs. The antimicrobial activity of the bacteria might be due to the presence of fatty acids and proteins which holds promise for the development of new drugs.

  9. Activation of ERK mitogen-activated protein kinase in human cells by the mycotoxin patulin

    International Nuclear Information System (INIS)

    Patulin (PAT), a mycotoxin produced by certain species of Penicillium and Aspergillus, is often detectable in moldy fruits and their derivative products. PAT led to a concentration-dependent and time-dependent increase in phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in human embryonic kidney (HEK293) cells, human peripheral blood mononuclear cells (PBMCs), and Madin-Darby canine kidney (MDCK) cells. Exposure of HEK293 cells to concentrations above 5 μM PAT for 30 min induced ERK1/2 phosphorylation; activation of ERK1/2 was also observed after 24 h incubation with 0.05 μM of PAT. Treatment of human PBMCs for 30 min with 30 μM PAT dramatically increased the phosphorylated ERK1/2 levels. Both MEK1/2 inhibitors, U0126 and PD98059, suppressed ERK1/2 activation in either HEK293 or MDCK cells. In HEK293 cells, U0126-mediated inhibition of PAT-induced ERK1/2 phosphorylation resulted in a significant decrease in levels of DNA damage, expressed as tail moment values, in the single cell gel electrophoresis assay. Conversely, U0126 did not affect cell viability, lactate dehydrogenase release, and the DNA synthesis rate in PAT-treated cultures. Exposure of HEK293 cells for 90 min to 15 μM PAT elevated the levels of early growth response gene-1 (egr-1) mRNA, but not of c-fos, fosB, and junB mRNAs. These results indicate that in human cells, PAT causes a rapid and persistent activation of ERK1/2 and this signaling pathway plays an important role in mediating PAT-induced DNA damage and egr-1 gene expression

  10. TOPICAL REVIEW: Protein stability and enzyme activity at extreme biological temperatures

    Science.gov (United States)

    Feller, Georges

    2010-08-01

    Psychrophilic microorganisms thrive in permanently cold environments, even at subzero temperatures. To maintain metabolic rates compatible with sustained life, they have improved the dynamics of their protein structures, thereby enabling appropriate molecular motions required for biological activity at low temperatures. As a consequence of this structural flexibility, psychrophilic proteins are unstable and heat-labile. In the upper range of biological temperatures, thermophiles and hyperthermophiles grow at temperatures > 100 °C and synthesize ultra-stable proteins. However, thermophilic enzymes are nearly inactive at room temperature as a result of their compactness and rigidity. At the molecular level, both types of extremophilic proteins have adapted the same structural factors, but in opposite directions, to address either activity at low temperatures or stability in hot environments. A model based on folding funnels is proposed accounting for the stability-activity relationships in extremophilic proteins.

  11. Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain

    DEFF Research Database (Denmark)

    Adari, H; Lowy, D R; Willumsen, B M;

    1988-01-01

    A cytoplasmic protein that greatly enhances the guanosine triphosphatase (GTPase) activity of N-ras protein but does not affect the activity of oncogenic ras mutants has been recently described. This protein (GAP) is shown here to be ubiquitous in higher eukaryotes and to interact with H-ras...... as well as with N-ras proteins. To identify the region of ras p21 with which GAP interacts, 21 H-ras mutant proteins were purified and tested for their ability to undergo stimulation of GTPase activity by GAP. Mutations in nonessential regions of H-ras p21 as well as mutations in its carboxyl....... Transforming mutations at positions 12, 59, and 61 (the phosphoryl binding region) abolished GTPase stimulation by GAP. Point mutations in the putative effector region of ras p21 (amino acids 35, 36, and 38) were also insensitive to GAP. However, a point mutation at position 39, shown previously not to impair...

  12. Oral glucose ingestion attenuates exercise-induced activation of 5'-AMP-activated protein kinase in human skeletal muscle

    DEFF Research Database (Denmark)

    Åkerström, Thorbjörn; Birk, Jesper Bratz; Klein, Ditte Kjærsgaard;

    2006-01-01

    5'-AMP-activated protein kinase (AMPK) has been suggested to be a 'metabolic master switch' regulating various aspects of muscle glucose and fat metabolism. In isolated rat skeletal muscle, glucose suppresses the activity of AMPK and in human muscle glycogen loading decreases exercise-induced AMPK...

  13. 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

  14. Two putative protein kinase CK2 phosphorylation sites are important for Myf-5 activity

    DEFF Research Database (Denmark)

    Winter, B; Kautzner, I; Issinger, O G;

    1997-01-01

    Myf-5, a member of a family of muscle-specific transcription factors, is important for myogenic cell determination and differentiation. Here, we report that Myf-5 protein constitutes a substrate for phosphorylation in vitro by protein kinase CK2. We identified two potential phosphorylation sites...... localization and/or protein stability. Our data suggest that CK2-mediated phosphorylation of Myf-5 is required for Myf-5 activity....

  15. Trans activation by the bovine papillomavirus E2 protein in Saccharomyces cerevisiae.

    OpenAIRE

    Morrissey, L C; Barsoum, J; Androphy, E J

    1989-01-01

    The papillomavirus E2 protein functions as an enhancer-binding factor to promote transcription in mammalian cells. We found that one copy of the E2 binding site acted as an E2 protein-dependent upstream activating sequence in Saccharomyces cerevisiae. Additional copies of the binding motif further augmented transcription. These results imply that the E2 protein functionally interacts with highly conserved transcriptional elements.

  16. The role of AMP-activated protein kinase in regulation of skeletal muscle metabolism

    OpenAIRE

    Anna Dziewulska; Paweł Dobrzyń; Agnieszka Dobrzyń

    2010-01-01

    AMP-activated protein kinase (AMPK) is a conserved, ubiquitously expressed eukaryotic enzyme that is activated in response to increasing AMP level. Regulation of AMPK activity in skeletal muscle is coordinated by contraction and phosphorylation by upstream kinases and a growing number of hormones and cytokines. Once activated, AMPK turns on catabolic, ATP-generating pathways, and turns off ATP-consuming metabolic processes such as biosynthesis and proliferation. Activation of AMPK promotes gl...

  17. Hsp90 protein interacts with phosphorothioate oligonucleotides containing hydrophobic 2'-modifications and enhances antisense activity.

    Science.gov (United States)

    Liang, Xue-Hai; Shen, Wen; Sun, Hong; Kinberger, Garth A; Prakash, Thazha P; Nichols, Joshua G; Crooke, Stanley T

    2016-05-01

    RNase H1-dependent antisense oligonucleotides (ASOs) are chemically modified to enhance pharmacological properties. Major modifications include phosphorothioate (PS) backbone and different 2'-modifications in 2-5 nucleotides at each end (wing) of an ASO. Chemical modifications can affect protein binding and understanding ASO-protein interactions is important for better drug design. Recently we identified many intracellular ASO-binding proteins and found that protein binding could affect ASO potency. Here, we analyzed the structure-activity-relationships of ASO-protein interactions and found 2'-modifications significantly affected protein binding, including La, P54nrb and NPM. PS-ASOs containing more hydrophobic 2'-modifications exhibit higher affinity for proteins in general, although certain proteins, e.g. Ku70/Ku80 and TCP1, are less affected by 2'-modifications. We found that Hsp90 protein binds PS-ASOs containing locked-nucleic-acid (LNA) or constrained-ethyl-bicyclic-nucleic-acid ((S)-cEt) modifications much more avidly than 2'-O-methoxyethyl (MOE). ASOs bind the mid-domain of Hsp90 protein. Hsp90 interacts with more hydrophobic 2' modifications, e.g. (S)-cEt or LNA, in the 5'-wing of the ASO. Reduction of Hsp90 protein decreased activity of PS-ASOs with 5'-LNA or 5'-cEt wings, but not with 5'-MOE wing. Together, our results indicate Hsp90 protein enhances the activity of PS/LNA or PS/(S)-cEt ASOs, and imply that altering protein binding of ASOs using different chemical modifications can improve therapeutic performance of PS-ASOs. PMID:26945041

  18. Regulation of AMP-activated protein kinase by LKB1 and CaMKK in adipocytes

    DEFF Research Database (Denmark)

    Gormand, Amélie; Henriksson, Emma; Ström, Kristoffer;

    2011-01-01

    AMP-activated protein kinase (AMPK) is a serine/threonine kinase that regulates cellular and whole body energy homeostasis. In adipose tissue, activation of AMPK has been demonstrated in response to a variety of extracellular stimuli. However, the upstream kinase that activates AMPK in adipocytes...

  19. 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, Saema; Edvinsson, Lars

    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...... enhanced phosphorylation only at 48 hours after SAH. The pattern was identical in large cerebral arteries and in intracerebral microvessels. Treatment with either the PKC (RO-31-7549) or the raf (SB386023-b) inhibitor prevented the kinase activation. CONCLUSIONS: The results show that specific subtypes...... 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...

  20. Methoden zur Immobilisierung von Proteinen auf Polyurethan- und Goldoberflächen und ihr Einfluss auf Konformation und Aktivität der Proteine

    OpenAIRE

    Kreider, Alexej

    2014-01-01

    In recent years anti-freeze proteins became the focus of interest for materials science due to their ice-crystall-growth inhibiting properties, recrystallisation properties and ice-crystall structuring properties. The transfer of these properties to surfaces by means of a molecular biomimetic approach is the challenge as well as motivation of this work. Here, the molecular bionic approach is based on chemical immobilization methods of proteins to solid surfaces. Thus, the first question of th...

  1. Complement activation and complement control proteins in acute pancreatitis.

    OpenAIRE

    Whicher, J T; Barnes, M. P.; Brown, A; Cooper, M J; Read, R; Walters, G; Williamson, R C

    1982-01-01

    Serum levels of the complement proteins C3, C4, C1 inhibitor (C1 INH), factor I (C3b inactivator) and factor H (BIH) and plasma levels of cleavage products of C3 (C3c) and factor B were measured in 26 patients with acute pancreatitis. Breakdown of C3 occurred in 19 patients, as shown by a reduction in C3 level and the presence of C3c. C4 levels, however, did not fall and factor B breakdown products were not detected, thus suggesting that enzymatic cleavage of C3 occurred without significant i...

  2. Croatian produced unifloral honey characterized according to the protein and proline content and enzyme activities

    Directory of Open Access Journals (Sweden)

    Flanjak Ivana

    2016-06-01

    Full Text Available In honey, the content of proteins, including the enzymes, is relatively low and has a minor nutritive significance. On the other hand, the proteins, including the enzymes, are usually used as honey quality evaluation parameters. This is because protein content and enzyme activities vary regarding the botanical origin of the honey. Since the results of protein content, glucose-oxidase, and acid phosphatase, for honeys produced in Croatia, are not available, four of the most abundant honey types produced in Croatia (black locust, sage, chestnut, and honeydew honey are characterised according to the protein and proline content and enzyme activities. The characterisation was done to determine specificities and contribute to the characterisation of unifloral honeys. Dark honey types (honeydew and chestnut honey had a higher proline content, and diastase, invertase, and glucose-oxidase activity than lighter sage and black locust honey. Black locust honey has a naturally low enzyme activity and showed the highest acid phosphatase activity among the analysed honey types, while honeydew honey, otherwise known to possess high proline content and enzyme activity, had a low protein content comparable to black locust honey. Statistically significant correlations were obtained between all analysed parameters, with the exception of acid phosphatase activity.

  3. Endosomal SNARE proteins regulate CFTR activity and trafficking in epithelial cells.

    Science.gov (United States)

    Bilan, Frédéric; Nacfer, Magali; Fresquet, Fleur; Norez, Caroline; Melin, Patricia; Martin-Berge, Alice; Costa de Beauregard, Marie-Alyette; Becq, Frédéric; Kitzis, Alain; Thoreau, Vincent

    2008-07-01

    The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) protein is a chloride channel localized at the apical plasma membrane of epithelial cells. We previously described that syntaxin 8, an endosomal SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) protein, interacts with CFTR and regulates its trafficking to the plasma membrane and hence its channel activity. Syntaxin 8 belongs to the endosomal SNARE complex which also contains syntaxin 7, vti1b and VAMP8. Here, we report that these four endosomal SNARE proteins physically and functionally interact with CFTR. In LLC-PK1 cells transfected with CFTR and in Caco-2 cells endogenously expressing CFTR, we demonstrated that endosomal SNARE protein overexpression inhibits CFTR activity but not swelling- or calcium-activated iodide efflux, indicating a specific effect upon CFTR activity. Moreover, co-immunoprecipitation experiments in LLC-PK1-CFTR cells showed that CFTR and SNARE proteins belong to a same complex and pull-down assays showed that VAMP8 and vti1b preferentially interact with CFTR N-terminus tail. By cell surface biotinylation and immunofluorescence experiments, we evidenced that endosomal SNARE overexpression disturbs CFTR apical targeting. Finally, we found a colocalization of CFTR and endosomal SNARE proteins in Rab11-positive recycling endosomes, suggesting a new role for endosomal SNARE proteins in CFTR trafficking in epithelial cells.

