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Sample records for protein synthetic activity

  1. Hepatic protein synthetic activity in vivo after ethanol administration

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  2. Effect of synthetic adjuvants of biological activity of spleen proteins

    International Nuclear Information System (INIS)

    Kartasheva, A.L.; Yuferova, N.V.; Drozhennikov, V.A.; Orlova, E.B.; Perevezentseva, O.S.; Filatov, P.P.

    1981-01-01

    Intraperitoneal administration to mice of synthetic adjuvants of a polyanion type increases the spleen mass by 500% and rises the content of proteins with activity of inhibitor of DNAase 1. A protein fraction isolated from the spleen of treated animals administered to exposed (7.7 Gy) mice alone or in a combination with exogenous DNA increases survival up to 61.1 and 80.5%, respectively, as opposed to 36.6% in the case of administration of proteins from intact animals, or 8.3% in the control (no treatment). The protein fraction from treated animals administered to mice exposed to 5.1-5.5 Gy accelerates the recovery of hemopoesis and immune response better than proteins of intact animals

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

    Directory of Open Access Journals (Sweden)

    David Grahame Hardie

    2016-01-01

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

  4. Changes in protein synthetic activity in early Drosophila embryos mutant for the segmentation gene Krueppel

    International Nuclear Information System (INIS)

    Bedian, V.; Summers, M.C.; Kauffman, S.A.

    1988-01-01

    We have identified early embryo proteins related to the segmentation gene Krueppel by [35S]methionine pulse labelling and two-dimensional gel electrophoresis. Protein synthesis differences shared by homozygous embryos of two Krueppel alleles when compared to heterozygous and wild-type embryos are reported. The study was extended to syncytial blastoderm stages by pulse labelling and gel analysis of single embryos, using Krueppel-specific proteins from gastrula stages as molecular markers for identifying homozygous Krueppel embryos. Localized expression of interesting proteins was examined in embryo fragments. The earliest differences detected at nuclear migration stages showed unregulated synthesis in mutant embryos of two proteins that have stage specific synthesis in normal embryos. At the cellular blastoderm stage one protein was not synthesized and two proteins showed apparent shifts in isoelectric point in mutant embryos. Differences observed in older embryos included additional proteins with shifted isoelectric points and a number of qualitative and quantitative changes in protein synthesis. Five of the proteins with altered rates of synthesis in mutant embryos showed localized synthesis in normal embryos. The early effects observed are consistent with the hypothesis that the Krueppel product can be a negative or positive regulator of expression of other loci, while blastoderm and gastrula stage shifts in isoelectric point indicate that a secondary effect of Krueppel function may involve post-translational modification of proteins

  5. Immunoregulatory activities of human immunodeficiency virus (HIV) proteins: Effect of HIV recombinant and synthetic peptides on immunoglobulin synthesis and proliferative responses by normal lymphocytes

    International Nuclear Information System (INIS)

    Nair, M.P.N.; Pottathil, R.; Heimer, E.P.; Schwartz, S.A.

    1988-01-01

    Recombinant and synthetic peptides corresponding to envelope proteins of the human immunodeficiency virus (HIV) were examined for their effects on the activities of lymphocytes from normal donors in vitro. Although lymphocytes cultured with env-gag peptides produced significant amounts of IgG, addition of env-gag peptides to a pokeweed mitogen-induced B-cell activation system resulted in suppression of immunoglobulin synthesis by normal lymphocytes. Recombinant antigens, env-gag and env-80 dihydrofolate reductase (DHFR), produced a substantial proliferative response by peripheral blood mononuclear cells (PBMC) as determined by [ 3 H]thymidine incorporation. PBMC precultured with HIV synthetic peptide env 578-608 also manifested significant proliferative responses as compared to control cultures. CD3 + lymphocytes precultured with recombinant HIV antigens, env-gag and env-80 DHFR, and synthetic HIV peptide, env 487-511, showed moderate but significant proliferative responses. Both recombinant antigens and synthetic peptides also produced a dose-dependent stimulatory effect on proliferation by CD3 - lymphocytes. These studies demonstrate that recombinant and synthetic peptides of the HIV genome express immunoregulatory T- and B-cell epitopes. Identification of unique HIV epitopes with immunogenic and immunoregulatory activities is necessary for the development of an effective vaccine against HIV infection

  6. Reprogramming cells with synthetic proteins.

    Science.gov (United States)

    Yang, Xiaoxiao; Malik, Vikas; Jauch, Ralf

    2015-01-01

    Conversion of one cell type into another cell type by forcibly expressing specific cocktails of transcription factors (TFs) has demonstrated that cell fates are not fixed and that cellular differentiation can be a two-way street with many intersections. These experiments also illustrated the sweeping potential of TFs to "read" genetically hardwired regulatory information even in cells where they are not normally expressed and to access and open up tightly packed chromatin to execute gene expression programs. Cellular reprogramming enables the modeling of diseases in a dish, to test the efficacy and toxicity of drugs in patient-derived cells and ultimately, could enable cell-based therapies to cure degenerative diseases. Yet, producing terminally differentiated cells that fully resemble their in vivocounterparts in sufficient quantities is still an unmet clinical need. While efforts are being made to reprogram cells nongenetically by using drug-like molecules, defined TF cocktails still dominate reprogramming protocols. Therefore, the optimization of TFs by protein engineering has emerged as a strategy to enhance reprogramming to produce functional, stable and safe cells for regenerative biomedicine. Engineering approaches focused on Oct4, MyoD, Sox17, Nanog and Mef2c and range from chimeric TFs with added transactivation domains, designer transcription activator-like effectors to activate endogenous TFs to reprogramming TFs with rationally engineered DNA recognition principles. Possibly, applying the complete toolkit of protein design to cellular reprogramming can help to remove the hurdles that, thus far, impeded the clinical use of cells derived from reprogramming technologies.

  7. Reprogramming cells with synthetic proteins

    Directory of Open Access Journals (Sweden)

    Xiaoxiao Yang

    2015-06-01

    Full Text Available Conversion of one cell type into another cell type by forcibly expressing specific cocktails of transcription factors (TFs has demonstrated that cell fates are not fixed and that cellular differentiation can be a two-way street with many intersections. These experiments also illustrated the sweeping potential of TFs to "read" genetically hardwired regulatory information even in cells where they are not normally expressed and to access and open up tightly packed chromatin to execute gene expression programs. Cellular reprogramming enables the modeling of diseases in a dish, to test the efficacy and toxicity of drugs in patient-derived cells and ultimately, could enable cell-based therapies to cure degenerative diseases. Yet, producing terminally differentiated cells that fully resemble their in vivocounterparts in sufficient quantities is still an unmet clinical need. While efforts are being made to reprogram cells nongenetically by using drug-like molecules, defined TF cocktails still dominate reprogramming protocols. Therefore, the optimization of TFs by protein engineering has emerged as a strategy to enhance reprogramming to produce functional, stable and safe cells for regenerative biomedicine. Engineering approaches focused on Oct4, MyoD, Sox17, Nanog and Mef2c and range from chimeric TFs with added transactivation domains, designer transcription activator-like effectors to activate endogenous TFs to reprogramming TFs with rationally engineered DNA recognition principles. Possibly, applying the complete toolkit of protein design to cellular reprogramming can help to remove the hurdles that, thus far, impeded the clinical use of cells derived from reprogramming technologies.

  8. Reprogramming cells with synthetic proteins

    Science.gov (United States)

    Yang, Xiaoxiao; Malik, Vikas; Jauch, Ralf

    2015-01-01

    Conversion of one cell type into another cell type by forcibly expressing specific cocktails of transcription factors (TFs) has demonstrated that cell fates are not fixed and that cellular differentiation can be a two-way street with many intersections. These experiments also illustrated the sweeping potential of TFs to “read” genetically hardwired regulatory information even in cells where they are not normally expressed and to access and open up tightly packed chromatin to execute gene expression programs. Cellular reprogramming enables the modeling of diseases in a dish, to test the efficacy and toxicity of drugs in patient-derived cells and ultimately, could enable cell-based therapies to cure degenerative diseases. Yet, producing terminally differentiated cells that fully resemble their in vivo counterparts in sufficient quantities is still an unmet clinical need. While efforts are being made to reprogram cells nongenetically by using drug-like molecules, defined TF cocktails still dominate reprogramming protocols. Therefore, the optimization of TFs by protein engineering has emerged as a strategy to enhance reprogramming to produce functional, stable and safe cells for regenerative biomedicine. Engineering approaches focused on Oct4, MyoD, Sox17, Nanog and Mef2c and range from chimeric TFs with added transactivation domains, designer transcription activator-like effectors to activate endogenous TFs to reprogramming TFs with rationally engineered DNA recognition principles. Possibly, applying the complete toolkit of protein design to cellular reprogramming can help to remove the hurdles that, thus far, impeded the clinical use of cells derived from reprogramming technologies. PMID:25652623

  9. The muscle protein synthetic response to food ingestion.

    Science.gov (United States)

    Gorissen, Stefan H M; Rémond, Didier; van Loon, Luc J C

    2015-11-01

    Preservation of skeletal muscle mass is of great importance for maintaining both metabolic health and functional capacity. Muscle mass maintenance is regulated by the balance between muscle protein breakdown and synthesis rates. Both muscle protein breakdown and synthesis rates have been shown to be highly responsive to physical activity and food intake. Food intake, and protein ingestion in particular, directly stimulates muscle protein synthesis rates. The postprandial muscle protein synthetic response to feeding is regulated on a number of levels, including dietary protein digestion and amino acid absorption, splanchnic amino acid retention, postprandial insulin release, skeletal muscle tissue perfusion, amino acid uptake by muscle, and intramyocellular signaling. The postprandial muscle protein synthetic response to feeding is blunted in many conditions characterized by skeletal muscle loss, such as aging and muscle disuse. Therefore, it is important to define food characteristics that modulate postprandial muscle protein synthesis. Previous work has shown that the muscle protein synthetic response to feeding can be modulated by changing the amount of protein ingested, the source of dietary protein, as well as the timing of protein consumption. Most of this work has studied the postprandial response to the ingestion of isolated protein sources. Only few studies have investigated the postprandial muscle protein synthetic response to the ingestion of protein dense foods, such as dairy and meat. The current review will focus on the capacity of proteins and protein dense food products to stimulate postprandial muscle protein synthesis and identifies food characteristics that may modulate the anabolic properties. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Connective tissue-activating peptide III (CTAP-III): cloning the synthetic gene and characterization of the protein expressed in E. coli

    International Nuclear Information System (INIS)

    Johnson, P.H.; Castor, C.W.; Walz, D.A.