  4. 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...

  5. Generation of Fluorogen-Activating Designed Ankyrin Repeat Proteins (FADAs) as Versatile Sensor Tools.

    Science.gov (United States)

    Schütz, Marco; Batyuk, Alexander; Klenk, Christoph; Kummer, Lutz; de Picciotto, Seymour; Gülbakan, Basri; Wu, Yufan; Newby, Gregory A; Zosel, Franziska; Schöppe, Jendrik; Sedlák, Erik; Mittl, Peer R E; Zenobi, Renato; Wittrup, K Dane; Plückthun, Andreas

    2016-03-27

    Fluorescent probes constitute a valuable toolbox to address a variety of biological questions and they have become irreplaceable for imaging methods. Commonly, such probes consist of fluorescent proteins or small organic fluorophores coupled to biological molecules of interest. Recently, a novel class of fluorescence-based probes, fluorogen-activating proteins (FAPs), has been reported. These binding proteins are based on antibody single-chain variable fragments and activate fluorogenic dyes, which only become fluorescent upon activation and do not fluoresce when free in solution. Here we present a novel class of fluorogen activators, termed FADAs, based on the very robust designed ankyrin repeat protein scaffold, which also readily folds in the reducing environment of the cytoplasm. The FADA generated in this study was obtained by combined selections with ribosome display and yeast surface display. It enhances the fluorescence of malachite green (MG) dyes by a factor of more than 11,000 and thus activates MG to a similar extent as FAPs based on single-chain variable fragments. As shown by structure determination and in vitro measurements, this FADA was evolved to form a homodimer for the activation of MG dyes. Exploiting the favorable properties of the designed ankyrin repeat protein scaffold, we created a FADA biosensor suitable for imaging of proteins on the cell surface, as well as in the cytosol. Moreover, based on the requirement of dimerization for strong fluorogen activation, a prototype FADA biosensor for in situ detection of a target protein and protein-protein interactions was developed. Therefore, FADAs are versatile fluorescent probes that are easily produced and suitable for diverse applications and thus extend the FAP technology. PMID:26812208

  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. Antioxidant activity and functional properties of enzymatic protein hydrolysates from common carp (Cyprinus carpio) roe (egg).

    Science.gov (United States)

    Chalamaiah, M; Jyothirmayi, T; Diwan, Prakash V; Dinesh Kumar, B

    2015-09-01

    Previously, we have reported the composition, molecular mass distribution and in vivo immunomodulatory effects of common carp roe protein hydrolysates. In the current study, antioxidative activity and functional properties of common carp (Cyprinus carpio) roe (egg) protein hydrolysates, prepared by pepsin, trypsin and Alcalase, were evaluated. The three hydrolysates showed excellent antioxidant activities in a dose dependent manner in various in vitro models such as 2,2 diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, 2,2'-azino-bis(3-ethylbenzthiazoline-6)-sulfonic acid (ABTS(+)) radical scavenging activity, ferric reducing antioxidant power (FRAP) and ferrous ion (Fe(2+)) chelating ability. Enzymatic hydrolysis significantly increased protein solubility of the hydrolysates to above 62 % over a wide pH range (2-12). Carp roe hydrolysates exhibited good foaming and emulsification properties. The results suggest that bioactive carp roe protein hydrolysates (CRPHs) with good functional properties could be useful in health food/nutraceutical/pharmaceutical industry for various applications. PMID:26344996

  8. Cartilage oligomeric matrix protein in patients with juvenile idiopathic arthritis: relation to growth and disease activity

    DEFF Research Database (Denmark)

    Bjørnhart, Birgitte; Juul, Anders; Nielsen, Susan;

    2009-01-01

    OBJECTIVE: Cartilage oligomeric matrix protein (COMP) has been identified as a prognostic marker of progressive joint destruction in rheumatoid arthritis. In this population based study we evaluated associations between plasma concentrations of COMP, disease activity, and growth velocity in patie...

  9. Substrate-induced activation of a trapped IMC-mediated protein folding intermediate.

    Science.gov (United States)

    Inouye, M; Fu, X; Shinde, U

    2001-04-01

    While several unfolded proteins acquire native structures through distinct folding intermediates, the physiological relevance and importance of such states in the folding kinetics remain controversial. The intramolecular chaperone (IMC) of subtilisin was used to trap a partially folded, stable crosslinked intermediate conformer (CLIC) through a disulfide bond between mutated IMC and subtilisin. The trapped CLIC contains non-native interactions. Here we show that CLIC can be induced into a catalytically active form by incubating it with small peptide substrates. The structure and catalytic properties of the activated crosslinked intermediate conformer (A-CLIC) differ from those of the fully folded enzyme in that A-CLIC lacks any endopeptidase activity toward a large protein substrate. Our results show that a disulfide-linked partially folded protein can be induced to acquire catalytic activity with a substrate specificity that is different from completely folded subtilisin. These results also suggest that protein folding intermediates may also participate in catalytic reactions.

  10. An Expanded Role for AMP-activated Protein Kinase-Regulator of Myocardial Protein Degradation

    OpenAIRE

    Baskin, Kedryn K.; Taegtmeyer, Heinrich

    2011-01-01

    Rudolph Schoenheimer’s concept of the “dynamic state of body constituents” has existed since the 1940s, but the idea that heart muscle cells renew themselves from within is relatively new. Many studies have elucidated the interaction of metabolic pathways for energy provision and contraction of the heart, and work in the field has uncovered novel metabolic regulators of enzyme action. However, the impact of myocardial energy metabolism on myocardial protein turnover has received little attent...

  11. Activation of a mitogen-activated protein kinase pathway in Arabidopsis by chitin.

    Science.gov (United States)

    Wan, Jinrong; Zhang, Shuqun; Stacey, Gary

    2004-03-01

    SUMMARY Chitin, a polysaccharide composed of beta-1-->4-linked N-acetyl-d-glucosamine, has been shown or implicated as a signal in plant defence and development. However, the key components of chitin perception and downstream signalling in non-leguminous plants are largely unknown. In recent years, mitogen-activated protein kinases (MAPKs) and their cascades were shown to transduce various extracellular stimuli into internal cellular responses. To investigate the possible involvement of MAPKs in chitin signalling in plants, the model plant Arabidopsis thaliana was treated with crab-shell chitin and also with the purified chitin oligomers (degree of polymerization, d.p. = 2-8). Both mRNA levels and kinase activity of two MAPK genes, AtMPK6 and AtMPK3, were monitored after treatment. The mRNA of AtMPK3 was strongly up-regulated by both chitin and its larger oligomers (d.p. = 6-8), but the mRNA of AtMPK6 did not appear to be regulated by these treatments. However, the kinase activity of both MAPKs was induced by chitin and the larger oligomers (d.p. = 6-8), with AtMPK6 much more strongly induced. In addition, WRKY22, WRKY29, WRKY33 and WRKY53, which encode four WRKY transcription factors that recognize TTGAC(C/T) W-box elements in promoters of numerous plant defence-related genes, were up-regulated by these treatments. WRKY33 and WRKY53 expression was induced by the transgenic expression of the tobacco MAPKK NtMEK2 active mutant NtMEK2(DD), suggesting a potential role for these WRKY transcription factors in relaying the signal generated from the MAPK cascade to downstream genes. These data suggest that AtMPK6/AtMPK3 and WRKY transcription factors (such as WRKY33 and WRKY53) may be important components of a pathway involved in chitin signalling in Arabidopsis plants.

  12. 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.

  13. RBP1 Recruits Both Histone Deacetylase-Dependent and -Independent Repression Activities to Retinoblastoma Family Proteins

    OpenAIRE

    Lai, Albert; Lee, Joseph M; Yang, Wen-Ming; DeCaprio, James A.; William G Kaelin; Seto, Edward; Branton, Philip E.

    1999-01-01

    Retinoblastoma (RB) tumor suppressor family proteins block cell proliferation in part by repressing certain E2F-specific promoters. Both histone deacetylase (HDAC)-dependent and -independent repression activities are associated with the RB “pocket.” The mechanism by which these two repression functions occupy the pocket is unknown. A known RB-binding protein, RBP1, was previously found by our group to be an active corepressor which, if overexpressed, represses E2F-mediated transcription via i...

  14. Protein Hydrolysis from Catfish Prepared by Papain Enzyme and Antioxidant Activity of Hydrolyzate

    OpenAIRE

    Ace Baehaki1); Shanti Dwita Lestari; Achmad Rizky Romadhoni

    2015-01-01

    The objective of this research was to make a protein hydrolysates from catfish (Pangasius pangasius) enzymatically using papain enzyme and analyzed the antioxidant activity of protein hydrolysates produced. The research used the method completely randomized design with two replications the treatment were the difference concentration of the papain enzyme (0%, 1%, 2%, 3%, 4%, 5%, and 6%). The parameters of research were antioxidative activity using DPPH (2,2-difenil-1–pikrilhidra...

  15. Antioxidant Activities of Protein Hydrolysates from Little Hairtail (Trichiurus haumela) of East China Sea

    OpenAIRE

    Ting Jin; Yu-Xue Wu

    2015-01-01

    This study investigated antioxidant properties of the little hairtail (Trichiurus haumela) protein hydrolysates obtained by commercial protease of Alcalase through using various antioxidant assays, including reducing power and free radical scavenging activities. The molecular mass distribution of hydrolysates was also examined to evaluate their relationship with antioxidant activity. The results showed that little hairtail protein hydrolysates had good ability to donate electron or hydrogen a...

  16. Utility of a fluorescent vitamin E analog as a probe for tocopherol transfer protein activity

    OpenAIRE

    Morley, Samantha; Cross, Valerie; Cecchini, Matt; Nava, Phil; Atkinson, Jeffrey; Manor, Danny

    2006-01-01

    The tocopherol transfer protein (TTP1) is a member of the CRAL-TRIO family of lipid binding proteins that facilitates vitamin E transfer between membrane vesicles in vitro. In cultured hepatocytes, TTP enhances the secretion of tocopherol to the media; presumably, tocopherol transfer is at the basis of this biological activity. The mechanism underlying ligand transfer by TTP is presently unknown, and available tools for monitoring this activity suffer from complicated assay procedure and poor...

  17. Diacylglycerol kinase theta and zeta isoforms: regulation of activity, protein binding partners and physiological functions

    OpenAIRE

    Los, Alrik Pieter

    2007-01-01

    Diacylglycerol kinases (DGKs) phosphorylate the second messenger diacylglycerol (DAG) yielding phosphatidic acid (PA). In this thesis, we investigated which structural domains of DGKtheta are required for DGK activity. Furthermore, we showed that DGKzeta binds to and is activated by the Retinoblastoma tumour suppressor protein (pRB) and the pRB-related proteins p107 and p130, key regulators of the cell-cycle, differentiation and apoptosis. The interaction between pRB and DGKzeta is regulated ...

  18. Phospholipase D1 Mediates AMP-Activated Protein Kinase Signaling for Glucose Uptake

    OpenAIRE

    Jong Hyun Kim; Ji-Man Park; Kyungmoo Yea; Hyun Wook Kim; Pann-Ghill Suh; Sung Ho Ryu

    2010-01-01

    BACKGROUND: Glucose homeostasis is maintained by a balance between hepatic glucose production and peripheral glucose utilization. In skeletal muscle cells, glucose utilization is primarily regulated by glucose uptake. Deprivation of cellular energy induces the activation of regulatory proteins and thus glucose uptake. AMP-activated protein kinase (AMPK) is known to play a significant role in the regulation of energy balances. However, the mechanisms related to the AMPK-mediated control of glu...