    1986-01-01

    CTAP-III, an α-granule protein secreted by human platelets, is known to stimulate mitogenesis, extracellular matrix synthesis, and plasminogen activator synthesis in human fibroblast cultures. From its primary sequence, a synthetic gene was constructed to code for a methionine-free derivative (Leu substituted for Met-21), then cloned and expressed in E. coli using a new expression vector containing regulatory elements of the colicin E1 operon. Partially purified recombinant CTAP-III showed a line of identity with CTAP-III by immunodiffusion against rabbit antibody to platelet-derived CTAP-III. Immunodetection of the reduced protein after SDS-PAGE electrophoresis showed a molecular weight (mobility) in agreement with the natural form. Biologic activity of rCTAP-III eluted from an antiCTAP-III immunoaffinity column was measured in human synovial cell bioassay systems. rCTAP-III stimulated synovial cell synthesis of 14 C-hyaluronic acid approximately 13-fold; significant (P < 0.001) mitogenesis was also observed. These studies indicate that a sufficient quantity of bioactive peptide can be obtained for a more comprehensive study of its biologic properties

  11. Computational protein design-the next generation tool to expand synthetic biology applications.

    Science.gov (United States)

    Gainza-Cirauqui, Pablo; Correia, Bruno Emanuel

    2018-05-02

    One powerful approach to engineer synthetic biology pathways is the assembly of proteins sourced from one or more natural organisms. However, synthetic pathways often require custom functions or biophysical properties not displayed by natural proteins, limitations that could be overcome through modern protein engineering techniques. Structure-based computational protein design is a powerful tool to engineer new functional capabilities in proteins, and it is beginning to have a profound impact in synthetic biology. Here, we review efforts to increase the capabilities of synthetic biology using computational protein design. We focus primarily on computationally designed proteins not only validated in vitro, but also shown to modulate different activities in living cells. Efforts made to validate computational designs in cells can illustrate both the challenges and opportunities in the intersection of protein design and synthetic biology. We also highlight protein design approaches, which although not validated as conveyors of new cellular function in situ, may have rapid and innovative applications in synthetic biology. We foresee that in the near-future, computational protein design will vastly expand the functional capabilities of synthetic cells. Copyright © 2018. Published by Elsevier Ltd.

  12. Synthetic multicellular oscillatory systems: controlling protein dynamics with genetic circuits

    International Nuclear Information System (INIS)

    Koseska, Aneta; Volkov, Evgenii; Kurths, Juergen

    2011-01-01

    Synthetic biology is a relatively new research discipline that combines standard biology approaches with the constructive nature of engineering. Thus, recent efforts in the field of synthetic biology have given a perspective to consider cells as 'programmable matter'. Here, we address the possibility of using synthetic circuits to control protein dynamics. In particular, we show how intercellular communication and stochasticity can be used to manipulate the dynamical behavior of a population of coupled synthetic units and, in this manner, finely tune the expression of specific proteins of interest, e.g. in large bioreactors.

  13. Rewiring protein synthesis: From natural to synthetic amino acids.

    Science.gov (United States)

    Fan, Yongqiang; Evans, Christopher R; Ling, Jiqiang

    2017-11-01

    The protein synthesis machinery uses 22 natural amino acids as building blocks that faithfully decode the genetic information. Such fidelity is controlled at multiple steps and can be compromised in nature and in the laboratory to rewire protein synthesis with natural and synthetic amino acids. This review summarizes the major quality control mechanisms during protein synthesis, including aminoacyl-tRNA synthetases, elongation factors, and the ribosome. We will discuss evolution and engineering of such components that allow incorporation of natural and synthetic amino acids at positions that deviate from the standard genetic code. The protein synthesis machinery is highly selective, yet not fixed, for the correct amino acids that match the mRNA codons. Ambiguous translation of a codon with multiple amino acids or complete reassignment of a codon with a synthetic amino acid diversifies the proteome. Expanding the genetic code with synthetic amino acids through rewiring protein synthesis has broad applications in synthetic biology and chemical biology. Biochemical, structural, and genetic studies of the translational quality control mechanisms are not only crucial to understand the physiological role of translational fidelity and evolution of the genetic code, but also enable us to better design biological parts to expand the proteomes of synthetic organisms. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Synthetic protease substrate n-benzoyl-L-argininyl-p-nitroanilide activates specific binding of [3H]estradiol to a protein in rat pancreas: relationship of structure to activity

    International Nuclear Information System (INIS)

    Grossman, A.

    1984-01-01

    N-benzoyl-L-argininyl-p-nitroanilide (BAN), a synthetic substrate for trypsin-like proteolytic enzymes, is a potent activator of [ 3 H]estradiol-binding to a protein present in rat pancreas. When partially purified, this protein is almost devoid of [ 3 H]estradiol-binding activity in the absence of an endogenous accessory factor. BAN can mimic the natural coligand in this steroid binding reaction. The effect of BAN is specific since a number of derivatives of this substance are inactive or may even inhibit steroid binding. It is unlikely that BAN exerts this stimulatory action indirectly, possibly by preventing proteolytic inactivation of the [ 3 H]estradiol-binding protein, since preincubation of the protein in the absence of BAN resulted neither in reduced rate, nor extent, of steroid binding following BAN addition. Also, a number of protease inhibitors had no effect on the binding reaction. Of those inhibitors tested, only antipain significantly enhanced binding of [ 3 H]estradiol, but only about 20 percent as effectively as BAN. 13 references, 1 figure, 2 tables

  15. Activation of Fetal γ-globin Gene Expression via Direct Protein Delivery of Synthetic Zinc-finger DNA-Binding Domains

    Directory of Open Access Journals (Sweden)

    Mir A Hossain

    2016-01-01

    Full Text Available Reactivation of γ-globin expression has been shown to ameliorate disease phenotypes associated with mutations in the adult β-globin gene, including sickle cell disease. Specific mutations in the promoter of the γ-globin genes are known to prevent repression of the genes in the adult and thus lead to hereditary persistence of fetal hemoglobin. One such hereditary persistence of fetal hemoglobin is associated with a sequence located 567 bp upstream of the Gγ-globin gene which assembles a GATA-containing repressor complex. We generated two synthetic zinc-finger DNA-binding domains (ZF-DBDs targeting this sequence. The -567Gγ ZF-DBDs associated with high affinity and specificity with the target site in the γ-globin gene promoter. We delivered the -567Gγ ZF-DBDs directly to primary erythroid cells. Exposure of these cells to the recombinant -567Gγ ZF-DBDs led to increased expression of the γ-globin gene. Direct protein delivery of ZF-DBDs that compete with transcription regulatory proteins will have broad implications for modulating gene expression in analytical or therapeutic settings.

  16. Immunomodulating activities of soluble synthetic polymer-bound drugs.

    Science.gov (United States)

    Ríhová, Blanka

    2002-09-13

    The introduction of a synthetic material into the body always affects different body systems, including the defense system. Synthetic polymers are usually thymus-independent antigens with only a limited ability to elicit antibody formation or to induce a cellular immune response against them. However, there are many other ways that they influence or can be used to influence the immune system of the host. Low-immunogenic water-soluble synthetic polymers sometimes exhibit significant immunomodulating activity, mainly concerning the activation/suppression of NK cells, LAK cells and macrophages. Some of them, such as poly(ethylene glycol) and poly[N-(2-hydroxypropyl)methacrylamide], can be used as effective protein carriers, as they are able to reduce the immunogenicity of conjugated proteins and/or to reduce non-specific uptake of liposome/nanoparticle-entrapped drugs and other therapeutic agents. Recently, the development of vaccine delivery systems prepared from biodegradable and biocompatible water-soluble synthetic polymers, microspheres, liposomes and/or nanoparticles has received considerable attention, as they can be tailored to meet the specific physical, chemical, and immunogenic requirements of a particular antigen and some of them can also act as adjuvants. Copyright 2002 Elsevier Science B.V.

  17. Peptide/protein-polymer conjugates: synthetic strategies and design concepts.

    Science.gov (United States)

    Gauthier, Marc A; Klok, Harm-Anton

    2008-06-21

    This feature article provides a compilation of tools available for preparing well-defined peptide/protein-polymer conjugates, which are defined as hybrid constructs combining (i) a defined number of peptide/protein segments with uniform chain lengths and defined monomer sequences (primary structure) with (ii) a defined number of synthetic polymer chains. The first section describes methods for post-translational, or direct, introduction of chemoselective handles onto natural or synthetic peptides/proteins. Addressed topics include the residue- and/or site-specific modification of peptides/proteins at Arg, Asp, Cys, Gln, Glu, Gly, His, Lys, Met, Phe, Ser, Thr, Trp, Tyr and Val residues and methods for producing peptides/proteins containing non-canonical amino acids by peptide synthesis and protein engineering. In the second section, methods for introducing chemoselective groups onto the side-chain or chain-end of synthetic polymers produced by radical, anionic, cationic, metathesis and ring-opening polymerization are described. The final section discusses convergent and divergent strategies for covalently assembling polymers and peptides/proteins. An overview of the use of chemoselective reactions such as Heck, Sonogashira and Suzuki coupling, Diels-Alder cycloaddition, Click chemistry, Staudinger ligation, Michael's addition, reductive alkylation and oxime/hydrazone chemistry for the convergent synthesis of peptide/protein-polymer conjugates is given. Divergent approaches for preparing peptide/protein-polymer conjugates which are discussed include peptide synthesis from synthetic polymer supports, polymerization from peptide/protein macroinitiators or chain transfer agents and the polymerization of peptide side-chain monomers.

  18. Development and optimization of a cell-based assay for the selection of synthetic compounds that potentiate bone morphogenetic protein-2 activity.

    Science.gov (United States)

    Okada, Motohiro; Sangadala, Sreedhara; Liu, Yunshan; Yoshida, Munehito; Reddy, Boojala Vijay B; Titus, Louisa; Boden, Scott D

    2009-12-01

    The requirement of large amounts of the recombinant human bone morphogenetic protein-2 (BMP-2) produces a huge translational barrier for its routine clinical use due to high cost. This leads to an urgent need to develop alternative methods to lower costs and/or increase efficacies for using BMP-2. In this study, we describe the development and optimization of a cell-based assay that is sensitive, reproducible, and reliable in identifying reagents that potentiate the effects of BMP-2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. The assay is based on a BMP-responsive Smad1-driven luciferase reporter gene. LIM mineralization protein-1 (LMP-1) is a novel intracellular LIM domain protein that has been shown by our group to enhance cellular responsiveness to BMP-2. Our previous report elucidated that the binding of LMP-1 with the WW2 domain in Smad ubiquitin regulatory factor-1 (Smurf1) rescues the osteogenic Smads from degradation. Here, using the optimized cell-based assay, we first evaluated the activity of the recombinantly prepared proteins, LMP-1, and its mutant (LMP-1DeltaSmurf1) that lacks the Smurf1-WW2 domain-binding motif. Both the wild type and the mutant proteins were engineered to contain an 11-amino acid HIV-TAT protein derived membrane transduction domain to aid the cellular delivery of recombinant proteins. The cell-based reporter assay confirmed that LMP-1 potentiates the BMP-induced stimulation of C2C12 cells towards the osteoblastic phenotype. The potentiating effect of LMP-1 was significantly reduced when a specific-motif known to interact with Smurf1 was mutated. We validated the results obtained in the reporter assay by also monitoring the expression of mRNA for osteocalcin and alkaline phosphatase (ALP) which is widely accepted osteoblast differentiation marker genes. Finally, we provide further confirmation of our results by measuring the activity of alkaline phosphatase in support of the accuracy and

  19. Rationally designed synthetic protein hydrogels with predictable mechanical properties.

    Science.gov (United States)

    Wu, Junhua; Li, Pengfei; Dong, Chenling; Jiang, Heting; Bin Xue; Gao, Xiang; Qin, Meng; Wang, Wei; Bin Chen; Cao, Yi

    2018-02-12

    Designing synthetic protein hydrogels with tailored mechanical properties similar to naturally occurring tissues is an eternal pursuit in tissue engineering and stem cell and cancer research. However, it remains challenging to correlate the mechanical properties of protein hydrogels with the nanomechanics of individual building blocks. Here we use single-molecule force spectroscopy, protein engineering and theoretical modeling to prove that the mechanical properties of protein hydrogels are predictable based on the mechanical hierarchy of the cross-linkers and the load-bearing modules at the molecular level. These findings provide a framework for rationally designing protein hydrogels with independently tunable elasticity, extensibility, toughness and self-healing. Using this principle, we demonstrate the engineering of self-healable muscle-mimicking hydrogels that can significantly dissipate energy through protein unfolding. We expect that this principle can be generalized for the construction of protein hydrogels with customized mechanical properties for biomedical applications.

  20. Activity of synthetic peptides against Chlamydia.

    Science.gov (United States)

    Donati, Manuela; Cenacchi, Giovanna; Biondi, Roberta; Papa, Valentina; Borel, Nicole; Vecchio Nepita, Edoardo; Magnino, Simone; Pasquinelli, Gianandrea; Levi, Aurora; Franco, Octavio L

    2017-11-01

    The in vitro activity of six synthetic peptides against 36 strains of Chlamydia from different origins was investigated. Clavanin MO (CMO) proved to be the most active peptide, reducing the inclusion number of all Chlamydia strains from eight different species tested by ≥50% at 10 µg mL -1 . Mastoparan L showed an equal activity against C. trachomatis, C. pneumoniae, C. suis, and C. muridarum, but did not exert any inhibitory effect against C. psittaci, C. pecorum, C. abortus, and C. avium even at 80 µg mL -1 . These data suggest that CMO could be a promising compound in the prevention and treatment of chlamydial infections. © 2017 Wiley Periodicals, Inc.

  1. Protein scaffolds and higher-order complexes in synthetic biology

    NARCIS (Netherlands)

    den Hamer, A.; Rosier, B.J.H.M.; Brunsveld, L.; de Greef, T.F.A.; Ryadnov, M.; Brunsveld, L.; Suga, H.

    2017-01-01

    Interactions between proteins control molecular functions such as signalling or metabolic activity. Assembly of proteins via scaffold proteins or in higher-order complexes is a key regulatory mechanism. Understanding and functionally applying this concept requires the construction, study, and

  2. Protein engineering techniques gateways to synthetic protein universe

    CERN Document Server

    Poluri, Krishna Mohan

    2017-01-01

    This brief provides a broad overview of protein-engineering research, offering a glimpse of the most common experimental methods. It also presents various computational programs with applications that are widely used in directed evolution, computational and de novo protein design. Further, it sheds light on the advantages and pitfalls of existing methodologies and future perspectives of protein engineering techniques.

  3. A homogeneous fluorometric assay platform based on novel synthetic proteins

    International Nuclear Information System (INIS)

    Vardar-Schara, Goenuel; Krab, Ivo M.; Yi, Guohua; Su, Wei Wen

    2007-01-01

    Novel synthetic recombinant sensor proteins have been created to detect analytes in solution, in a rapid single-step 'mix and read' noncompetitive homogeneous assay process, based on modulating the Foerster resonance energy transfer (FRET) property of the sensor proteins upon binding to their targets. The sensor proteins comprise a protein scaffold that incorporates a specific target-capturing element, sandwiched by genetic fusion between two molecules that form a FRET pair. The utility of the sensor proteins was demonstrated via three examples, for detecting an anti-biotin Fab antibody, a His-tagged recombinant protein, and an anti-FLAG peptide antibody, respectively, all done directly in solution. The diversity of sensor-target interactions that we have demonstrated in this study points to a potentially universal applicability of the biosensing concept. The possibilities for integrating a variety of target-capturing elements with a common sensor scaffold predict a broad range of practical applications

  4. Synthetic peptides and ribosomal proteins as substrate for 60S ribosomal protein kinase from yeast cells

    DEFF Research Database (Denmark)

    Grankowski, N; Gasior, E; Issinger, O G

    1993-01-01

    Kinetic studies on the 60S protein kinase were conducted with synthetic peptides and ribosomal proteins as substrate. Peptide RRREEESDDD proved to be the best synthetic substrate for this enzyme. The peptide has a sequence of amino acids which most closely resembles the structure of potential...... phosphorylation sites in natural substrates, i.e., acidic ribosomal proteins. The superiority of certain kinetic parameters for 60S kinase obtained with the native whole 80S ribosomes over those of the isolated fraction of acidic ribosomal proteins indicates that the affinity of 60S kinase to the specific protein...

  5. Hybrid protein-synthetic polymer nanoparticles for drug delivery.

    Science.gov (United States)

    Koseva, Neli S; Rydz, Joanna; Stoyanova, Ekaterina V; Mitova, Violeta A

    2015-01-01

    Among the most common nanoparticulate systems, the polymeric nanocarriers have a number of key benefits, which give a great choice of delivery platforms. Nevertheless, polymeric nanoparticles possess some limitations that include use of toxic solvents in the production process, polymer degradation, drug leakage outside the diseased tissue, and polymer cytotoxicity. The combination of polymers of biological and synthetic origin is an appealing modern strategy for the production of novel nanocarriers with unprecedented properties. Proteins' interface can play an important role in determining bioactivity and toxicity and gives perspective for future development of the polymer-based nanoparticles. The design of hybrid constructs composed of synthetic polymer and biological molecules such as proteins can be considered as a straightforward tool to integrate a broad spectrum of properties and biofunctions into a single device. This review discusses hybrid protein-synthetic polymer nanoparticles with different structures and levels in complexity and functionality, in view of their applications as drug delivery systems. © 2015 Elsevier Inc. All rights reserved.

  6. Synthetic mRNA devices that detect endogenous proteins and distinguish mammalian cells.

    Science.gov (United States)

    Kawasaki, Shunsuke; Fujita, Yoshihiko; Nagaike, Takashi; Tomita, Kozo; Saito, Hirohide

    2017-07-07

    Synthetic biology has great potential for future therapeutic applications including autonomous cell programming through the detection of protein signals and the production of desired outputs. Synthetic RNA devices are promising for this purpose. However, the number of available devices is limited due to the difficulty in the detection of endogenous proteins within a cell. Here, we show a strategy to construct synthetic mRNA devices that detect endogenous proteins in living cells, control translation and distinguish cell types. We engineered protein-binding aptamers that have increased stability in the secondary structures of their active conformation. The designed devices can efficiently respond to target proteins including human LIN28A and U1A proteins, while the original aptamers failed to do so. Moreover, mRNA delivery of an LIN28A-responsive device into human induced pluripotent stem cells (hiPSCs) revealed that we can distinguish living hiPSCs and differentiated cells by quantifying endogenous LIN28A protein expression level. Thus, our endogenous protein-driven RNA devices determine live-cell states and program mammalian cells based on intracellular protein information. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. Development of a Synthetic Switch to Control Protein Stability in Eukaryotic Cells with Light.

    Science.gov (United States)

    Taxis, Christof

    2017-01-01

    In eukaryotic cells, virtually all regulatory processes are influenced by proteolysis. Thus, synthetic control of protein stability is a powerful approach to influence cellular behavior. To achieve this, selected target proteins are modified with a conditional degradation sequence (degron) that responds to a distinct signal. For development of a synthetic degron, an appropriate sensor domain is fused with a degron such that activity of the degron is under control of the sensor. This chapter describes the development of a light-activated, synthetic degron in the model organism Saccharomyces cerevisiae. This photosensitive degron module is composed of the light-oxygen-voltage (LOV) 2 photoreceptor domain of Arabidopsis thaliana phototropin 1 and a degron derived from murine ornithine decarboxylase (ODC). Excitation of the photoreceptor with blue light induces a conformational change that leads to exposure and activation of the degron. Subsequently, the protein is targeted for degradation by the proteasome. Here, the strategy for degron module development and optimization is described in detail together with experimental aspects, which were pivotal for successful implementation of light-controlled proteolysis. The engineering of the photosensitive degron (psd) module may well serve as a blueprint for future development of sophisticated synthetic switches.

  8. Libraries of Synthetic TALE-Activated Promoters: Methods and Applications.

    Science.gov (United States)

    Schreiber, T; Tissier, A

    2016-01-01

    The discovery of proteins with programmable DNA-binding specificities triggered a whole array of applications in synthetic biology, including genome editing, regulation of transcription, and epigenetic modifications. Among those, transcription activator-like effectors (TALEs) due to their natural function as transcription regulators, are especially well-suited for the development of orthogonal systems for the control of gene expression. We describe here the construction and testing of libraries of synthetic TALE-activated promoters which are under the control of a single TALE with a given DNA-binding specificity. These libraries consist of a fixed DNA-binding element for the TALE, a TATA box, and variable sequences of 19 bases upstream and 43 bases downstream of the DNA-binding element. These libraries were cloned using a Golden Gate cloning strategy making them usable as standard parts in a modular cloning system. The broad range of promoter activities detected and the versatility of these promoter libraries make them valuable tools for applications in the fine-tuning of expression in metabolic engineering projects or in the design and implementation of regulatory circuits. © 2016 Elsevier Inc. All rights reserved.