  19. 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

    2016-01-01

    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 (Pmechanism of fibrosis. PMID:27385084

  20. Erythrocyte-derived microparticles supporting activated protein C-mediated regulation of blood coagulation.

    Directory of Open Access Journals (Sweden)

    Ruzica Livaja Koshiar

    Full Text Available Elevated levels of erythrocyte-derived microparticles are present in the circulation in medical conditions affecting the red blood cells. Erythrocyte-derived microparticles expose phosphatidylserine thus providing a suitable surface for procoagulant reactions leading to thrombin formation via the tenase and prothrombinase complexes. Patients with elevated levels of circulating erythrocyte-derived microparticles have increased thrombin generation in vivo. The aim of the present study was to investigate whether erythrocyte-derived microparticles are able to support the anticoagulant reactions of the protein C system. Erythrocyte-derived microparticles were isolated using ultracentrifugation after incubation of freshly prepared erythrocytes with the ionophore A23187 or from outdated erythrocyte concentrates, the different microparticles preparations yielding similar results. According to flow cytometry analysis, the microparticles exposed phoshatidylserine and bound lactadherin, annexin V, and protein S, which is a cofactor to activated protein C. The microparticles were able to assemble the tenase and prothrombinase complexes and to stimulate the formation of thrombin in plasma-based thrombin generation assay both in presence and absence of added tissue factor. The addition of activated protein C in the thrombin generation assay inhibited thrombin generation in a dose-dependent fashion. The anticoagulant effect of activated protein C in the thrombin generation assay was inhibited by a monoclonal antibody that prevents binding of protein S to microparticles and also attenuated by anti-TFPI antibodies. In the presence of erythrocyte-derived microparticles, activated protein C inhibited tenase and prothrombinase by degrading the cofactors FVIIIa and FVa, respectively. Protein S stimulated the Arg306-cleavage in FVa, whereas efficient inhibition of FVIIIa depended on the synergistic cofactor activity of protein S and FV. In summary, the erythrocyte

  1. Protein Function Prediction Based on Active Semi-sup ervised Learning

    Institute of Scientific and Technical Information of China (English)

    WANG Xuesong,CHENG Yuhu; LI Lijing

    2016-01-01

    In our study, the active learning and semi-supervised learning methods are comprehensively used for label delivery of proteins with known functions in Protein-protein interaction (PPI) network so as to predict the func-tions of unknown proteins. Because the real PPI network is generally observed with overlapping protein nodes with multiple functions, the mislabeling of overlapping protein may result in accumulation of prediction errors. For this reason, prior to executing the label delivery process of semi-supervised learning, the adjacency matrix is used to detect overlapping proteins. As the topological structure description of interactive relation between proteins, PPI network is observed with party hub protein nodes that play an important role, in co-expression with its neighborhood. Therefore, to reduce the manual labeling cost, party hub proteins most beneficial for improvement of prediction ac-curacy are selected for class labeling and the labeled party hub proteins are added into the labeled sample set for semi-supervised learning later. As the experimental results of real yeast PPI network show, the proposed algorithm can achieve high prediction accuracy with few labeled samples.

  2. Laser-activated protein bands for peripheral nerve repair

    Science.gov (United States)

    Lauto, Antonio; Trickett, Rodney I.; Malik, Richard; Dawes, Judith M.; Owen, Earl R.

    1996-01-01

    A 100 micrometer core optical fiber-coupled 75 mW diode laser operating at a wavelength of 800 nm has been used in conjunction with a protein solder to stripe weld severed rat tibial nerves, reducing the long operating time required for microsurgical nerve repair. Welding is produced by selective laser denaturation of the protein based solder which contains the dye indocyanine green. Operating time for laser soldering was 10 plus or minus 5 min. (n equals 24) compared to 23 plus or minus 9 min (n equals 13) for microsuturing. The laser solder technique resulted in patent welds with a tensile strength of 15 plus or minus 5 g, while microsutured nerves had a tensile strength of 40 plus or minus 10 g. Histopathology of the laser soldered nerves, conducted immediately after surgery, displayed solder adhesion to the outer membrane with minimal damage to the inner axons of the nerves. An in vivo study, with a total of fifty-seven adult male wistar rats, compared laser solder repaired tibial nerves to conventional microsuture repair. Twenty-four laser soldered nerves and thirteen sutured nerves were characterized at three months and showed successful regeneration with average compound muscle action potentials (CMAP) of 2.4 plus or minus 0.7 mV and 2.7 plus or minus 0.8 mV respectively. Histopathology of the in vivo study, confirmed the comparable regeneration of axons in laser and suture operated nerves. A faster, less damaging and long lasting laser based anastomotic technique is presented.

  3. AMP-activated protein kinase downregulates Kv7.1 cell surface expression

    DEFF Research Database (Denmark)

    Andersen, Martin N; Krzystanek, Katarzyna; Jespersen, Thomas;

    2012-01-01

    in response to polarization of the epithelial Madin-Darby canine kidney (MDCK) cell line and that this was mediated by activation of protein kinase C (PKC). In this study, the pathway downstream of PKC, which leads to internalization of Kv7.1 upon cell polarization, is elucidated. We show by confocal...... microscopy that Kv7.1 is endocytosed upon initiation of the polarization process and sent for degradation by the lysosomal pathway. The internalization could be mimicked by pharmacological activation of the AMP-activated protein kinase (AMPK) using three different AMPK activators. We demonstrate...

  4. Identification of active pocket and protein druggability within envelope glycoprotein GP2 from Ebola virus

    Institute of Scientific and Technical Information of China (English)

    Beuy; Joob; Viroj; Wiwanitkit

    2014-01-01

    The drug searching for combating the present outbreak of Ebola virus infection is the urgent activity at present.Finding the new effective drug at present must base on the molecular analysis of the pathogenic virus.The in-depth analysis of the viral protein to find the binding site,active pocket is needed.Here,the authors analyzed the envelope glycoprotein GP2 from Ebola virus.Identification of active pocket and protein draggability within envelope glycoprotein GP2 from Ebola virus was done.According to this assessment,7 active pockets with varied draggability could be identified.

  5. Identification of active pocket and protein druggability within envelope glycoprotein GP2 from Ebola virus

    Institute of Scientific and Technical Information of China (English)

    Beuy Joob; Viroj Wiwanitkit

    2014-01-01

    The drug searching for combating the present outbreak of Ebola virus infection is the urgent activity at present. Finding the new effective drug at present must base on the molecular analysis of the pathogenic virus. The in-depth analysis of the viral protein to find the binding site, active pocket is needed. Here, the authors analyzed the envelope glycoprotein GP2 from Ebola virus. Identification of active pocket and protein druggability within envelope glycoprotein GP2 from Ebola virus was done. According to this assessment, 7 active pockets with varied druggability could be identified.

  6. 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

  7. Activation of protein kinase C inhibits synthesis and release of decidual prolactin

    Energy Technology Data Exchange (ETDEWEB)

    Harman, I.; Costello, A.; Ganong, B.; Bell, R.M.; Handwerger, S.

    1986-08-01

    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 (diC8), 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 M. Diolein (100 M), 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 4US -phorbol 12,13-dibutyrate, which activate protein kinase C in other systems, also inhibited the release of prolactin, which the protein kinase C inactivate 4 -phorbol-12,13-didecanoate was without effect. The inhibition of prolactin release was secondary to a decrease in prolactin synthesis. Although diC8 and PMA inhibited the synthesis and release of prolactin, these agents had no effect on the synthesis or release of trichloroacetic acid-precipitable (TVS)methionine-labeled decidual proteins and did not cause the release of the cytosolic enzymes lactic dehydrogenase and alkaline phosphatase. DiC8 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 diC8 and phorbol esters strongly implicates protein kinase C in the regulation of the production and release of prolactin from the decidua.

  8. Motifs with potential physiological activity in food proteins – BIOPEP database

    Directory of Open Access Journals (Sweden)

    Bartłomiej Dziuba

    2009-09-01

    Full Text Available Proteins are the multifunctional food components affecting the living organisms. One of the proteins function is the impact on the body due to the presence of motifs that show specific physiological and biological activities. Due to the worldwide growth of demand for the food containing bioactive components, increasing attention has been paid recently to the use of bioactive peptides as physiologically active food ingredients. They are important elements of the prevention and treatment of various lifestyle diseases. In addition to its primary function and according to current knowledge, each protein may be a reserve source of peptides controlling the life processes of organisms. For this reason, in this work, application of a new, additional criterion for evaluating proteins as a potential source of biologically active peptides, contributes to a more comprehensive and objective definition of their biological value. A complementary part of such research is the strategy for evaluation of the food proteins as precursors of biologically active peptides which involves the database of proteins and bioactive peptides – BIOPEP (available online at: http://www.uwm.edu.pl/biochemia. The database contains information on 2123 peptides representing 48 types of bioactivities, their EC50 values and source of origin. Proteins (706 sequences are considered as bioactive peptide precursors based on newly introduced criteria: the profile of potential biological activity, the frequency of bioactive fragments occurrence and potential biological protein activity. This original and unprecedented so far approach, started to be successfully and more widely applied by other authors. BIOPEP can be interfaced with global databases such as e.g. TrEMBL, SWISS-PROT, EROP and PepBank. Recently the BIOPEP database was enlarged with the data about allergenic proteins, including information about structure of their epitopes and molecular markers.  

  9. Identification of a New Pyk2 Target Protein with Arf-GAP Activity

    OpenAIRE

    Andreev, J; Simon, J.-P.; Sabatini, D D; J. Kam; Plowman, G; Randazzo, P. A.; Schlessinger, J

    1999-01-01

    Protein tyrosine kinase Pyk2 is activated by a variety of G-protein-coupled receptors and by extracellular signals that elevate intracellular Ca2+ concentration. We have identified a new Pyk2 binding protein designated Pap. Pap is a multidomain protein composed of an N-terminal α-helical region with a coiled-coil motif, followed by a pleckstrin homology domain, an Arf-GAP domain, an ankyrin homology region, a proline-rich region, and a C-terminal SH3 domain. We demonstrate that Pap forms a st...

  10. Proteins, peptides, polysaccharides, and nucleotides with inhibitory activity on human immunodeficiency virus and its enzymes.

    Science.gov (United States)

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

    2015-12-01

    Human immunodeficiency virus (HIV), the causative agent of acquired immune deficiency syndrome, has claimed innumerable lives in the past. Many biomolecules which suppress HIV replication and also other biomolecules that inhibit enzymes essential to HIV replication have been reported. Proteins including a variety of milk proteins, ribosome-inactivating proteins, ribonucleases, antifungal proteins, and trypsin inhibitors; peptides comprising cathelicidins, defensins, synthetic peptides, and others; polysaccharides and polysaccharopeptides; nucleosides, nucleotides, and ribozymes, demonstrated anti-HIV activity. In many cases, the mechanism of anti-HIV action has been elucidated. Strategies have been devised to augment the anti-HIV potency of these compounds.

  11. A Bacillus thuringiensis insecticidal crystal protein with a high activity against members of the family Noctuidae.

    OpenAIRE

    Lambert, B.; Buysse, L; Decock, C.; Jansens, S.; Piens, C; Saey, B; Seurinck, J; Van Audenhove, K; Van Rie, J.; A. van Vliet; Peferoen, M.

    1996-01-01

    The full characterization of a novel insecticidal crystal protein, named Cry9Ca1 according to the revised nomenclature for Cry proteins, from Bacillus thuringiensis serovar tolworthi is reported. The crystal protein has 1,157 amino acids and a molecular mass of 129.8 kDa. It has the typical features of the Lepidoptera-active crystal proteins such as five conserved sequence blocks. Also, it is truncated upon trypsin digestion to a toxic fragment of 68.7 kDa by removal of 43 amino acids at the ...

  12. Antifreeze Proteins Enhance Survival of Cells in Cryopreservation - Substituting DMSO with RmAFP#1 in cryopreservation of cells

    OpenAIRE

    Henriksen, Beatriche L. E.; Kofod, Lotte; Gammeltoft, Karen A.; Christensen, Erik; Khan, Omar J.

    2015-01-01

    Cryopreservation is a useful method for preserving living cells and biological tissues. Dimethyl sulfoxide (DMSO) is considered the most effective cryoprotective agent (CPA) used in cryopreservation. DMSO helps to reduce ice crystallization within the cell and thus preventing cell death during the freezing and thawing process. However, DMSO has toxic effects on cells which are not only concentration dependent, but also temperature dependent. In this study, DMSO was substituted with an ins...