  9. Yeast synthetic biology for the production of recombinant therapeutic proteins.

    Science.gov (United States)

    Kim, Hyunah; Yoo, Su Jin; Kang, Hyun Ah

    2015-02-01

    The production of recombinant therapeutic proteins is one of the fast-growing areas of molecular medicine and currently plays an important role in treatment of several diseases. Yeasts are unicellular eukaryotic microbial host cells that offer unique advantages in producing biopharmaceutical proteins. Yeasts are capable of robust growth on simple media, readily accommodate genetic modifications, and incorporate typical eukaryotic post-translational modifications. Saccharomyces cerevisiae is a traditional baker's yeast that has been used as a major host for the production of biopharmaceuticals; however, several nonconventional yeast species including Hansenula polymorpha, Pichia pastoris, and Yarrowia lipolytica have gained increasing attention as alternative hosts for the industrial production of recombinant proteins. In this review, we address the established and emerging genetic tools and host strains suitable for recombinant protein production in various yeast expression systems, particularly focusing on current efforts toward synthetic biology approaches in developing yeast cell factories for the production of therapeutic recombinant proteins. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

  10. Combined protein construct and synthetic gene engineering for heterologous protein expression and crystallization using Gene Composer

    Directory of Open Access Journals (Sweden)

    Walchli John

    2009-04-01

    Full Text Available Abstract Background With the goal of improving yield and success rates of heterologous protein production for structural studies we have developed the database and algorithm software package Gene Composer. This freely available electronic tool facilitates the information-rich design of protein constructs and their engineered synthetic gene sequences, as detailed in the accompanying manuscript. Results In this report, we compare heterologous protein expression levels from native sequences to that of codon engineered synthetic gene constructs designed by Gene Composer. A test set of proteins including a human kinase (P38α, viral polymerase (HCV NS5B, and bacterial structural protein (FtsZ were expressed in both E. coli and a cell-free wheat germ translation system. We also compare the protein expression levels in E. coli for a set of 11 different proteins with greatly varied G:C content and codon bias. Conclusion The results consistently demonstrate that protein yields from codon engineered Gene Composer designs are as good as or better than those achieved from the synonymous native genes. Moreover, structure guided N- and C-terminal deletion constructs designed with the aid of Gene Composer can lead to greater success in gene to structure work as exemplified by the X-ray crystallographic structure determination of FtsZ from Bacillus subtilis. These results validate the Gene Composer algorithms, and suggest that using a combination of synthetic gene and protein construct engineering tools can improve the economics of gene to structure research.

  11. Bringing the science of proteins into the realm of organic chemistry: total chemical synthesis of SEP (synthetic erythropoiesis protein).

    Science.gov (United States)

    Kent, Stephen B H

    2013-11-11

    Erythropoietin, commonly known as EPO, is a glycoprotein hormone that stimulates the production of red blood cells. Recombinant EPO has been described as "arguably the most successful drug spawned by the revolution in recombinant DNA technology". Recently, the EPO glycoprotein molecule has re-emerged as a major target of synthetic organic chemistry. In this article I will give an account of an important body of earlier work on the chemical synthesis of a designed EPO analogue that had full biological activity and improved pharmacokinetic properties. The design and synthesis of this "synthetic erythropoiesis protein" was ahead of its time, but has gained new relevance in recent months. Here I will document the story of one of the major accomplishments of synthetic chemistry in a more complete way than is possible in the primary literature, and put the work in its contemporaneous context. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Applications of cell-free protein synthesis in synthetic biology: Interfacing bio-machinery with synthetic environments.

    Science.gov (United States)

    Lee, Kyung-Ho; Kim, Dong-Myung

    2013-11-01

    Synthetic biology is built on the synthesis, engineering, and assembly of biological parts. Proteins are the first components considered for the construction of systems with designed biological functions because proteins carry out most of the biological functions and chemical reactions inside cells. Protein synthesis is considered to comprise the most basic levels of the hierarchical structure of synthetic biology. Cell-free protein synthesis has emerged as a powerful technology that can potentially transform the concept of bioprocesses. With the ability to harness the synthetic power of biology without many of the constraints of cell-based systems, cell-free protein synthesis enables the rapid creation of protein molecules from diverse sources of genetic information. Cell-free protein synthesis is virtually free from the intrinsic constraints of cell-based methods and offers greater flexibility in system design and manipulability of biological synthetic machinery. Among its potential applications, cell-free protein synthesis can be combined with various man-made devices for rapid functional analysis of genomic sequences. This review covers recent efforts to integrate cell-free protein synthesis with various reaction devices and analytical platforms. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Investigation of PVdF active diaphragms for synthetic jets

    Science.gov (United States)

    Bailo, Kelly C.; Brei, Diann E.; Calkins, Frederick T.

    2000-06-01

    Current research has shown that aircraft can gain significant aerodynamic performance benefits by employing active flow control (AFC). One of the enabling technologies of AFC is the synthetic jet. Synthetic jets, also known as zero-net-mass flux actuators, act as bi-directional pumps injecting high momentum air into the local aerodynamic flow. Previous work has concentrated on high frequency synthetic jets based on piezoelectric active diaphragms such as Thunder actuators. Low frequency synthetic jets present a unique challenge requiring large displacements, which current technology has difficulty meeting. Boeing is investigating novel shaped low frequency synthetic jets that can modify the flow over fixed aircraft wings. This paper present the initial study of two promising active diaphragm concepts: a crescent shape and an opposing bender shape. These active diaphragms were numerically modeled utilizing the general-purpose finite element code ABAQUS. Using the ABAQUS results, the dynamic volume change within each jet was calculated and incorporated into an analytical linear Bernoulli model to predict the velocities and pressures at the nozzle. Simulations were performed to determine trends to assist in selection of prototype configurations. Prototypes of both diaphragm concepts were constructed from polyvinylidene fluoride and experimentally tested at Boeing with promising results.

  14. Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently

    Science.gov (United States)

    Currin, Andrew; Swainston, Neil; Day, Philip J.

    2015-01-01

    The amino acid sequence of a protein affects both its structure and its function. Thus, the ability to modify the sequence, and hence the structure and activity, of individual proteins in a systematic way, opens up many opportunities, both scientifically and (as we focus on here) for exploitation in biocatalysis. Modern methods of synthetic biology, whereby increasingly large sequences of DNA can be synthesised de novo, allow an unprecedented ability to engineer proteins with novel functions. However, the number of possible proteins is far too large to test individually, so we need means for navigating the ‘search space’ of possible protein sequences efficiently and reliably in order to find desirable activities and other properties. Enzymologists distinguish binding (K d) and catalytic (k cat) steps. In a similar way, judicious strategies have blended design (for binding, specificity and active site modelling) with the more empirical methods of classical directed evolution (DE) for improving k cat (where natural evolution rarely seeks the highest values), especially with regard to residues distant from the active site and where the functional linkages underpinning enzyme dynamics are both unknown and hard to predict. Epistasis (where the ‘best’ amino acid at one site depends on that or those at others) is a notable feature of directed evolution. The aim of this review is to highlight some of the approaches that are being developed to allow us to use directed evolution to improve enzyme properties, often dramatically. We note that directed evolution differs in a number of ways from natural evolution, including in particular the available mechanisms and the likely selection pressures. Thus, we stress the opportunities afforded by techniques that enable one to map sequence to (structure and) activity in silico, as an effective means of modelling and exploring protein landscapes. Because known landscapes may be assessed and reasoned about as a whole

  15. Synthetic

    Directory of Open Access Journals (Sweden)

    Anna Maria Manferdini

    2010-06-01

    Full Text Available Traditionally materials have been associated with a series of physical properties that can be used as inputs to production and manufacturing. Recently we witnessed an interest in materials considered not only as ‘true matter’, but also as new breeds where geometry, texture, tooling and finish are able to provoke new sensations when they are applied to a substance. These artificial materials can be described as synthetic because they are the outcome of various qualities that are not necessarily true to the original matter, but they are the combination of two or more parts, whether by design or by natural processes. The aim of this paper is to investigate the potential of architectural surfaces to produce effects through the invention of new breeds of artificial matter, using micro-scale details derived from Nature as an inspiration.

  16. Protein cages and synthetic polymers: a fruitful symbiosis for drug delivery applications, bionanotechnology and materials science.

    Science.gov (United States)

    Rother, Martin; Nussbaumer, Martin G; Renggli, Kasper; Bruns, Nico

    2016-11-07

    Protein cages are hollow protein nanoparticles, such as viral capsids, virus-like particles, ferritin, heat-shock proteins and chaperonins. They have well-defined capsule-like structures with a monodisperse size. Their protein subunits can be modified by genetic engineering at predetermined positions, allowing for example site-selective introduction of attachment points for functional groups, catalysts or targeting ligands on their outer surface, in their interior and between subunits. Therefore, protein cages have been extensively explored as functional entities in bionanotechnology, as drug-delivery or gene-delivery vehicles, as nanoreactors or as templates for the synthesis of organic and inorganic nanomaterials. The scope of functionalities and applications of protein cages can be significantly broadened if they are combined with synthetic polymers on their surface or within their interior. For example, PEGylation reduces the immunogenicity of protein cage-based delivery systems and active targeting ligands can be attached via polymer chains to favour their accumulation in diseased tissue. Polymers within protein cages offer the possibility of increasing the loading density of drug molecules, nucleic acids, magnetic resonance imaging contrast agents or catalysts. Moreover, the interaction of protein cages and polymers can be used to modulate the size and shape of some viral capsids to generate structures that do not occur with native viruses. Another possibility is to use the interior of polymer cages as a confined reaction space for polymerization reactions such as atom transfer radical polymerization or rhodium-catalysed polymerization of phenylacetylene. The protein nanoreactors facilitate a higher degree of control over polymer synthesis. This review will summarize the hybrid structures that have been synthesized by polymerizing from protein cage-bound initiators, by conjugating polymers to protein cages, by embedding protein cages into bulk polymeric

  17. Improved protein quality in transgenic soybean expressing a de novo synthetic protein, MB-16.

    Science.gov (United States)

    Zhang, Yunfang; Schernthaner, Johann; Labbé, Natalie; Hefford, Mary A; Zhao, Jiping; Simmonds, Daina H

    2014-06-01

    To improve soybean [Glycine max (L.) Merrill] seed nutritional quality, a synthetic gene, MB-16 was introduced into the soybean genome to boost seed methionine content. MB-16, an 11 kDa de novo protein enriched in the essential amino acids (EAAs) methionine, threonine, lysine and leucine, was originally developed for expression in rumen bacteria. For efficient seed expression, constructs were designed using the soybean codon bias, with and without the KDEL ER retention sequence, and β-conglycinin or cruciferin seed specific protein storage promoters. Homozygous lines, with single locus integrations, were identified for several transgenic events. Transgene transmission and MB-16 protein expression were confirmed to the T5 and T7 generations, respectively. Quantitative RT-PCR analysis of developing seed showed that the transcript peaked in growing seed, 5-6 mm long, remained at this peak level to the full-sized green seed and then was significantly reduced in maturing yellow seed. Transformed events carrying constructs with the rumen bacteria codon preference showed the same transcription pattern as those with the soybean codon preference, but the transcript levels were lower at each developmental stage. MB-16 protein levels, as determined by immunoblots, were highest in full-sized green seed but the protein virtually disappeared in mature seed. However, amino acid analysis of mature seed, in the best transgenic line, showed a significant increase of 16.2 and 65.9 % in methionine and cysteine, respectively, as compared to the parent. This indicates that MB-16 elevated the sulfur amino acids, improved the EAA seed profile and confirms that a de novo synthetic gene can enhance the nutritional quality of soybean.

  18. Synthetic Promoters and Transcription Factors for Heterologous Protein Expression in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Fabian Machens

    2017-10-01

    Full Text Available Orthogonal systems for heterologous protein expression as well as for the engineering of synthetic gene regulatory circuits in hosts like Saccharomyces cerevisiae depend on synthetic transcription factors (synTFs and corresponding cis-regulatory binding sites. We have constructed and characterized a set of synTFs based on either transcription activator-like effectors or CRISPR/Cas9, and corresponding small synthetic promoters (synPs with minimal sequence identity to the host’s endogenous promoters. The resulting collection of functional synTF/synP pairs confers very low background expression under uninduced conditions, while expression output upon induction of the various synTFs covers a wide range and reaches induction factors of up to 400. The broad spectrum of expression strengths that is achieved will be useful for various experimental setups, e.g., the transcriptional balancing of expression levels within heterologous pathways or the construction of artificial regulatory networks. Furthermore, our analyses reveal simple rules that enable the tuning of synTF expression output, thereby allowing easy modification of a given synTF/synP pair. This will make it easier for researchers to construct tailored transcriptional control systems.

  19. Application of Minicircle Technology of Self-Reproducing Synthetic Protein Drugs in Preventing Skin Allograft Rejection.

    Science.gov (United States)

    Lim, Sun Woo; Kim, Young Kyun; Park, Narae; Jin, Long; Jin, Jian; Doh, Kyoung Chan; Ju, Ji Hyeon; Yang, Chul Woo

    2015-07-30

    Recently, it has been reported that minicircle vectors could allow the expression of transgenes using the protein synthesis system of the host. Here, we tested a novel strategy to permit the production of synthetic biologics using minicircle technology and evaluated their feasibility as a therapeutic tool in a skin allograft model. We engineered vectors to carry cassette sequences for tocilizumab [anti-soluble interleukin-6 receptor (sIL-6R) antibody] and/or etanercept [tumor necrosis factor receptor 2 (TNFR2)-Fc fusion protein], and then isolated minicircle vectors from the parent vectors. We verified the production of proteins from minicircles and their duration in HEK293T cells and mice. We also evaluated whether these proteins were expressed at levels sufficient to ameliorate skin allograft rejection in mice. Each minicircle transfected into cells was detectable for at least 30 days. In mice, the drugs were mainly expressed in the liver and were detectable for at least 10 days after a single injection. These drugs were also detected in the blood. Treatment of mice with minicircles prolonged skin allograft survival, which was accompanied by a reduction of the number of interferon-γ+ or interleukin-17+ lymphocytes and an induction of forkhead box P3 expression. These findings suggest that blocking of sIL-6R and/or TNF-α using minicircles encoding tocilizumab and/or etanercept was functionally active and relevant for preventing acute allograft rejection. Self-reproducing synthetic protein drugs produced using minicircle technology are potentially powerful tools for preventing acute rejection in transplantation.

  20. Antimicrobial activity of new porphyrins of synthetic and natural origin

    Science.gov (United States)

    Gyulkhandanyan, Grigor V.; Ghazaryan, Robert K.; Paronyan, Marina H.; Ulikhanyan, Ghukas I.; Gyulkhandanyan, Aram G.; Sahakyan, Lida A.

    2012-03-01

    Antimicrobial photodynamic inactivation has been successfully used against Gram (+) microorganisms, but most of the photosensitizers (PSs) on Gram (-) bacteria acts weakly. PSs are the natural or synthetic origin dyes, mainly porphyrins. We have synthesized more than 100 new cationic porphyrins and metalloporphyrins with different functional groups (hydroxyethyl, butyl, allyl, methallyl) and metals (cobalt, iron, copper, zinc, silver and other); from the nettle have also been purified pheophytin (a+b) and pheophytin (a) and have synthesized their Ag-and Zn-metalloporphyrins. It was found that in the dark (cytotoxic) mode, the most highly efficiency against microorganisms showed Agmetalloporphyrins of both types of porphyrins (synthetic and natural). Metalloporphyrin of natural origin Ag-pheophytin (a + b) is a strong antibacterial agent and causes 100% death as the Gram (+) microorganisms (St. aureus and MRSA) and the Gram (-) microorganisms (E.coli and Salmonella). It is established that for the destruction of Gram (+) and Gram (-) microorganisms in photodynamic mode cationic water-soluble synthetic metalloporphyrins, especially Zn-TBut4PyP, many times more effective than pheophytins. In vivo conditions on mice established that the best therapeutic activity against various strains of the microorganism St. aureus has the synthetic metalloporphyrin Ag-TBut4PyP. It is significantly more efficient than known drug "Chlorophyllipt" (2.5-3 times) and leads the survival rate of animals up to 50-60%.

  1. Monoclonal antibodies against a synthetic peptide from human immunodeficiency virus type 1 Nef protein

    DEFF Research Database (Denmark)

    Steinaa, L; Wulff, A M; Saermark, T

    1994-01-01

    Monoclonal antibodies against a synthetic peptide (aa 138-152) from HIV-1 Nef protein were produced and characterized. Three hybridoma lines producing monoclonal antibodies (MAbs) against the synthetic peptide were generated by fusion between P3-X63 Ag8.653 myeloma cells and BALB/c splenocytes from...... mice immunized with the synthetic peptide coupled to keyhole limpet hemocyanin (KLH). The hybridomas were screened and selected by ELISA with the peptide coupled to bovine serum albumin (BSA) immobilized to the polystyrene surface and specificity for the peptide was confirmed by competitive ELISA...... with the peptide free in solution. The reactions of the MAbs with a 5-aa motif (WCYKL) included in the sequence were examined with synthetic peptides and two of the MAbs reacted with the motif. The recognitions of recombinant full-length Nef protein were also tested. One MAb reacted with the protein in both ELISA...

  2. The cell shape proteins MreB and MreC control cell morphogenesis by positioning cell wall synthetic complexes.

    Science.gov (United States)

    Divakaruni, Arun V; Baida, Cyril; White, Courtney L; Gober, James W

    2007-10-01

    MreB, the bacterial actin homologue, is thought to function in spatially co-ordinating cell morphogenesis in conjunction with MreC, a protein that wraps around the outside of the cell within the periplasmic space. In Caulobacter crescentus, MreC physically associates with penicillin-binding proteins (PBPs) which catalyse the insertion of intracellularly synthesized precursors into the peptidoglycan cell wall. Here we show that MreC is required for the spatial organization of components of the peptidoglycan-synthesizing holoenzyme in the periplasm and MreB directs the localization of a peptidoglycan precursor synthesis protein in the cytosol. Additionally, fluorescent vancomycin (Van-FL) labelling revealed that the bacterial cytoskeletal proteins MreB and FtsZ, as well as MreC and RodA, were required for peptidoglycan synthetic activity. MreB and FtsZ were found to be required for morphogenesis of the polar stalk. FtsZ was required for a cell cycle-regulated burst of peptidoglycan synthesis early in the cell cycle resulting in the synthesis of cross-band structures, whereas MreB was required for lengthening of the stalk. Thus, the bacterial cytoskeleton and cell shape-determining proteins such as MreC, function in concert to orchestrate the localization of cell wall synthetic complexes resulting in spatially co-ordinated and efficient peptidoglycan synthetic activity.

  3. Dietary protein considerations to support active aging.

    Science.gov (United States)

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

    2014-11-01

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

  4. Antiproliferative activity of synthetic fatty acid amides from renewable resources.

    Science.gov (United States)

    dos Santos, Daiane S; Piovesan, Luciana A; D'Oca, Caroline R Montes; Hack, Carolina R Lopes; Treptow, Tamara G M; Rodrigues, Marieli O; Vendramini-Costa, Débora B; Ruiz, Ana Lucia T G; de Carvalho, João Ernesto; D'Oca, Marcelo G Montes

    2015-01-15

    In the work, the in vitro antiproliferative activity of a series of synthetic fatty acid amides were investigated in seven cancer cell lines. The study revealed that most of the compounds showed antiproliferative activity against tested tumor cell lines, mainly on human glioma cells (U251) and human ovarian cancer cells with a multiple drug-resistant phenotype (NCI-ADR/RES). In addition, the fatty methyl benzylamide derived from ricinoleic acid (with the fatty acid obtained from castor oil, a renewable resource) showed a high selectivity with potent growth inhibition and cell death for the glioma cell line-the most aggressive CNS cancer. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Study of the peptide length and amino acid specific substitution in the antigenic activity of the chimeric synthetic peptides, containing the p19 core and gp46 envelope proteins of the HTLV-I virus.

    Science.gov (United States)

    Marin, Milenen Hernández; Rodríguez-Tanty, Chryslaine; Higginson-Clarke, David; Bocalandro, Yadaris Márquez; Peña, Lilliam Pozo

    2005-10-28

    Four chimeric synthetic peptides (Q5, Q6, Q7(multiply sign in circle), and Q8(multiply sign in circle)), incorporating immunodominant epitopes of the core p19 (105-124 a.a.) and envelope gp46 proteins (175-205 a.a.), of HTLV-I were obtained. Also, two gp46 monomeric peptides M4 and M5(multiply sign in circle) (Ser at position 192) were synthesized. The analysis of the influence of the peptide lengths and the proline to serine substitution on the chimeric and monomeric peptides' antigenicity, with regard to the chimeric peptides Q1, Q2, Q3(multiply sign in circle), and Q4(multiply sign in circle), reported previously, for HTLV-I was carried out. The peptides' antigenicity was evaluated in an ultramicroenzyme-linked immunosorbent assay (UMELISA) using sera of HTLV-I/II. The peptides' antigenicity was affected appreciably by the change of the peptide length and amino acid substitutions into the immunodominant sequence of gp46 peptide.

  6. Biocontainment of genetically modified organisms by synthetic protein design

    Science.gov (United States)

    Mandell, Daniel J.; Lajoie, Marc J.; Mee, Michael T.; Takeuchi, Ryo; Kuznetsov, Gleb; Norville, Julie E.; Gregg, Christopher J.; Stoddard, Barry L.; Church, George M.

    2015-02-01

    Genetically modified organisms (GMOs) are increasingly deployed at large scales and in open environments. Genetic biocontainment strategies are needed to prevent unintended proliferation of GMOs in natural ecosystems. Existing biocontainment methods are insufficient because they impose evolutionary pressure on the organism to eject the safeguard by spontaneous mutagenesis or horizontal gene transfer, or because they can be circumvented by environmentally available compounds. Here we computationally redesign essential enzymes in the first organism possessing an altered genetic code (Escherichia coli strain C321.ΔA) to confer metabolic dependence on non-standard amino acids for survival. The resulting GMOs cannot metabolically bypass their biocontainment mechanisms using known environmental compounds, and they exhibit unprecedented resistance to evolutionary escape through mutagenesis and horizontal gene transfer. This work provides a foundation for safer GMOs that are isolated from natural ecosystems by a reliance on synthetic metabolites.

  7. Synthetic strategies for efficient conjugation of organometallic complexes with pendant protein reactive markers

    KAUST Repository

    Jantke, Dominik

    2013-11-01

    Site-directed conjugation of metal centers to proteins is fundamental for biological and bioinorganic applications of transition metals. However, methods for the site-selective introduction of metal centers remain scarce. Herein, we present broadly applicable synthetic strategies for the conjugation of bioactive molecules with a range of organometallic complexes. Following three different synthetic strategies, we were able to synthesize a small library of metal conjugated protein markers featuring different types of protein reactive sites (epoxides, phenylphosphonates, fluorosulfonates and fluorophosphonate groups) as well as different late transition metals (iron, ruthenium, rhodium, palladium and platinum). The products were isolated in moderate to excellent yields and high purity. Furthermore, X-ray diffraction of the metalated protein markers corroborates structural integrity of the metal complex and the protein reactive site. © 2013 Elsevier B.V. All rights reserved.