  13. Anticancer Activity of Chamaejasmine: Effect on Tubulin Protein

    Directory of Open Access Journals (Sweden)

    Yingkun Nie

    2011-07-01

    Full Text Available In this work, the anticancer activity of chamaejasmine was studied by evaluating its in vitro cytotoxicity against several human cancer cell lines (MCF-7, A549, SGC-7901, HCT-8, HO-4980, Hela, HepG2, PC-3, LNCap, Vero and MDCK using the MTT assay. Results indicated chamaejasmine showed more notable anticancer activity than taxol against PC-3 cells, with IC50 values of 2.28 and 3.98 µM, respectively. Furthermore, Western blot analysis showed that chamaejasmine was able to increase the expression of β-tubulin, but not α-tubulin. In silico simulations indicated that chamaejasmine specifically interacts with the active site which is located at the top of β-tubulin, thanks to the presence of strong hydrophobic effects between the core templates and the hydrophobic surface of the TB active site. The binding energy (Einter was calculated to be −164.77 kcal·mol−1. Results presented here suggest that chamaejasmine possesses anti-cancer properties relating to β-tubulin depolymerization inhibition, and therefore is a potential source of anticancer leads for the pharmaceutical industry.

  14. 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), c

  15. Determination of CKMB Activity and Protein Concentration and Their Application in the Diagnosis of AMI

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The activity and protein concentration of CKMB in 19 patients with AMI, 17 non-AMI patients and 26 normal persons. It was found that both peak times in patients with AMI and nonAMI patients were similar but the peak values were different. At peak values, the F value of CKMB (5. 3) was much lower than that of CKMB protein concentration (50. 1). We are led to conclude that the measurement of CKMB protein level can identify AMI much earlier than that of CKMB activity.

  16. 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

    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 invol...... 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....

  17. 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

    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......) 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......(2)) may fully substitute for the cAMP-elevating agent isobutylmethylxanthine (IBMX). Moreover, selective activation of Epac-dependent signaling promoted adipocyte differentiation when the Rho-associated kinase (ROCK) was inhibited. Unlike the case for murine preadipocytes cell lines, long...

  18. Menin-MLL inhibitors reverse oncogenic activity of MLL fusion proteins in leukemia.

    Science.gov (United States)

    Grembecka, Jolanta; He, Shihan; Shi, Aibin; Purohit, Trupta; Muntean, Andrew G; Sorenson, Roderick J; Showalter, Hollis D; Murai, Marcelo J; Belcher, Amalia M; Hartley, Thomas; Hess, Jay L; Cierpicki, Tomasz

    2012-03-01

    Translocations involving the mixed lineage leukemia (MLL) gene result in human acute leukemias with very poor prognosis. The leukemogenic activity of MLL fusion proteins is critically dependent on their direct interaction with menin, a product of the multiple endocrine neoplasia (MEN1) gene. Here we present what are to our knowledge the first small-molecule inhibitors of the menin-MLL fusion protein interaction that specifically bind menin with nanomolar affinities. These compounds effectively reverse MLL fusion protein-mediated leukemic transformation by downregulating the expression of target genes required for MLL fusion protein oncogenic activity. They also selectively block proliferation and induce both apoptosis and differentiation of leukemia cells harboring MLL translocations. Identification of these compounds provides a new tool for better understanding MLL-mediated leukemogenesis and represents a new approach for studying the role of menin as an oncogenic cofactor of MLL fusion proteins. Our findings also highlight a new therapeutic strategy for aggressive leukemias with MLL rearrangements.

  19. Corticosterone activates Erk1/2 mitogen-activated protein kinase in primary hippocampal cells through rapid nongenomic mechanism

    Institute of Scientific and Technical Information of China (English)

    QI Aiqun; QIU Jian; XIAO Lin; CHEN Yizhang

    2005-01-01

    Nongenomic effects of glucocorticoids (GC) in various cell types have been well documented, but it still remains unknown whether the mechanism also works in hippocampus which is a crucial target of glucocorticoids in neural system during physiological and/or pathophysiological processes. We present here that corticosterone (B) could rapidly activate Erk1/2 mitogen-activated protein kinase (MAPK) in primarily cultured hippocampal cells within minutes, with a bell-shaped time dependent curve which peaked at 15min and then went down to normal level in 30 min. This activation was blocked by protein kinase C (PKC) inhibitor (Go6976), G protein inhibitor (GDPβs), and MEK(MAPK/extracellular signal-regulated kinase kinase) inhibitor(PD98059), but not by protein kinase A (PKA) inbibitor (H89), tyrosine kinase inhibitor (genistein), and glucocorticoid receptor ( GR ) antagonist (RU38486). Thus, the rapid activation of Erk1/2 MAPK in primary hippocampal cells induced by B was likely mediated by a G protein coupled receptor (GPCR) pathway with involvement of PKC, which belonged to the nongenomic rather than genomic mechanism of GC' s effects.

  20. A Conserved Hydrophobic Core in Gαi1 Regulates G Protein Activation and Release from Activated Receptor.

    Science.gov (United States)

    Kaya, Ali I; Lokits, Alyssa D; Gilbert, James A; Iverson, T M; Meiler, Jens; Hamm, Heidi E

    2016-09-01

    G protein-coupled receptor-mediated heterotrimeric G protein activation is a major mode of signal transduction in the cell. Previously, we and other groups reported that the α5 helix of Gαi1, especially the hydrophobic interactions in this region, plays a key role during nucleotide release and G protein activation. To further investigate the effect of this hydrophobic core, we disrupted it in Gαi1 by inserting 4 alanine amino acids into the α5 helix between residues Gln(333) and Phe(334) (Ins4A). This extends the length of the α5 helix without disturbing the β6-α5 loop interactions. This mutant has high basal nucleotide exchange activity yet no receptor-mediated activation of nucleotide exchange. By using structural approaches, we show that this mutant loses critical hydrophobic interactions, leading to significant rearrangements of side chain residues His(57), Phe(189), Phe(191), and Phe(336); it also disturbs the rotation of the α5 helix and the π-π interaction between His(57) and Phe(189) In addition, the insertion mutant abolishes G protein release from the activated receptor after nucleotide binding. Our biochemical and computational data indicate that the interactions between α5, α1, and β2-β3 are not only vital for GDP release during G protein activation, but they are also necessary for proper GTP binding (or GDP rebinding). Thus, our studies suggest that this hydrophobic interface is critical for accurate rearrangement of the α5 helix for G protein release from the receptor after GTP binding.

  1. Regulation of protein kinase B/Akt activity and Ser473 phosphorylation by protein kinase Calpha in endothelial cells.

    Science.gov (United States)

    Partovian, Chohreh; Simons, Michael

    2004-08-01

    Protein kinase Balpha (PKBalpha/Akt-1) is a key mediator of multiple signaling pathways involved in angiogenesis, cell proliferation and apoptosis among others. The unphosphorylated form of Akt-1 is virtually inactive and its full activation requires two phosphatidylinositol-3,4,5-triphosphate-dependent phosphorylation events, Thr308 by 3-phosphoinositide-dependent kinase-1 (PDK1) and Ser473 by an undefined kinase that has been termed PDK2. Recent studies have suggested that the Ser473 kinase is a plasma membrane raft-associated kinase. In this study we show that protein kinase Calpha (PKCalpha) translocates to the membrane rafts in response to insulin growth factor-1 (IGF-1) stimulation. Overexpression of PKCalpha increases Ser473 phosphorylation and Akt-1 activity, while inhibition of its activity or expression decreases IGF-1-dependent activation of Akt-1. Furthermore, in vitro, in the presence of phospholipids and calcium, PKCalpha directly phosphorylates Akt-1 at the Ser473 site. We conclude, therefore, that PKCalpha regulates Akt-1 activity via Ser473 phosphorylation and may function as PDK2 in endothelial cells. PMID:15157674

  2. 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.

  3. Laser-activated protein solder for peripheral nerve repair

    Science.gov (United States)

    Trickett, Rodney I.; Lauto, Antonio; Dawes, Judith M.; Owen, Earl R.

    1995-05-01

    A 100 micrometers core optical fiber-coupled 75 mW diode laser operating at a wavelength of 800 nm has been used in conjunction with a protein solder to stripe weld severed rat tibial nerves, reducing the long operating time required for microsurgical nerve repair. Welding is produced by selective laser denaturation of the albumin based solder which contains the dye indocyanine green. Operating time for laser soldering was 10 +/- 5 min. (n equals 20) compared to 23 +/- 9 min. (n equals 10) for microsuturing. The laser solder technique resulted in patent welds with a tensile strength of 15 +/- 5 g, while microsutured nerves had a tensile strength of 40 +/- 10 g. Histopathology of the laser soldered nerves, conducted immediately after surgery, displayed solder adhesion to the outer membrane with minimal damage to the inner axons of the nerves. An in vivo study is under way comparing laser solder repaired tibial nerves to conventional microsuture repair. At the time of submission 15 laser soldered nerves and 7 sutured nerves were characterized at 3 months and showed successful regeneration with compound muscle action potentials of 27 +/- 8 mV and 29 +/- 8 mW respectively. A faster, less damaging and long lasting laser based anastomotic technique is presented.

  4. Cell type-specific neuroprotective activity of untranslocated prion protein.

    Directory of Open Access Journals (Sweden)

    Elena Restelli

    Full Text Available BACKGROUND: 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. PRINCIPAL FINDINGS: 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. SIGNIFICANCE: 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.

  5. 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

  6. Leptin stimulates protein synthesis-activating translation machinery in human trophoblastic cells.

    Science.gov (United States)

    Pérez-Pérez, Antonio; Maymó, Julieta; Gambino, Yésica; Dueñas, José L; Goberna, Raimundo; Varone, Cecilia; Sánchez-Margalet, Víctor

    2009-11-01

    Leptin was originally considered as an adipocyte-derived signaling molecule for the central control of metabolism. However, pleiotropic effects of leptin have been identified in reproduction and pregnancy, particularly in placenta, where it may work as an autocrine hormone, mediating angiogenesis, growth, and immunomodulation. Leptin receptor (LEPR, also known as Ob-R) shows sequence homology to members of the class I cytokine receptor (gp130) superfamily. In fact, leptin may function as a proinflammatory cytokine. We have previously found that leptin is a trophic and mitogenic factor for trophoblastic cells. In order to further investigate the mechanism by which leptin stimulates cell growth in JEG-3 cells and trophoblastic cells, we studied the phosphorylation state of different proteins of the initiation stage of translation and the total protein synthesis by [(3)H]leucine incorporation in JEG-3 cells. We have found that leptin dose-dependently stimulates the phosphorylation and activation of the translation initiation factor EIF4E as well as the phosphorylation of the EIF4E binding protein EIF4EBP1 (PHAS-I), which releases EIF4E to form active complexes. Moreover, leptin dose-dependently stimulates protein synthesis, and this effect can be partially prevented by blocking mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 kinase (PIK3) pathways. In conclusion, leptin stimulates protein synthesis, at least in part activating the translation machinery, via the activation of MAPK and PIK3 pathways.

  7. 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

  8. Controlled localization of functionally active proteins to inclusion bodies using leucine zippers.

    Directory of Open Access Journals (Sweden)

    Su-Lim Choi

    Full Text Available Inclusion bodies (IBs are typically non-functional particles of aggregated proteins. However, some proteins in fusion with amyloid-like peptides, viral coat proteins, and cellulose binding domains (CBDs generate IB particles retaining the original functions in cells. Here, we attempted to generate CBD IBs displaying functional leucine zipper proteins (LZs as bait for localizing cytosolic proteins in E. coli. When a red fluorescent protein was tested as a target protein, microscopic observations showed that the IBs red-fluoresced strongly. When different LZ pairs with KDs of 8-1,000 µM were tested as the bait and prey, the localization of the red fluorescence appeared to change following the affinities between the LZs, as observed by fluorescence imaging and flow cytometry. This result proposed that LZ-tagged CBD IBs can be applied as an in vivo matrix to entrap cytosolic proteins in E. coli while maintaining their original activities. In addition, easy detection of localization to IBs provides a unique platform for the engineering and analyses of protein-protein interactions in E. coli.