  8. Synthetic strategies for efficient conjugation of organometallic complexes with pendant protein reactive markers

    KAUST Repository

    Jantke, Dominik; Marziale, Alexander N.; Reiner, Thomas; Kraus, Florian; Herdtweck, Eberhardt; Raba, Andreas; Eppinger, Jö rg

    2013-01-01

    Site-directed conjugation of metal centers to proteins is fundamental for biological and bioinorganic applications of transition metals. However, methods for the site-selective introduction of metal centers remain scarce. Herein, we present broadly applicable synthetic strategies for the conjugation of bioactive molecules with a range of organometallic complexes. Following three different synthetic strategies, we were able to synthesize a small library of metal conjugated protein markers featuring different types of protein reactive sites (epoxides, phenylphosphonates, fluorosulfonates and fluorophosphonate groups) as well as different late transition metals (iron, ruthenium, rhodium, palladium and platinum). The products were isolated in moderate to excellent yields and high purity. Furthermore, X-ray diffraction of the metalated protein markers corroborates structural integrity of the metal complex and the protein reactive site. © 2013 Elsevier B.V. All rights reserved.

  9. A synthetic prestin reveals protein domains and molecular operation of outer hair cell piezoelectricity.

    Science.gov (United States)

    Schaechinger, Thorsten J; Gorbunov, Dmitry; Halaszovich, Christian R; Moser, Tobias; Kügler, Sebastian; Fakler, Bernd; Oliver, Dominik

    2011-06-24

    Prestin, a transporter-like protein of the SLC26A family, acts as a piezoelectric transducer that mediates the fast electromotility of outer hair cells required for cochlear amplification and auditory acuity in mammals. Non-mammalian prestin orthologues are anion transporters without piezoelectric activity. Here, we generated synthetic prestin (SynPres), a chimera of mammalian and non-mammalian prestin exhibiting both, piezoelectric properties and anion transport. SynPres delineates two distinct domains in the protein's transmembrane core that are necessary and sufficient for generating electromotility and associated non-linear charge movement (NLC). Functional analysis of SynPres showed that the amplitude of NLC and hence electromotility are determined by the transport of monovalent anions. Thus, prestin-mediated electromotility is a dual-step process: transport of anions by an alternate access cycle, followed by an anion-dependent transition generating electromotility. The findings define structural and functional determinants of prestin's piezoelectric activity and indicate that the electromechanical process evolved from the ancestral transport mechanism.

  10. Cell-free protein synthesis enabled rapid prototyping for metabolic engineering and synthetic biology

    Directory of Open Access Journals (Sweden)

    Lihong Jiang

    2018-06-01

    Full Text Available Advances in metabolic engineering and synthetic biology have facilitated the manufacturing of many valuable-added compounds and commodity chemicals using microbial cell factories in the past decade. However, due to complexity of cellular metabolism, the optimization of metabolic pathways for maximal production represents a grand challenge and an unavoidable barrier for metabolic engineering. Recently, cell-free protein synthesis system (CFPS has been emerging as an enabling alternative to address challenges in biomanufacturing. This review summarizes the recent progresses of CFPS in rapid prototyping of biosynthetic pathways and genetic circuits (biosensors to speed up design-build-test (DBT cycles of metabolic engineering and synthetic biology. Keywords: Cell-free protein synthesis, Metabolic pathway optimization, Genetic circuits, Metabolic engineering, Synthetic biology

  11. Structure and biological activity of endogenous and synthetic agonists of GPR119

    Science.gov (United States)

    Tyurenkov, I. N.; Ozerov, A. A.; Kurkin, D. V.; Logvinova, E. O.; Bakulin, D. A.; Volotova, E. V.; Borodin, D. D.

    2018-02-01

    A G-protein-coupled receptor, GPR119, is a promising pharmacological target for a new class of hypoglycaemic drugs with an original mechanism of action, namely, increase in the glucose-dependent incretin and insulin secretion. In 2005, the first ligands were found and in the subsequent years, a large number of GPR119 agonists were synthesized in laboratories in various countries; the safest and most promising agonists have entered phase I and II clinical trials as agents for the treatment of type 2 diabetes mellitus and obesity. The review describes the major endogenous GPR119 agonists and the main trends in the design and modification of synthetic structures for increasing the hypoglycaemic activity. The data on synthetic agonists are arranged according to the type of the central core of the molecules. The bibliography includes 104 references.

  12. Isolation and Identification of Proteins Secreted by Cells Cultured within Synthetic Hydrogel-Based Matrices.

    Science.gov (United States)

    Sawicki, Lisa A; Choe, Leila H; Wiley, Katherine L; Lee, Kelvin H; Kloxin, April M

    2018-03-12

    Cells interact with and remodel their microenvironment, degrading large extracellular matrix (ECM) proteins (e.g., fibronectin, collagens) and secreting new ECM proteins and small soluble factors (e.g., growth factors, cytokines). Synthetic mimics of the ECM have been developed as controlled cell culture platforms for use in both fundamental and applied studies. However, how cells broadly remodel these initially well-defined matrices remains poorly understood and difficult to probe. In this work, we have established methods for widely examining both large and small proteins that are secreted by cells within synthetic matrices. Specifically, human mesenchymal stem cells (hMSCs), a model primary cell type, were cultured within well-defined poly(ethylene glycol) (PEG)-peptide hydrogels, and these cell-matrix constructs were decellularized and degraded for subsequent isolation and analysis of deposited proteins. Shotgun proteomics using liquid chromatography and mass spectrometry identified a variety of proteins, including the large ECM proteins fibronectin and collagen VI. Immunostaining and confocal imaging confirmed these results and provided visualization of protein organization within the synthetic matrices. Additionally, culture medium was collected from the encapsulated hMSCs, and a Luminex assay was performed to identify secreted soluble factors, including vascular endothelial growth factor (VEGF), endothelial growth factor (EGF), basic fibroblast growth factor (FGF-2), interleukin 8 (IL-8), and tumor necrosis factor alpha (TNF-α). Together, these methods provide a unique approach for studying dynamic reciprocity between cells and synthetic microenvironments and have the potential to provide new biological insights into cell responses during three-dimensional (3D) controlled cell culture.

  13. Antimycobacterial and cytotoxicity activity of synthetic and natural compounds

    Directory of Open Access Journals (Sweden)

    Ana O. de Souza

    2007-01-01

    Full Text Available Antimycobacterial and cytotoxicity activity of synthetic and natural compounds. Secondary metabolites from Curvularia eragrostidis and Drechslera dematioidea, Clusia sp. floral resin, alkaloids from Pilocarpus alatus, salicylideneanilines, piperidine amides, the amine 1-cinnamylpiperazine and chiral pyridinium salts were assayed on Mycobacterium tuberculosis H37Rv. N-(salicylidene-2-hydroxyaniline was the most effective compound with a minimal inhibitory concentration (MIC of 8 µmol/L. Dihydrocurvularin was moderately effective with a MIC of 40 µmol/L. Clusia sp. floral resin and a gallocatechin-epigallocatechin mixture showed MIC of 0.02 g/L and 38 µmol/L, respectively. The cytotoxicity was evaluated for N-(salicylidene-2-hydroxyaniline, curvularin, dihydrocurvularin and Clusia sp. floral resin, and the selectivity indexes were > 125, 0.47, 0.75 and 5, respectively.

  14. TARSyn: Tunable Antibiotic Resistance Devices Enabling Bacterial Synthetic Evolution and Protein Production

    DEFF Research Database (Denmark)

    Rennig, Maja; Martinez, Virginia; Mirzadeh, Kiavash

    2018-01-01

    Evolution can be harnessed to optimize synthetic biology designs. A prominent example is recombinant protein production-a dominating theme in biotechnology for more than three decades. Typically, a protein coding sequence (cds) is recombined with genetic elements, such as promoters, ribosome...... and allows expression levels in large clone libraries to be probed using a simple cell survival assay on the respective antibiotic. The power of the approach is demonstrated by substantially increasing production of two commercially interesting proteins, a Nanobody and an Affibody. The method is a simple......-level expression-an example of synthetic evolution. However, manual screening limits the ability to assay expression levels of all putative sequences in the libraries. Here we have solved this bottleneck by designing a collection of translational coupling devices based on a RNA secondary structure. Exchange...

  15. From Never Born Proteins to Minimal Living Cells: two projects in synthetic biology.

    Science.gov (United States)

    Luisi, Pier Luigi; Chiarabelli, Cristiano; Stano, Pasquale

    2006-12-01

    The Never Born Proteins (NBPs) and the Minimal Cell projects are two currently developed research lines belonging to the field of synthetic biology. The first deals with the investigation of structural and functional properties of de novo proteins with random sequences, selected and isolated using phage display methods. The minimal cell is the simplest cellular construct which displays living properties, such as self-maintenance, self-reproduction and evolvability. The semi-synthetic approach to minimal cells involves the use of extant genes and proteins in order to build a supramolecular construct based on lipid vesicles. Results and outlooks on these two research lines are shortly discussed, mainly focusing on their relevance to the origin of life studies.

  16. Production and characterization of polyclonal antibody against a synthetic peptide from β-actin protein

    Directory of Open Access Journals (Sweden)

    Nazila Amini

    2014-06-01

    Full Text Available Objective(s:Antibodies against actin, as one of the most widely studied structural and multifunctional housekeeping proteins in eukaryotic cells, are used as internal loading controls in western blot analyses. The aim of this study was to produce polyclonal antibody against a synthetic peptide derived from N-terminal region of β-actin protein to be used as a protein loading control in western blot and other assay systems. Materials and Methods: A synthetic peptide derived from β-actin protein was designed and conjugated to Keyhole limpet hemocyanin (KLH (and used to immunize a white New Zealand rabbit. The antibody was purified from serum by affinity chromatography column. The purity of the antibody was determined by SDS-PAGE and its ability to recognize the immunizing peptide was measured by ELISA. The reactivity of the antibody with β-actin protein in a panel of different cell lysates was then evaluated by western blot. In addition, the reactivity of the antibody with the corresponding protein was also evaluated by Immunocytochemistry and Immunohistochemistry in different samples. Results: The antibody could recognize the immunizing peptide in ELISA. It could also recognize            β-actin protein in western blot as well as in immunocytochemistry and immunohistochemistry. Conclusion: Our data suggest that this antibody may be used as an internal control in western blot analyses as well as in other immunological applications such as ELISA,immunocytochemistry and immunohistochemistry.

  17. Fast and easy protocol for the purification of recombinant S-layer protein for synthetic biology applications

    KAUST Repository

    Norville, Julie E.

    2011-06-17

    A goal of synthetic biology is to make biological systems easier to engineer. One of the aims is to design, with nanometer-scale precision, biomaterials with well-defined properties. The surface-layer protein SbpA forms 2D arrays naturally on the cell surface of Lysinibacillus sphaericus, but also as the purified protein in solution upon the addition of divalent cations. The high propensity of SbpA to form crystalline arrays, which can be simply controlled by divalent cations, and the possibility to genetically alter the protein, make SbpA an attractive molecule for synthetic biology. To be a useful tool, however, it is important that a simple protocol can be used to produce recombinant wild-type and modified SbpA in large quantities and in a biologically active form. The present study addresses this requirement by introducing a mild and non-denaturing purification protocol to produce milligram quantities of recombinant, active SbpA.

  18. Glutarimides: Biological activity, general synthetic methods and physicochemical properties

    Directory of Open Access Journals (Sweden)

    Popović-Đorđević Jelena B.

    2015-01-01

    Full Text Available Glutarimides, 2,6-dioxopiperidines are compounds that rarely occur in natural sources, but so far isolated ones exert widespread pharmacological activities, which makes them valuable as potential pharmacotherapeutics. Glutarimides act as androgen receptor antagonists, anti-inflammatory, anxiolytics, antibacterials, and tumor suppressing agents. Some synthetic glutarimide derivatives are already in use as immunosuppressive and sedative (e.g., thalidomide or anxiolytics (buspirone drugs. The wide applicability of this class of compounds, justify the interest of scientists to explore new pathways for its syntheses. General methods for synthesis of six-membered imide ring, are presented in this paper. These methods include: a reaction of dicarboxylic acids with ammonia or primary amine, b reactions of cyclization: amido-acids, diamides, dinitriles, nitrilo-acids, amido-nitriles, amido-esters, amidoacyl-chlorides or diacyl-chlorides, c adition of carbon-monoxide on a,b-unsaturated amides, d oxidation reactions, e Michael adition of active methylen compounds on methacrylamide or conjugated amides. Some of the described methods are used for closing glutarimide ring in syntheses of farmacological active compounds sesbanimide and aldose reductase inhibitors (ARI. Analyses of the geometry, as well as, the spectroscopic analyses (NMR and FT-IR of some glutarimides are presented because of their broad spectrum of pharmacological activity. To elucidate structures of glutarimides, geometrical parameters of newly synthesized tert-pentyl-1-benzyl-4-methyl-glutarimide-3-carboxylate (PBMG are analyzed and compared with the experimental data from X-ray analysis for glutarimide. Moreover, molecular electrostatic potential (MEP surface which is plotted over the optimized geometry to elucidate the reactivity of PBMG molecule is analyzed. The electronic properties of glutarimide derivatives are explained on the example of thalidomide. The Frontier Molecular Orbital

  19. Grafting synthetic transmembrane units to the engineered low-toxicity α-hemolysin to restore its hemolytic activity.

    Science.gov (United States)

    Ui, Mihoko; Harima, Kousuke; Takei, Toshiaki; Tsumoto, Kouhei; Tabata, Kazuhito V; Noji, Hiroyuki; Endo, Sumire; Akiyama, Kimio; Muraoka, Takahiro; Kinbara, Kazushi

    2014-12-01

    The chemical modification of proteins to provide desirable functions and/or structures broadens their possibilities for use in various applications. Usually, proteins can acquire new functions and characteristics, in addition to their original ones, via the introduction of synthetic functional moieties. Here, we adopted a more radical approach to protein modification, i.e., the replacement of a functional domain of proteins with alternative chemical compounds to build "cyborg proteins." As a proof of concept model, we chose staphylococcal α-hemolysin (Hla), which is a well-studied, pore-forming toxin. The hemolytic activity of Hla mutants was dramatically decreased by truncation of the stem domain, which forms a β-barrel pore in the membrane. However, the impaired hemolytic activity was significantly restored by attaching a pyrenyl-maleimide unit to the cysteine residue that was introduced in the remaining stem domain. In contrast, negatively charged fluorescein-maleimide completely abolished the remaining activity of the mutants.

  20. Active control of continuous air jet with bifurcated synthetic jets

    Directory of Open Access Journals (Sweden)

    Dančová Petra

    2017-01-01

    Full Text Available The synthetic jets (SJs have many significant applications and the number of applications is increasing all the time. In this research the main focus is on the primary flow control which can be used effectively for the heat transfer increasing. This paper deals with the experimental research of the effect of two SJs worked in the bifurcated mode used for control of an axisymmetric air jet. First, the control synthetic jets were measured alone. After an adjustment, the primary axisymmetric jet was added in to the system. For comparison, the primary flow without synthetic jets control was also measured. All experiments were performed using PIV method whereby the synchronization between synthetic jets and PIV system was necessary to do.

  1. De Novo Construction of Redox Active Proteins.

    Science.gov (United States)

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

    2016-01-01

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

  2. Antifouling Activity of Synthetic Alkylpyridinium Polymers Using the Barnacle Model

    Science.gov (United States)

    Piazza, Veronica; Dragić, Ivanka; Sepčić, Kristina; Faimali, Marco; Garaventa, Francesca; Turk, Tom; Berne, Sabina

    2014-01-01

    Polymeric alkylpyridinium salts (poly-APS) isolated from the Mediterranean marine sponge, Haliclona (Rhizoniera) sarai, effectively inhibit barnacle larva settlement and natural marine biofilm formation through a non-toxic and reversible mechanism. Potential use of poly-APS-like compounds as antifouling agents led to the chemical synthesis of monomeric and oligomeric 3-alkylpyridinium analogues. However, these are less efficient in settlement assays and have greater toxicity than the natural polymers. Recently, a new chemical synthesis method enabled the production of poly-APS analogues with antibacterial, antifungal and anti-acetylcholinesterase activities. The present study examines the antifouling properties and toxicity of six of these synthetic poly-APS using the barnacle (Amphibalanus amphitrite) as a model (cyprids and II stage nauplii larvae) in settlement, acute and sub-acute toxicity assays. Two compounds, APS8 and APS12-3, show antifouling effects very similar to natural poly-APS, with an anti-settlement effective concentration that inhibits 50% of the cyprid population settlement (EC50) after 24 h of 0.32 mg/L and 0.89 mg/L, respectively. The toxicity of APS8 is negligible, while APS12-3 is three-fold more toxic (24-h LC50: nauplii, 11.60 mg/L; cyprids, 61.13 mg/L) than natural poly-APS. This toxicity of APS12-3 towards nauplii is, however, 60-fold and 1200-fold lower than that of the common co-biocides, Zn- and Cu-pyrithione, respectively. Additionally, exposure to APS12-3 for 24 and 48 h inhibits the naupliar swimming ability with respective IC50 of 4.83 and 1.86 mg/L. PMID:24699112

  3. Antifouling Activity of Synthetic Alkylpyridinium Polymers Using the Barnacle Model

    Directory of Open Access Journals (Sweden)

    Veronica Piazza

    2014-04-01

    Full Text Available Polymeric alkylpyridinium salts (poly-APS isolated from the Mediterranean marine sponge, Haliclona (Rhizoniera sarai, effectively inhibit barnacle larva settlement and natural marine biofilm formation through a non-toxic and reversible mechanism. Potential use of poly-APS-like compounds as antifouling agents led to the chemical synthesis of monomeric and oligomeric 3-alkylpyridinium analogues. However, these are less efficient in settlement assays and have greater toxicity than the natural polymers. Recently, a new chemical synthesis method enabled the production of poly-APS analogues with antibacterial, antifungal and anti-acetylcholinesterase activities. The present study examines the antifouling properties and toxicity of six of these synthetic poly-APS using the barnacle (Amphibalanus amphitrite as a model (cyprids and II stage nauplii larvae in settlement, acute and sub-acute toxicity assays. Two compounds, APS8 and APS12-3, show antifouling effects very similar to natural poly-APS, with an anti-settlement effective concentration that inhibits 50% of the cyprid population settlement (EC50 after 24 h of 0.32 mg/L and 0.89 mg/L, respectively. The toxicity of APS8 is negligible, while APS12-3 is three-fold more toxic (24-h LC50: nauplii, 11.60 mg/L; cyprids, 61.13 mg/L than natural poly-APS. This toxicity of APS12-3 towards nauplii is, however, 60-fold and 1200-fold lower than that of the common co-biocides, Zn- and Cu-pyrithione, respectively. Additionally, exposure to APS12-3 for 24 and 48 h inhibits the naupliar swimming ability with respective IC50 of 4.83 and 1.86 mg/L.

  4. The Current Case of Quinolones: Synthetic Approaches and Antibacterial Activity.

    Science.gov (United States)

    Naeem, Abdul; Badshah, Syed Lal; Muska, Mairman; Ahmad, Nasir; Khan, Khalid

    2016-03-28

    Quinolones are broad-spectrum synthetic antibacterial drugs first obtained during the synthesis of chloroquine. Nalidixic acid, the prototype of quinolones, first became available for clinical consumption in 1962 and was used mainly for urinary tract infections caused by Escherichia coli and other pathogenic Gram-negative bacteria. Recently, significant work has been carried out to synthesize novel quinolone analogues with enhanced activity and potential usage for the treatment of different bacterial diseases. These novel analogues are made by substitution at different sites--the variation at the C-6 and C-8 positions gives more effective drugs. Substitution of a fluorine atom at the C-6 position produces fluroquinolones, which account for a large proportion of the quinolones in clinical use. Among others, substitution of piperazine or methylpiperazine, pyrrolidinyl and piperidinyl rings also yields effective analogues. A total of twenty six analogues are reported in this review. The targets of quinolones are two bacterial enzymes of the class II topoisomerase family, namely gyrase and topoisomerase IV. Quinolones increase the concentration of drug-enzyme-DNA cleavage complexes and convert them into cellular toxins; as a result they are bactericidal. High bioavailability, relative low toxicity and favorable pharmacokinetics have resulted in the clinical success of fluoroquinolones and quinolones. Due to these superior properties, quinolones have been extensively utilized and this increased usage has resulted in some quinolone-resistant bacterial strains. Bacteria become resistant to quinolones by three mechanisms: (1) mutation in the target site (gyrase and/or topoisomerase IV) of quinolones; (2) plasmid-mediated resistance; and (3) chromosome-mediated quinolone resistance. In plasmid-mediated resistance, the efflux of quinolones is increased along with a decrease in the interaction of the drug with gyrase (topoisomerase IV). In the case of chromosome

  5. The Current Case of Quinolones: Synthetic Approaches and Antibacterial Activity

    Directory of Open Access Journals (Sweden)