  9. The SARS coronavirus spike glycoprotein is selectively recognized by lung surfactant protein D and activates macrophages

    DEFF Research Database (Denmark)

    Leth-Larsen, Rikke; Zhong, Fei; Chow, Vincent T K;

    2007-01-01

    The severe acute respiratory syndrome coronavirus (SARS-CoV) infects host cells with its surface glycosylated spike-protein (S-protein). Here we expressed the SARS-CoV S-protein to investigate its interactions with innate immune mechanisms in the lung. The purified S-protein was detected as a 210 k......Da glycosylated protein. It was not secreted in the presence of tunicamycin and was detected as a 130 kDa protein in the cell lysate. The purified S-protein bound to Vero but not 293T cells and was itself recognized by lung surfactant protein D (SP-D), a collectin found in the lung alveoli. The binding required...... Ca(2+) and was inhibited by maltose. The serum collectin, mannan-binding lectin (MBL), exhibited no detectable binding to the purified S-protein. S-protein binds and activates macrophages but not dendritic cells (DCs). It suggests that SARS-CoV interacts with innate immune mechanisms in the lung...

  10. 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

  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. Mitogen-activated protein kinase-dependent apoptosis in norcan-tharidin-treated A375-S2 cells is proceeded by the activation of protein kinase C

    Institute of Scientific and Technical Information of China (English)

    AN Wei-wei; WANG Min-wei; Tashiro Shin-ichi; Onodera Satoshi; Ikejima Takashi

    2005-01-01

    Background We have reported that norcantharidin (NCTD) induces human melanoma A375-S2 cell apoptosis and that the activation of caspase and the mitochondrial pathway are involved in the apoptotic process. This study aimed at investigating the roles of mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) in A375-S2 cell apoptosis induced by NCTD. Methods We assessed the effects of NCTD on cell growth inhibition using the 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheyltetrazolium bromide (MTT) assay, DNA fragmentation (DNA agarose gel electrophoresis), and MAPK protein levels (Western blot analysis) in A375-S2 cells. Photomicroscopic data were also collected.Results The NCTD inhibitory effect on A375-S2 cells was partially reversed by MAPK and PKC inhibitors. The expression of phosphorylated JNK and p38 also increased after the treatment with NCTD, and inhibitors of c-Jun NH2-terminal kinase (JNK) and p38 (SP600125 and SB203580, respectively) had significant inhibitory effects on the upregulation of phosphorylated JNK and p38 expression. Simultaneously, the PKC inhibitor staurosporine blocked the upregulation of phosphorylated JNK and phosphorylated p38, but had little effect on extracellular signal-regulated kinase (ERK) expression. Conclusion These results suggest that the activation of JNK and p38 MAPK promotes the process of NCTD-induced A375-S2 cell apoptosis and that PKC plays an important regulation role in the activation of MAPKs.

  13. AMP-activated protein kinase phosphorylation in brain is dependent on method of sacrifice and tissue preparation

    OpenAIRE

    Scharf, Matthew T.; Mackiewicz, Miroslaw; Naidoo, Nirinjini; O'Callaghan, James P.; Pack, Allan I.

    2007-01-01

    AMP-activated protein kinase is activated when the catalytic α subunit is phosphorylated on Thr172 and therefore, phosphorylation of the α subunit is used as a measure of activation. However, measurement of α-AMP-activated protein kinase phosphorylation in vivo can be technically challenging. To determine the most accurate method for measuring α-AMP-activated protein kinase phosphorylation in the mouse brain, we compared different methods of sacrifice and tissue preparation. We found that fre...

  14. The effects of adiponectin and metformin on prostate and colon neoplasia involve activation of AMP-activated protein kinase.

    Science.gov (United States)

    Zakikhani, Mahvash; Dowling, Ryan J O; Sonenberg, Nahum; Pollak, Michael N

    2008-10-01

    Population studies provide evidence that obesity and insulin resistance are associated not only with elevated serum insulin levels and reduced serum adiponectin levels but also with increased risk of aggressive prostate and colon cancer. We show here that adiponectin activates AMP-activated protein kinase (AMPK) in colon (HT-29) and prostate (PC-3) cancer cells. These results are consistent with prior observations in myocytes, but we show that in epithelial cancer cells AMPK activation is associated with reduction in mammalian target of rapamycin activation as estimated by Ser(2448) phosphorylation, with reduction in p70S6 kinase activation as estimated by Thr(389) phosphorylation, with ribosomal protein S6 activation as estimated by Ser(235/236) phosphorylation, with reduction in protein translation as estimated by [(35)S]methionine incorporation, and with growth inhibition. Adiponectin-induced growth inhibition is significantly attenuated when AMPK level is reduced using small interfering RNA, indicating that AMPK is involved in mediating the antiproliferative action of this adipokine. Thus, adiponectin has the characteristics of a AMPK-dependent growth inhibitor that is deficient in obesity, and this may contribute to the adverse effects of obesity on neoplastic disease. Furthermore, metformin was observed to activate AMPK and to have growth inhibitory actions on prostate and colon cancer cells, suggesting that this compound may be of particular value in attenuating the adverse effects of obesity on neoplasia. PMID:19138981

  15. A quantitative analysis of contractility in active cytoskeletal protein networks.

    Science.gov (United States)

    Bendix, Poul M; Koenderink, Gijsje H; Cuvelier, Damien; Dogic, Zvonimir; Koeleman, Bernard N; Brieher, William M; Field, Christine M; Mahadevan, L; Weitz, David A

    2008-04-15

    Cells actively produce contractile forces for a variety of processes including cytokinesis and motility. Contractility is known to rely on myosin II motors which convert chemical energy from ATP hydrolysis into forces on actin filaments. However, the basic physical principles of cell contractility remain poorly understood. We reconstitute contractility in a simplified model system of purified F-actin, muscle myosin II motors, and alpha-actinin cross-linkers. We show that contractility occurs above a threshold motor concentration and within a window of cross-linker concentrations. We also quantify the pore size of the bundled networks and find contractility to occur at a critical distance between the bundles. We propose a simple mechanism of contraction based on myosin filaments pulling neighboring bundles together into an aggregated structure. Observations of this reconstituted system in both bulk and low-dimensional geometries show that the contracting gels pull on and deform their surface with a contractile force of approximately 1 microN, or approximately 100 pN per F-actin bundle. Cytoplasmic extracts contracting in identical environments show a similar behavior and dependence on myosin as the reconstituted system. Our results suggest that cellular contractility can be sensitively regulated by tuning the (local) activity of molecular motors and the cross-linker density and binding affinity. PMID:18192374

  16. Hormonal activation of a kinase cascade localized at the mitochondria is required for StAR protein activity.

    Science.gov (United States)

    Poderoso, Cecilia; Maloberti, Paula; Duarte, Alejandra; Neuman, Isabel; Paz, Cristina; Cornejo Maciel, Fabiana; Podesta, Ernesto J

    2009-03-01

    It is known that ERK1/2 and MEK1/2 participate in the regulation of Star gene transcription. However, their role in StAR protein post-transcriptional regulation is not described yet. In this study we analyzed the relationship between the MAPK cascade and StAR protein phosphorylation and function. We have demonstrated that (a) steroidogenesis in MA-10 Leydig cells depends on the specific of ERK1/2 activation at the mitochondria; (b) ERK1/2 phosphorylation is driven by mitochondrial PKA and constitutive MEK1/2 in this organelle; (c) active ERK1/2 interacts with StAR protein, leads to StAR protein phosphorylation at Ser(232) only in the presence of cholesterol; (d) directed mutagenesis of Ser(232) (S232A) inhibited in vitro StAR protein phosphorylation by ERK1; (e) transient transfection of MA-10 cells with StAR S232A cDNA markedly reduced the yield of progesterone production. We show that StAR protein is a substrate of ERK1/2, and that mitochondrial ERK1/2 is part of a multimeric complex that regulates cholesterol transport.

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. Protein turnover, amino acid requirements and recommendations for athletes and active populations

    International Nuclear Information System (INIS)

    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

  2. Arabidopsis resistance protein SNC1 activates immune responses through association with a transcriptional corepressor

    Science.gov (United States)

    Zhu, Zhaohai; Xu, Fang; Zhang, Yaxi; Cheng, Yu Ti; Wiermer, Marcel; Li, Xin; Zhang, Yuelin

    2010-01-01

    In both plants and animals, nucleotide-binding (NB) domain and leucine-rich repeat (LRR)-containing proteins (NLR) function as sensors of pathogen-derived molecules and trigger immune responses. Although NLR resistance (R) proteins were first reported as plant immune receptors more than 15 years ago, how these proteins activate downstream defense responses is still unclear. Here we report that the Toll-like/interleukin-1 receptor (TIR)-NB-LRR R protein, suppressor of npr1-1, constitutive 1 (SNC1) functions through its associated protein, Topless-related 1 (TPR1). Knocking out TPR1 and its close homologs compromises immunity mediated by SNC1 and several other TIR-NB-LRR–type R proteins, whereas overexpression of TPR1 constitutively activates SNC1-mediated immune responses. TPR1 functions as a transcriptional corepressor and associates with histone deacetylase 19 in vivo. Among the target genes of TPR1 are Defense no Death 1 (DND1) and Defense no Death 2 (DND2), two known negative regulators of immunity that are repressed during pathogen infection, suggesting that TPR1 activates R protein-mediated immune responses through repression of negative regulators. PMID:20647385

  3. Selective radiolabeling of cell surface proteins to a high specific activity

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, J.A.; Lau, A.L.; Cunningham, D.D.

    1987-02-10

    A procedure was developed for selective radiolabeling of membrane proteins on cells to higher specific activities than possible with available techniques. Cell surface amino groups were derivatized with /sup 125/I-(hydroxyphenyl)propionyl groups via /sup 125/I-sulfosuccinimidyl (hydroxyphenyl)propionate (/sup 125/II-sulfo-SHPP). This reagent preferentially labeled membrane proteins exposed at the cell surface of erythrocytes as assessed by the degree of radiolabel incorporation into erythrocyte ghost proteins and hemoglobin. Comparison with the lactoperoxidase-(/sup 125/I)iodide labeling technique revealed that /sup 125/I-sulfo-SHPP labeled cell surface proteins to a much higher specific activity and hemoglobin to a much lower specific activity. Additionally, this reagent was used for selective radiolabeling of membrane proteins on the cytoplasmic face of the plasma membrane by blocking exofacial amino groups with uniodinated sulfo-SHPP, lysing the cells, and then incubating them with /sup 125/I-sulfo-SHPP. Exclusive labeling of either side of the plasma membrane was demonstrated by the labeling of some marker proteins with well-defined spacial orientations on erythroctyes. Transmembrane proteins such as the epidermal growth factor receptor on cultured cells could also be labeled differentially from either side of the plasma membrane.

  4. 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. PMID:26848951

  5. Protein Dynamics in Organic Media at Varying Water Activity Studied by Molecular Dynamics Simulation

    DEFF Research Database (Denmark)

    Wedberg, Nils Hejle Rasmus Ingemar; Abildskov, Jens; Peters, Günther H.J.

    2012-01-01

    In nonaqueous enzymology, control of enzyme hydration is commonly approached by fixing the thermodynamic water activity of the medium. In this work, we present a strategy for evaluating the water activity in molecular dynamics simulations of proteins in water/organic solvent mixtures. The method...

  6. DEVELOPMENTAL REGULATION OF PROTEIN KINASE B ACTIVATION IS ISOFORM SPECIFIC IN SKELETAL MUSCLE OF NEONATAL PIGS

    Science.gov (United States)

    The postprandial activation of the insulin signaling pathway that leads to translation initiation is enhanced in skeletal muscle of the neonate and decreases with development in parallel with the developmental decline in muscle protein synthesis. Our previous study showed that the activity of protei...

  7. Inflammation-associated activation of coagulation and immune regulation by the protein C pathway.

    Science.gov (United States)

    Weiler, Hartmut

    2014-05-01

    The inflammation-induced activation of the protein C pathway provides negative feedback inhibition of coagulation and exerts coagulation-independent anti-inflammatory and cytoprotective effects. The balance between these activities of aPC modulates the outcome of diverse inflammatory diseases such as encephalitis, diabetes, and sepsis; and is affected by naturally occurring aPC-resistance of coagulation factor V Leiden.

  8. Conserved hypothetical BB0462 protein enhances the transcription activity of oppAV promoter

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Borrelia burgdorferi BB0462 ORF encodes an unknown functional protein with 110 amino acids.A BLAST search in protein databases and the secondary structure being predicted by the program JUFO showed that the conserved hypothetical BB0462 protein was similar to the members of the YbaB protein family in both amino acid composition and protein structure.The co-transformation of BB0462 ORF and oppA upstream regulation DNA into E.coli host cells and β-galactosidase activity assay demonstrated that the BB0462 protein enhanced the transcriptional activity of the oppAV promoter,but does not affect those of oppAⅠ,Ⅱ,Ⅲ and Ⅳ promoters.Analysis of DNA retardation and competitive repression also confirmed that the BB0462 protein bound to the 409 bp upstream regulation DNA fragment close to the initiation codon of the oppAV gene.All data in our study suggested that the BB0462 protein was involved in the transcriptional regulation of the oppAV gene

  9. 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.

  10. Activation of transcriptional activity of HSE by a novel mouse zinc finger protein ZNFD specifically expressed in testis.

    Science.gov (United States)

    Xu, Fengqin; Wang, Weiping; Lei, Chen; Liu, Qingmei; Qiu, Hao; Muraleedharan, Vinaydhar; Zhou, Bin; Cheng, Hongxia; Huang, Zhongkai; Xu, Weian; Li, Bichun; Wang, Minghua

    2012-04-01

    Zinc finger proteins (ZFPs) that contain multiple cysteine and/or histidine residues perform important roles in various cellular functions, including transcriptional regulation, cell proliferation, differentiation, and apoptosis. The Cys-Cys-His-His (C(2)H(2)) type of ZFPs are the well-defined members of this super family and are the largest and most complex proteins in eukaryotic genomes. In this study, we identified a novel C(2)H(2) type of zinc finger gene ZNFD from mice which has a 1,002 bp open reading frame and encodes a protein with 333 amino acid residues. The predicted 37.4 kDa protein contains a C(2)H(2) zinc finger domain. ZNFD gene is located on chromosome 18qD1. RT-PCR analysis revealed that the ZNFD gene was specifically expressed in mouse testis but not in other tissues. Subcellular localization analysis demonstrated that ZNFD was localized in the nucleus. Reporter gene assays showed that overexpression of ZNFD in the COS7 cells activates the transcriptional activities of heat shock element (HSE). Overall, these results suggest that ZNFD is a member of the zinc finger transcription factor family and it participates in the transcriptional regulation of HSE. Many heat shock proteins regulated by HSE are involved in testicular development. Therefore, our results suggest that ZNFD may probably participate in the development of mouse testis and function as a transcription activator in HSE-mediated gene expression and signaling pathways.

  11. Probing the Biomimetic Ice Nucleation Inhibition Activity of Poly(vinyl alcohol) and Comparison to Synthetic and Biological Polymers.

    Science.gov (United States)

    Congdon, Thomas; Dean, Bethany T; Kasperczak-Wright, James; Biggs, Caroline I; Notman, Rebecca; Gibson, Matthew I

    2015-09-14

    Nature has evolved many elegant solutions to enable life to flourish at low temperatures by either allowing (tolerance) or preventing (avoidance) ice formation. These processes are typically controlled by ice nucleating proteins or antifreeze proteins, which act to either promote nucleation, prevent nucleation or inhibit ice growth depending on the specific need, respectively. These proteins can be expensive and their mechanisms of action are not understood, limiting their translation, especially into biomedical cryopreservation applications. Here well-defined poly(vinyl alcohol), synthesized by RAFT/MADIX polymerization, is investigated for its ice nucleation inhibition (INI) activity, in contrast to its established ice growth inhibitory properties and compared to other synthetic polymers. It is shown that ice nucleation inhibition activity of PVA has a strong molecular weight dependence; polymers with a degree of polymerization below 200 being an effective inhibitor at just 1 mg.mL(-1). Other synthetic and natural polymers, both with and without hydroxyl-functional side chains, showed negligible activity, highlighting the unique ice/water interacting properties of PVA. These findings both aid our understanding of ice nucleation but demonstrate the potential of engineering synthetic polymers as new biomimetics to control ice formation/growth processes. PMID:26258729

  12. The use of activated protein C in severe Plasmodium falciparum malaria.

    Science.gov (United States)

    Rankin, L G; Austin, D L H

    2007-06-01

    A 56-year-old man presented to a peripheral hospital in New Zealand with severe Plasmodium falciparum malaria with cerebral involvement and subsequently developed multi-system organ failure. Activated protein C was used in an attempt to stop the cascade of events into multi-organ failure. Severe infection with P. falciparum is life-threatening and appears to activate a hypercoagulable state similar to that of severe sepsis. Activated protein C is currently used in the treatment of severe sepsis and may provide a new adjuvant therapy for severe P. falciparum malaria.

  13. Creative elements: network-based predictions of active centres in proteins, cellular and social networks

    CERN Document Server

    Csermely, Peter

    2008-01-01

    Active centres and hot spots of proteins have a paramount importance in enzyme action, protein complex formation and drug design. Recently a number of publications successfully applied the analysis of residue networks to predict active centres in proteins. Most real-world networks show a number of properties, such as small-worldness or scale-free degree distribution, which are rather general features of networks from molecules to the society. Based on extensive analogies I propose that the existing findings and methodology enable us to detect active centres in cells, social networks and ecosystems. Members of these active centres are creative elements of the respective networks, which may help them to survive unprecedented, novel challenges, and play a key role in the development, survival and evolvability of complex systems.

  14. 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.

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

    Institute of Scientific and Technical Information of China (English)

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

    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 maltosebinding protein (MBP) and to evaluate its immunoreactivity and immunogenicity.METHODS: Neutrophil-activating protein gene of H pylori (HP-napA) was subcloned from the recombinant plasmid pNEB-napA, and fused to MalE gene of expressing vector pMAL-c2x. The recombinant plasmid pMAL-c2x-napA was confirmed by restriction enzyme digestion, and then transformed into E. coli TB1. Fusion protein rMBP-NAP was induced by IPTG and identified by SDS-PAGE analysis.Soluble rMBP-NAP was purified by amylose affinity chromatography. Immunoreactivity and immunogenicity of the fusion protein were evaluated by animal experiment,Western blotting with human H pylori anti-sera.RESULTS: E.coli TB1 carrying recombinant plasmid pMAL-c2x-napA was constructed and led to a high efficiency cytosol expression of fusion protein rBMP -NAP when induced by IPTG.The molecular weight of rBMP-NAP was about 57 kD,accounting for 37.55% of the total protein in the sonicated supematant of E. coli TB1 (pMAL-c2x-napA). The purity of the fusion protein after one-step affinity chromatography was 94% and the yield was 100 mg per liter of bacterial culture.The purified fusion protein could be specifically recognized by both human anti-sera from clinical patients with H pylori infection and rabbit sera immunized by rMBP-NAP itself.CONCLUSION: Recombinant protein rMBP-NAP might be a novel antigen for vaccine development against H pylori.

  16. Differential activities of cellular and viral macro domain proteins in binding of ADP-ribose metabolites.

    Science.gov (United States)

    Neuvonen, Maarit; Ahola, Tero

    2009-01-01

    Macro domain is a highly conserved protein domain found in both eukaryotes and prokaryotes. Macro domains are also encoded by a set of positive-strand RNA viruses that replicate in the cytoplasm of animal cells, including coronaviruses and alphaviruses. The functions of the macro domain are poorly understood, but it has been suggested to be an ADP-ribose-binding module. We have here characterized three novel human macro domain proteins that were found to reside either in the cytoplasm and nucleus [macro domain protein 2 (MDO2) and ganglioside-induced differentiation-associated protein 2] or in mitochondria [macro domain protein 1 (MDO1)], and compared them with viral macro domains from Semliki Forest virus, hepatitis E virus, and severe acute respiratory syndrome coronavirus, and with a yeast macro protein, Poa1p. MDO2 specifically bound monomeric ADP-ribose with a high affinity (K(d)=0.15 microM), but did not bind poly(ADP-ribose) efficiently. MDO2 also hydrolyzed ADP-ribose-1'' phosphate, resembling Poa1p in all these properties. Ganglioside-induced differentiation-associated protein 2 did not show affinity for ADP-ribose or its derivatives, but instead bound poly(A). MDO1 was generally active in these reactions, including poly(A) binding. Individual point mutations in MDO1 abolished monomeric ADP-ribose binding, but not poly(ADP-ribose) binding; in poly(ADP-ribose) binding assays, the monomer did not compete against polymer binding. The viral macro proteins bound poly(ADP-ribose) and poly(A), but had a low affinity for monomeric ADP-ribose. Thus, the viral proteins do not closely resemble any of the human proteins in their biochemical functions. The differential activity profiles of the human proteins implicate them in different cellular pathways, some of which may involve RNA rather than ADP-ribose derivatives.

  17. 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.

  18. Platelet-activating factor (PAF)-dependent biochemical, morphologic, and physiologic responses of human platelets: Demonstration of translocation of protein kinase C associated with protein phosphorylation

    International Nuclear Information System (INIS)

    Platelet-activating factor (PAF) is a potent stimulus for platelet aggregation and secretion. PAF has been shown to stimulate the phosphatidylinositol (PI) pathway in platelets, which implies that PAF should activate protein kinase C. In this study, measurements of PI metabolites, the elevation of intracellular free calcium concentration, (Ca2+)i, the activation of protein kinase C, and the phosphorylation of platelet proteins (using a two-dimensional gel electrophoretic technique) were performed before and after the addition of 10(-8) M PAF to human platelets. These findings were correlated with morphologic changes in the platelets as determined by immunoelectron microscopic studies on the cytoskeleton and by X-ray analysis of dense bodies. The results show that PAF stimulates the production of PI metabolites and causes an increase in the membrane-associated activity of protein kinase C. These changes are accompanied by a rise in the (Ca2+)i and protein phosphorylation. The increase in protein kinase C activity reaches a maximum at approximately 60 s, a time frame that is consistent with the protein phosphorylation and the subsequent morphologic and secretory events. X-ray analysis revealed two types of dense bodies containing various amounts of calcium which appeared to be released sequentially after PAF activation. These results suggest that the protein phosphorylation that controls the physiologic events resulting from PAF activation of human platelets is catalyzed by protein kinase C

  19. A bacterial ATP-dependent, enhancer binding protein that activates the housekeeping RNA polymerase

    Science.gov (United States)

    Bowman, William C.; Kranz, Robert G.

    1998-01-01

    A commonly accepted view of gene regulation in bacteria that has emerged over the last decade is that promoters are transcriptionally activated by one of two general mechanisms. The major type involves activator proteins that bind to DNA adjacent to where the RNA polymerase (RNAP) holoenzyme binds, usually assisting in recruitment of the RNAP to the promoter. This holoenzyme uses the housekeeping ς70 or a related factor, which directs the core RNAP to the promoter and assists in melting the DNA near the RNA start site. A second type of mechanism involves the alternative sigma factor (called ς54 or ςN) that directs RNAP to highly conserved promoters. In these cases, an activator protein with an ATPase function oligomerizes at tandem sites far upstream from the promoter. The nitrogen regulatory protein (NtrC) from enteric bacteria has been the model for this family of activators. Activation of the RNAP/ς54 holoenzyme to form the open complex is mediated by the activator, which is tethered upstream. Hence, this class of protein is sometimes called the enhancer binding protein family or the NtrC class. We describe here a third system that has properties of each of these two types. The NtrC enhancer binding protein from the photosynthetic bacterium, Rhodobacter capsulatus, is shown in vitro to activate the housekeeping RNAP/ς70 holoenzyme. Transcriptional activation by this NtrC requires ATP binding but not hydrolysis. Oligomerization at distant tandem binding sites on a supercoiled template is also necessary. Mechanistic and evolutionary questions of these systems are discussed. PMID:9637689

  20. Activation of the A2A adenosine G-protein-coupled receptor by conformational selection.

    Science.gov (United States)

    Ye, Libin; Van Eps, Ned; Zimmer, Marco; Ernst, Oliver P; Prosser, R Scott

    2016-05-12

    Conformational selection and induced fit are two prevailing mechanisms to explain the molecular basis for ligand-based activation of receptors. G-protein-coupled receptors are the largest class of cell surface receptors and are important drug targets. A molecular understanding of their activation mechanism is critical for drug discovery and design. However, direct evidence that addresses how agonist binding leads to the formation of an active receptor state is scarce. Here we use (19)F nuclear magnetic resonance to quantify the conformational landscape occupied by the adenosine A2A receptor (A2AR), a prototypical class A G-protein-coupled receptor. We find an ensemble of four states in equilibrium: (1) two inactive states in millisecond exchange, consistent with a formed (state S1) and a broken (state S2) salt bridge (known as 'ionic lock') between transmembrane helices 3 and 6; and (2) two active states, S3 and S3', as identified by binding of a G-protein-derived peptide. In contrast to a recent study of the β2-adrenergic receptor, the present approach allowed identification of a second active state for A2AR. Addition of inverse agonist (ZM241385) increases the population of the inactive states, while full agonists (UK432097 or NECA) stabilize the active state, S3', in a manner consistent with conformational selection. In contrast, partial agonist (LUF5834) and an allosteric modulator (HMA) exclusively increase the population of the S3 state. Thus, partial agonism is achieved here by conformational selection of a distinct active state which we predict will have compromised coupling to the G protein. Direct observation of the conformational equilibria of ligand-dependent G-protein-coupled receptor and deduction of the underlying mechanisms of receptor activation will have wide-reaching implications for our understanding of the function of G-protein-coupled receptor in health and disease. PMID:27144352

  1. Antimicrobial activity of peptides derived from olive flounder lipopolysaccharide binding protein/bactericidal permeability-increasing protein (LBP/BPI).

    Science.gov (United States)

    Nam, Bo-Hye; Moon, Ji-Young; Park, Eun-Hee; Kim, Young-Ok; Kim, Dong-Gyun; Kong, Hee Jeong; Kim, Woo-Jin; Jee, Young Ju; An, Cheul Min; Park, Nam Gyu; Seo, Jung-Kil

    2014-10-17

    We describe the antimicrobial function of peptides derived from the C-terminus of the olive flounder LBP BPI precursor protein. The investigated peptides, namely, ofLBP1N, ofLBP2A, ofLBP4N, ofLBP5A, and ofLBP6A, formed α-helical structures, showing significant antimicrobial activity against several Gram-negative bacteria, Gram-positive bacteria, and the yeast Candida albicans, but very limited hemolytic activities. The biological activities of these five analogs were evaluated against biomembranes or artificial membranes for the development of candidate therapeutic agents. Gel retardation studies revealed that peptides bound to DNA and inhibited migration on an agarose gel. In addition, we demonstrated that ofLBP6A inhibited polymerase chain reaction. These results suggested that the ofLBP-derived peptide bactericidal mechanism may be related to the interaction with intracellular components such as DNA or polymerase.

  2. Antimicrobial Activity of Peptides Derived from Olive Flounder Lipopolysaccharide Binding Protein/Bactericidal Permeability-Increasing Protein (LBP/BPI

    Directory of Open Access Journals (Sweden)

    Bo-Hye Nam

    2014-10-01

    Full Text Available We describe the antimicrobial function of peptides derived from the C-terminus of the olive flounder LBP BPI precursor protein. The investigated peptides, namely, ofLBP1N, ofLBP2A, ofLBP4N, ofLBP5A, and ofLBP6A, formed α-helical structures, showing significant antimicrobial activity against several Gram-negative bacteria, Gram-positive bacteria, and the yeast Candida albicans, but very limited hemolytic activities. The biological activities of these five analogs were evaluated against biomembranes or artificial membranes for the development of candidate therapeutic agents. Gel retardation studies revealed that peptides bound to DNA and inhibited migration on an agarose gel. In addition, we demonstrated that ofLBP6A inhibited polymerase chain reaction. These results suggested that the ofLBP-derived peptide bactericidal mechanism may be related to the interaction with intracellular components such as DNA or polymerase.

  3. The oxygen evolving enhancer protein 1 (OEE) of photosystem II in green algae exhibits thioredoxin activity.

    Science.gov (United States)

    Heide, Heinrich; Kalisz, Henryk M; Follmann, Hartmut

    2004-02-01

    A thioredoxin-like chloroplast protein of the fructosebisphosphatase-stimulating f-type, but with an unusually high molecular mass of 28 kDa has previously been identified and purified to homogeneity in a fractionation scheme for resolution of the acid- and heat-stable, regular-size (12kDa) thioredoxins of the unicellular green algae, Scenedesmus obliquus. An apparently analogous protein of 26 kDa was described in a cyanobacterium, Anabaena sp., but no such large thioredoxin species f exists in the thioredoxin profiles of higher plants. The structure of the 28 kDa protein, which had been envisaged to represent a precursor, or fusion product of the two more specialized, common chloroplast thioredoxins f and m has now been determined by amino acid sequencing. Although it exhibits virtually all the properties and enzyme-modulating activities of a thioredoxin proper this algal protein, surprisingly, does not belong to the thioredoxin family of small redox proteins but is identical with OEE (oxygen evolving enhancer) protein 1, an auxiliary component of the photosystem II manganese cluster. Extracts of Chlorella vulgaris and Chlamydomonas reinhardtii also contain heat-stable protein fractions of 23-26 kDa capable of specifically stimulating chloroplast fructosebisphosphatase in vitro. In contrast, OEE protein 1 from spinach is not able to modulate FbPase or NADP malate dehydrogenase from spinach chloroplasts. A dual function of the OEE protein in algal photosynthesis is envisaged. PMID:15022827

  4. Reversible thermal unfolding of a yfdX protein with chaperone-like activity

    Science.gov (United States)

    Saha, Paramita; Manna, Camelia; Chakrabarti, Jaydeb; Ghosh, Mahua

    2016-01-01

    yfdX proteins are ubiquitously present in a large number of virulent bacteria. A member of this family of protein in E. coli is known to be up-regulated by the multidrug response regulator. Their abundance in such bacteria suggests some important yet unidentified functional role of this protein. Here, we study the thermal response and stability of yfdX protein STY3178 from Salmonella Typhi using circular dichroism, steady state fluorescence, dynamic light scattering and nuclear magnetic resonance experiments. We observe the protein to be stable up to a temperature of 45 °C. It folds back to the native conformation from unfolded state at temperature as high as 80 °C. The kinetic measurements of unfolding and refolding show Arrhenius behavior where the refolding involves less activation energy barrier than that of unfolding. We propose a homology model to understand the stability of the protein. Our molecular dynamic simulation studies on this model structure at high temperature show that the structure of this protein is quite stable. Finally, we report a possible functional role of this protein as a chaperone, capable of preventing DTT induced aggregation of insulin. Our studies will have broader implication in understanding the role of yfdX proteins in bacterial function and virulence. PMID:27404435

  5. Negative regulation of caspase 3-cleaved PAK2 activity by protein phosphatase 1

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The p21-activated kinase 2 (PAK2) is activated by binding of small G proteins, Cdc42 and Rac, or through proteolytic cleavage by caspases or caspase-like proteases. Activation by both small G protein and caspase requires autophosphorylation at Thr-402 of PAK2. Although activation of PAK2 has been investigated for nearly a decade, the mechanism of PAK2 downregulation is unclear. In this study, we have applied the kinetic theory of substrate reaction during modification of enzyme activity to study the regulation mechanism of PAK2 activity by the catalytic subunit of protein phosphatase 1 (PP1α). On the basis of the kinetic equation of the substrate reaction during the reversible phosphorylation of PAK2, all microscopic kinetic constants for the free enzyme and enzyme-substrate(s) complexes have been determined. The results indicate that (1) PP1α can act directly on phosphorylated Thr-402 in the activation loop of PAK2 and down-regulate its kinase activity; (2) binding of the exogenous protein/peptide substrates at the active site of PAK2 decreases both the rates of PAK2 autoactivation and inactivation. The present method provides a novel approach for studying reversible phosphorylation reactions. The advantage of this method is not only its usefulness in study of substrate effects on enzyme modification but also its convenience in study of modification reaction directly involved in regulation of enzyme activity. This initial study should provide a foundation for future structural and mechanistic work of protein kinases and phosphatases.

  6. Negative regulation of caspase 3-cleaved PAK2 activity by protein phosphatase 1

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The p21-activated kinase 2 (PAK2) is activated by binding of small G proteins, Cdc42 and Rac, or through proteolytic cleavage by caspases or caspase-like proteases. Activation by both small G protein and caspase requires autophosphorylation at Thr-402 of PAK2. Although activation of PAK2 has been investigated for nearly a decade, the mechanism of PAK2 downregulation is unclear. In this study, we have applied the kinetic theory of substrate reaction during modification of enzyme activity to study the regulation mechanism of PAK2 activity by the catalytic subunit of protein phosphatase 1 (PP1α). On the basis of the kinetic equation of the substrate reaction during the reversible phosphorylation of PAK2, all microscopic kinetic constants for the free enzyme and enzyme-substrate(s) complexes have been determined. The results indicate that (1) PP1α can act directly on phosphorylated Thr-402 in the acti-vation loop of PAK2 and down-regulate its kinase activity; (2) binding of the exogenous protein/peptide substrates at the active site of PAK2 decreases both the rates of PAK2 autoactivation and inactivation. The present method provides a novel approach for studying reversible phosphorylation reactions. The advantage of this method is not only its usefulness in study of substrate effects on enzyme modifica-tion but also its convenience in study of modification reaction directly involved in regulation of enzyme activity. This initial study should provide a foundation for future structural and mechanistic work of protein kinases and phosphatases.

  7. Biocatalyst activity in nonaqueous environments correlates with centisecond-range protein motions.

    Science.gov (United States)

    Eppler, Ross K; Hudson, Elton P; Chase, Shannon D; Dordick, Jonathan S; Reimer, Jeffrey A; Clark, Douglas S

    2008-10-14

    Recent studies exploring the relationship between enzymatic catalysis and protein dynamics in the aqueous phase have yielded evidence that dynamics and enzyme activity are strongly correlated. Given that protein dynamics are significantly attenuated in organic solvents and that proteins exhibit a wide range of motions depending on the specific solvent environment, the nonaqueous milieu provides a unique opportunity to examine the role of protein dynamics in enzyme activity. Variable-temperature kinetic measurements, X-band electron spin resonance spectroscopy, (1)H NMR relaxation, and (19)F NMR spectroscopy experiments were performed on subtilisin Carlsberg colyophilized with several inorganic salts and suspended in organic solvents. The results indicate that salt activation induces a greater degree of transition-state flexibility, reflected by a more positive DeltaDeltaS(dagger), for the more active biocatalyst preparations in organic solvents. In contrast, DeltaDeltaH(dagger) was negligible regardless of salt type or salt content. Electron spin resonance spectroscopy and (1)H NMR relaxation measurements, including spin-lattice relaxation, spin-lattice relaxation in the rotating frame, and longitudinal magnetization exchange, revealed that the enzyme's turnover number (k(cat)) was strongly correlated with protein motions in the centisecond time regime, weakly correlated with protein motions in the millisecond regime, and uncorrelated with protein motions on the piconanosecond timescale. In addition, (19)F chemical shift measurements and hyperfine tensor measurements of biocatalyst formulations inhibited with 4-fluorobenzenesulfonyl fluoride and 4-ethoxyfluorophosphinyl-oxy-TEMPO, respectively, suggest that enzyme activation was only weakly affected by changes in active-site polarity. PMID:18840689

  8. Ca2+/calmodulin-dependent protein kinase kinase is not involved in hypothalamic AMP-activated protein kinase activation by neuroglucopenia.

    Directory of Open Access Journals (Sweden)

    Junji Kawashima

    Full Text Available Hypoglycemia and neuroglucopenia stimulate AMP-activated protein kinase (AMPK activity in the hypothalamus and this plays an important role in the counterregulatory responses, i.e. increased food intake and secretion of glucagon, corticosterone and catecholamines. Several upstream kinases that activate AMPK have been identified including Ca(2+/Calmodulin-dependent protein kinase kinase (CaMKK, which is highly expressed in neurons. However, the involvement of CaMKK in neuroglucopenia-induced activation of AMPK in the hypothalamus has not been tested. To determine whether neuroglucopenia-induced AMPK activation is mediated by CaMKK, we tested whether STO-609 (STO, a CaMKK inhibitor, would block the effects of 2-deoxy-D-glucose (2DG-induced neuroglucopenia both ex vivo on brain sections and in vivo. Preincubation of rat brain sections with STO blocked KCl-induced α1 and α2-AMPK activation but did not affect AMPK activation by 2DG in the medio-basal hypothalamus. To confirm these findings in vivo, STO was pre-administrated intracerebroventricularly (ICV in rats 30 min before 2DG ICV injection (40 µmol to induce neuroglucopenia. 2DG-induced neuroglucopenia lead to a significant increase in glycemia and food intake compared to saline-injected control rats. ICV pre-administration of STO (5, 20 or 50 nmol did not affect 2DG-induced hyperglycemia and food intake. Importantly, activation of hypothalamic α1 and α2-AMPK by 2DG was not affected by ICV pre-administration of STO. In conclusion, activation of hypothalamic AMPK by 2DG-induced neuroglucopenia is not mediated by CaMKK.

  9. Antioxidant Activities of Protein Hydrolysates from Little Hairtail (Trichiurus haumela of East China Sea

    Directory of Open Access Journals (Sweden)

    Ting Jin

    2015-02-01

    Full Text Available This study investigated antioxidant properties of the little hairtail (Trichiurus haumela protein hydrolysates obtained by commercial protease of Alcalase through using various antioxidant assays, including reducing power and free radical scavenging activities. The molecular mass distribution of hydrolysates was also examined to evaluate their relationship with antioxidant activity. The results showed that little hairtail protein hydrolysates had good ability to donate electron or hydrogen and scavenge DPPH, hydroxyl and superoxide anion radicals. The highest value of reducing power and radical scavenging activities was 1.89, 46.15% (DPPH radical, 75.65% (hydroxyl radical and 82.5% (superoxide anion radical, respectively. The reducing power and free radical scavenging activities of little hairtail protein hydrolysates were related to hydrolysis time to some extent. The molecular mass distribution of hydrolysates showed that their molecular mass was between 337 and 6007Da, which indicated that little hairtail protein hydrolysates were mainly composed of low molecular peptides with antioxidant activity. Conclusively, the little hairtail protein was a good natural source for producing antioxidants, which could be used as antioxidant ingredient with potential applications in various food products.

  10. Structural and functional diversity in the activity and regulation of DAPK-related protein kinases.

    Science.gov (United States)

    Temmerman, Koen; Simon, Bertrand; Wilmanns, Matthias

    2013-11-01

    Within the large group of calcium/calmodulin-dependent protein kinases (CAMKs) of the human kinome, there is a distinct branch of highly related kinases that includes three families: death-associated protein-related kinases, myosin light-chain-related kinases and triple functional domain protein-related kinases. In this review, we refer to these collectively as DMT kinases. There are several functional features that span the three families, such as a broad involvement in apoptotic processes, cytoskeletal association and cellular plasticity. Other CAMKs contain a highly conserved HRD motif, which is a prerequisite for kinase regulation through activation-loop phosphorylation, but in all 16 members of the DMT branch, this is replaced by an HF/LD motif. This DMT kinase signature motif substitutes phosphorylation-dependent active-site interactions with a local hydrophobic core that maintains an active kinase conformation. Only about half of the DMT kinases have an additional autoregulatory domain, C-terminal to the kinase domain that binds calcium/calmodulin in order to regulate kinase activity. Protein substrates have been identified for some of the DMT kinases, but little is known about the mechanism of recognition. Substrate conformation could be an equally important parameter in substrate recognition as specific preferences in sequence position. Taking the data together, this kinase branch encapsulates a treasure trove of features that renders it distinct from many other protein kinases and calls for future research activities in this field. PMID:23745726

  11. Activation of Neutrophils via IP3 Pathway Following Exposure to Demodex-Associated Bacterial Proteins.

    Science.gov (United States)

    McMahon, Fred; Banville, Nessa; Bergin, David A; Smedman, Christian; Paulie, Staffan; Reeves, Emer; Kavanagh, Kevin

    2016-02-01

    Rosacea is a chronic inflammatory condition that predominantly affects the skin of the face. Sera from rosacea patients display elevated reactivity to proteins from a bacterium (Bacillus oleronius) originally isolated from a Demodex mite from a rosacea patient suggesting a possible role for bacteria in the induction and persistence of this condition. This work investigated the ability of B. oleronius proteins to activate neutrophils and demonstrated activation via the IP3 pathway. Activated neutrophils displayed increased levels of IP1 production, F-actin formation, chemotaxis, and production of the pro-inflammatory cytokines IL-1β and IL-6 following stimulation by pure and crude B. oleronius protein preparations (2 μg/ml), respectively. In addition, neutrophils exposed to pure and crude B. oleronius proteins (2 μg/ml) demonstrated increased release of internally stored calcium (Ca(2+)), a hallmark of the IP3 pathway of neutrophil activation. Neutrophils play a significant role in the inflammation associated with rosacea, and this work demonstrates how B. oleronius proteins can induce neutrophil recruitment and activation. PMID:26433579

  12. 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.

  13. Downregulation of cold-inducible RNA-binding protein activates mitogen-activated protein kinases and impairs spermatogenic function in mouse testes

    Institute of Scientific and Technical Information of China (English)

    Zhi-Ping Xia; Xin-Min Zheng; Hang Zheng; Xiao-Jun Liu; Gui-Yong Liu; Xing-Huan Wang

    2012-01-01

    Cold-inducible RNA-binding protein (CIRP) is an RNA-binding protein that is expressed in normal testes and downregulated after heat stress caused by cryptorchidism,varicocele or environmental temperatures.The purpose of this study was to investigate the functions of CIRP in the testes.We employed RNAi technique to knock down the expression of CIRP in the testes,and performed haematoxylin and eosin staining to evaluate morphological changes following knockdown.Germ cell apoptosis was examined by terminal deoxynucleotidal transferase-mediated dUTP nick end labelling (TUNEL) assay,and mitogen-activated protein kinase (MAPK)signalling pathways were investigated by Western blotting to determine the possible mechanism of apoptosis.We found that using siRNA is a feasible and reliable method for knocking down gene expression in the testes.Compared to controls,the mean seminiferous tubule diameter (MSTD) and the thickness of the germ cell layers decreased following siRNA treatment,whereas the percentage of apoptotic seminiferous tubules increased.The p44/p42,p38 and SAPK/JNK MAPK pathways were activated after downregulation of CIRP.In conclusion,we discovered that downregulation of CIRP resulted in increased germ cell apoptosis,possibly viathe activation of the p44/p42,p38 and SAPK/JNK MAPK pathways.

  14. Activation of AMP-Activated Protein Kinase Is Required for Berberine-Induced Reduction of Atherosclerosis in Mice: The Role of Uncoupling Protein 2

    OpenAIRE

    Qilong Wang; Miao Zhang; Bin Liang; Najeeb Shirwany; Yi Zhu; Ming-Hui Zou

    2011-01-01

    AIMS: Berberine, a botanical alkaloid purified from Coptidis rhizoma, is reported to activate the AMP-activated protein kinase (AMPK). Whether AMPK is required for the protective effects of berberine in cardiovascular diseases remains unknown. This study was designed to determine whether AMPK is required for berberine-induced reduction of oxidative stress and atherosclerosis in vivo. METHODS: ApoE (ApoE⁻/⁻) mice and ApoE⁻/⁻/AMPK alpha 2⁻/⁻ mice that were fed Western diets were treated with be...

  15. Role of p38 Mitogen-activated Protein Kinase in Mediating Monocyte Chemoattractant Protein-1 in Human Umbilical Vein Endothelial Cells

    Institute of Scientific and Technical Information of China (English)

    李艳波; 邓华聪; 郑丹; 李呼伦

    2004-01-01

    @@ p38 mitogen-activated protein kinase (p38MAPK)is a member of the mitogen-activated protein kinase (MAPK) family. p38MAPK pathway is one of the most widely studied signaling pathways involved in the transduction of intracellular signals including survival, growth,differentiation and death.

  16. Age-related changes in AMP-activated protein kinase after stroke

    OpenAIRE

    Liu, Fudong; Benashski, Sharon E; Persky, Rebecca; Xu, Yan; Li, Jun; McCullough, Louise D.

    2011-01-01

    Adenosine monophosphate-activated protein kinase (AMPK) is an evolutionary conserved energy sensor sensitive to changes in cellular AMP/ATP ratio which is activated by phosphorylation (pAMPK). pAMPK levels decrease in peripheral tissues with age, but whether this also occurs in the aged brain, and how this contributes to the ability of the aged brain to cope with ischemic stress is unknown. This study investigated the activation of AMPK and the response to AMPK inhibition after induced stroke...

  17. Surfactant Protein A integrates activation signal strength to differentially modulate T cell proliferation

    OpenAIRE

    Mukherjee, Sambuddho; Giamberardino, Charles; Thomas, Joseph; Evans, Kathy; GOTO, HISATSUGU; Ledford, Julie G.; Hsia, Bethany; Pastva, Amy M.; Wright, Jo Rae

    2012-01-01

    Pulmonary surfactant lipoproteins lower the surface tension at the alveolar:airway interface of the lung and participate in host defense. Previous studies reported that surfactant protein A (SP-A) inhibits lymphocyte proliferation. We hypothesized that SP-A mediated modulation of T cell activation depends upon the strength, duration and type of lymphocyte activating signals. Modulation of T cell signal strength imparted by different activating agents ex and in vivo in different mouse models, ...

  18. Interactions between Human Liver Fatty Acid Binding Protein and Peroxisome Proliferator Activated Receptor Selective Drugs

    OpenAIRE

    Tony Velkov

    2013-01-01

    Fatty acid binding proteins (FABPs) act as intracellular shuttles for fatty acids as well as lipophilic xenobiotics to the nucleus, where these ligands are released to a group of nuclear receptors called the peroxisome proliferator activated receptors (PPARs). PPAR mediated gene activation is ultimately involved in maintenance of cellular homeostasis through the transcriptional regulation of metabolic enzymes and transporters that target the activating ligand. Here we show that liver- (L-) FA...

  19. Dynamic ubiquitination of the mitogen-activated protein kinase kinase (MAPKK) Ste7 determines mitogen-activated protein kinase (MAPK) specificity.

    Science.gov (United States)

    Hurst, Jillian H; Dohlman, Henrik G

    2013-06-28

    Ubiquitination is a post-translational modification that tags proteins for proteasomal degradation. In addition, there is a growing appreciation that ubiquitination can influence protein activity and localization. Ste7 is a prototype MAPKK in yeast that participates in both the pheromone signaling and nutrient deprivation/invasive growth pathways. We have shown previously that Ste7 is ubiquitinated upon pheromone stimulation. Here, we show that the Skp1/Cullin/F-box ubiquitin ligase SCF(Cdc4) and the ubiquitin protease Ubp3 regulate Ste7 ubiquitination and signal specificity. Using purified components, we demonstrate that SCF(Cdc4) ubiquitinates Ste7 directly. Using gene deletion mutants, we show that SCF(Cdc4) and Ubp3 have opposing effects on Ste7 ubiquitination. Although SCF(Cdc4) is necessary for proper activation of the pheromone MAPK Fus3, Ubp3 is needed to limit activation of the invasive growth MAPK Kss1. Finally, we show that Fus3 phosphorylates Ubp3 directly and that phosphorylation of Ubp3 is necessary to limit Kss1 activation. These results reveal a feedback loop wherein one MAPK limits the ubiquitination of an upstream MAPKK and thereby prevents spurious activation of a second competing MAPK. PMID:23645675

  20. TGEV nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p53 signaling

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Li [College of Veterinary Medicine, Northwest A and F University, Yangling, Shaanxi 712100 (China); College of Life Sciences, Hainan Normal University, Haikou, Hainan 571158 (China); Huang, Yong; Du, Qian; Dong, Feng; Zhao, Xiaomin; Zhang, Wenlong; Xu, Xingang [College of Veterinary Medicine, Northwest A and F University, Yangling, Shaanxi 712100 (China); Tong, Dewen, E-mail: dwtong@nwsuaf.edu.cn [College of Veterinary Medicine, Northwest A and F University, Yangling, Shaanxi 712100 (China)

    2014-03-07

    Highlights: • TGEV N protein reduces cell viability by inducing cell cycle arrest and apoptosis. • TGEV N protein induces cell cycle arrest and apoptosis by regulating p53 signaling. • TGEV N protein plays important roles in TGEV-induced cell cycle arrest and apoptosis. - Abstract: 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.