    Abdul Naeem

    2016-03-01

    Full Text Available Quinolones are broad-spectrum synthetic antibacterial drugs first obtained during the synthesis of chloroquine. Nalidixic acid, the prototype of quinolones, first became available for clinical consumption in 1962 and was used mainly for urinary tract infections caused by Escherichia coli and other pathogenic Gram-negative bacteria. Recently, significant work has been carried out to synthesize novel quinolone analogues with enhanced activity and potential usage for the treatment of different bacterial diseases. These novel analogues are made by substitution at different sites—the variation at the C-6 and C-8 positions gives more effective drugs. Substitution of a fluorine atom at the C-6 position produces fluroquinolones, which account for a large proportion of the quinolones in clinical use. Among others, substitution of piperazine or methylpiperazine, pyrrolidinyl and piperidinyl rings also yields effective analogues. A total of twenty six analogues are reported in this review. The targets of quinolones are two bacterial enzymes of the class II topoisomerase family, namely gyrase and topoisomerase IV. Quinolones increase the concentration of drug-enzyme-DNA cleavage complexes and convert them into cellular toxins; as a result they are bactericidal. High bioavailability, relative low toxicity and favorable pharmacokinetics have resulted in the clinical success of fluoroquinolones and quinolones. Due to these superior properties, quinolones have been extensively utilized and this increased usage has resulted in some quinolone-resistant bacterial strains. Bacteria become resistant to quinolones by three mechanisms: (1 mutation in the target site (gyrase and/or topoisomerase IV of quinolones; (2 plasmid-mediated resistance; and (3 chromosome-mediated quinolone resistance. In plasmid-mediated resistance, the efflux of quinolones is increased along with a decrease in the interaction of the drug with gyrase (topoisomerase IV. In the case of

  6. Aging Is Accompanied by a Blunted Muscle Protein Synthetic Response to Protein Ingestion.

    Directory of Open Access Journals (Sweden)

    Benjamin Toby Wall

    Full Text Available Progressive loss of skeletal muscle mass with aging (sarcopenia forms a global health concern. It has been suggested that an impaired capacity to increase muscle protein synthesis rates in response to protein intake is a key contributor to sarcopenia. We assessed whether differences in post-absorptive and/or post-prandial muscle protein synthesis rates exist between large cohorts of healthy young and older men.We performed a cross-sectional, retrospective study comparing in vivo post-absorptive muscle protein synthesis rates determined with stable isotope methodologies between 34 healthy young (22±1 y and 72 older (75±1 y men, and post-prandial muscle protein synthesis rates between 35 healthy young (22±1 y and 40 older (74±1 y men.Post-absorptive muscle protein synthesis rates did not differ significantly between the young and older group. Post-prandial muscle protein synthesis rates were 16% lower in the older subjects when compared with the young. Muscle protein synthesis rates were >3 fold more responsive to dietary protein ingestion in the young. Irrespective of age, there was a strong negative correlation between post-absorptive muscle protein synthesis rates and the increase in muscle protein synthesis rate following protein ingestion.Aging is associated with the development of muscle anabolic inflexibility which represents a key physiological mechanism underpinning sarcopenia.

  7. Inheritance of Protein Patterns in a Synthetic Allopolyploid of Triticum Monococcum (AA) and Aegilops Ventricosa (DDMvMv)

    DEFF Research Database (Denmark)

    Siddiqui, K. A.; Ingversen, J.; Køie, B.

    1972-01-01

    Patterns of seed proteins in Triticum monococcum (2n = 2x = 14 = AA), Aegilops ventricosa (2n = 4x = 28 = DDMVMV), and their synthetic amphiploid were studied. The distribution of proteins in the individual Osborne protein fractions of the amphiploid was characterized by a 14 per cent increase...

  8. Characterization of a synthetic bacterial self-destruction device for programmed cell death and for recombinant proteins release

    Directory of Open Access Journals (Sweden)

    Lupotto Manuel

    2011-06-01

    Full Text Available Abstract Background Bacterial cell lysis is a widely studied mechanism that can be achieved through the intracellular expression of phage native lytic proteins. This mechanism can be exploited for programmed cell death and for gentle cell disruption to release recombinant proteins when in vivo secretion is not feasible. Several genetic parts for cell lysis have been developed and their quantitative characterization is an essential step to enable the engineering of synthetic lytic systems with predictable behavior. Results Here, a BioBrick™ lysis device present in the Registry of Standard Biological Parts has been quantitatively characterized. Its activity has been measured in E. coli by assembling the device under the control of a well characterized N-3-oxohexanoyl-L-homoserine lactone (HSL -inducible promoter and the transfer function, lysis dynamics, protein release capability and genotypic and phenotypic stability of the device have been evaluated. Finally, its modularity was tested by assembling the device to a different inducible promoter, which can be triggered by heat induction. Conclusions The studied device is suitable for recombinant protein release as 96% of the total amount of the intracellular proteins was successfully released into the medium. Furthermore, it has been shown that the device can be assembled to different input devices to trigger cell lysis in response to a user-defined signal. For this reason, this lysis device can be a useful tool for the rational design and construction of complex synthetic biological systems composed by biological parts with known and well characterized function. Conversely, the onset of mutants makes this device unsuitable for the programmed cell death of a bacterial population.

  9. Cleaved thioredoxin fusion protein enables the crystallization of poorly soluble ERα in complex with synthetic ligands

    International Nuclear Information System (INIS)

    Cura, Vincent; Gangloff, Monique; Eiler, Sylvia; Moras, Dino; Ruff, Marc

    2007-01-01

    A new crystallization strategy: the presence of cleaved thioredoxin fusion is critical for crystallization of the estrogen nuclear receptor ligand binding domain in complex with synthetic ligands. This novel technique should be regarded as an interesting alternative for crystallization of difficult proteins. The ligand-binding domain (LBD) of human oestrogen receptor α was produced in Escherichia coli as a cleavable thioredoxin (Trx) fusion in order to improve solubility. Crystallization trials with either cleaved and purified LBD or with the purified fusion protein both failed to produce crystals. In another attempt, Trx was not removed from the LBD after endoproteolytic cleavage and its presence promoted nucleation and subsequent crystal growth, which allowed the structure determination of two different LBD–ligand–coactivator peptide complexes at 2.3 Å resolution. This technique is likely to be applicable to other low-solubility proteins

  10. Curcumin and synthetic analogs induce reactive oxygen species and decreases specificity protein (Sp) transcription factors by targeting microRNAs

    International Nuclear Information System (INIS)

    Gandhy, Shruti U; Kim, KyoungHyun; Larsen, Lesley; Rosengren, Rhonda J; Safe, Stephen

    2012-01-01

    Curcumin inhibits growth of several cancer cell lines, and studies in this laboratory in bladder and pancreatic cancer cells show that curcumin downregulates specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 and pro-oncogenic Sp-regulated genes. In this study, we investigated the anticancer activity of curcumin and several synthetic cyclohexanone and piperidine analogs in colon cancer cells. The effects of curcumin and synthetic analogs on colon cancer cell proliferation and apoptosis were determined using standardized assays. The changes in Sp proteins and Sp-regulated gene products were analysed by western blots, and real time PCR was used to determine microRNA-27a (miR-27a), miR-20a, miR-17-5p and ZBTB10 and ZBTB4 mRNA expression. The IC 50 (half-maximal) values for growth inhibition (24 hr) of colon cancer cells by curcumin and synthetic cyclohexanone and piperidine analogs of curcumin varied from 10 μM for curcumin to 0.7 μM for the most active synthetic piperidine analog RL197, which was used along with curcumin as model agents in this study. Curcumin and RL197 inhibited RKO and SW480 colon cancer cell growth and induced apoptosis, and this was accompanied by downregulation of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 and Sp-regulated genes including the epidermal growth factor receptor (EGFR), hepatocyte growth factor receptor (c-MET), survivin, bcl-2, cyclin D1 and NFκB (p65 and p50). Curcumin and RL197 also induced reactive oxygen species (ROS), and cotreatment with the antioxidant glutathione significantly attenuated curcumin- and RL197-induced growth inhibition and downregulation of Sp1, Sp3, Sp4 and Sp-regulated genes. The mechanism of curcumin-/RL197-induced repression of Sp transcription factors was ROS-dependent and due to induction of the Sp repressors ZBTB10 and ZBTB4 and downregulation of microRNAs (miR)-27a, miR-20a and miR-17-5p that regulate these repressors. These results identify a new and highly potent

  11. Curcumin and synthetic analogs induce reactive oxygen species and decreases specificity protein (Sp transcription factors by targeting microRNAs

    Directory of Open Access Journals (Sweden)

    Gandhy Shruti U

    2012-11-01

    Full Text Available Abstract Background Curcumin inhibits growth of several cancer cell lines, and studies in this laboratory in bladder and pancreatic cancer cells show that curcumin downregulates specificity protein (Sp transcription factors Sp1, Sp3 and Sp4 and pro-oncogenic Sp-regulated genes. In this study, we investigated the anticancer activity of curcumin and several synthetic cyclohexanone and piperidine analogs in colon cancer cells. Methods The effects of curcumin and synthetic analogs on colon cancer cell proliferation and apoptosis were determined using standardized assays. The changes in Sp proteins and Sp-regulated gene products were analysed by western blots, and real time PCR was used to determine microRNA-27a (miR-27a, miR-20a, miR-17-5p and ZBTB10 and ZBTB4 mRNA expression. Results The IC50 (half-maximal values for growth inhibition (24 hr of colon cancer cells by curcumin and synthetic cyclohexanone and piperidine analogs of curcumin varied from 10 μM for curcumin to 0.7 μM for the most active synthetic piperidine analog RL197, which was used along with curcumin as model agents in this study. Curcumin and RL197 inhibited RKO and SW480 colon cancer cell growth and induced apoptosis, and this was accompanied by downregulation of specificity protein (Sp transcription factors Sp1, Sp3 and Sp4 and Sp-regulated genes including the epidermal growth factor receptor (EGFR, hepatocyte growth factor receptor (c-MET, survivin, bcl-2, cyclin D1 and NFκB (p65 and p50. Curcumin and RL197 also induced reactive oxygen species (ROS, and cotreatment with the antioxidant glutathione significantly attenuated curcumin- and RL197-induced growth inhibition and downregulation of Sp1, Sp3, Sp4 and Sp-regulated genes. The mechanism of curcumin-/RL197-induced repression of Sp transcription factors was ROS-dependent and due to induction of the Sp repressors ZBTB10 and ZBTB4 and downregulation of microRNAs (miR-27a, miR-20a and miR-17-5p that regulate these repressors

  12. Biological activity and toxicitiy of imported and synthetic metal ...

    African Journals Online (AJOL)

    ... of green alga Scendesmus obliquus. The toxicity of surfactants to Scendesmus obliquus are arranged in the order: imported fluid > Synthetic fluid > S+ D > I+A> S+B> I+ C> I+B > I+D > I+D >S+A > I+4. These results prove that, the toxicity of fluids depends on its chemical structure. Egyptian Journal of Biotechnology Vol.

  13. Generation and analyses of human synthetic antibody libraries and their application for protein microarrays.

    Science.gov (United States)

    Säll, Anna; Walle, Maria; Wingren, Christer; Müller, Susanne; Nyman, Tomas; Vala, Andrea; Ohlin, Mats; Borrebaeck, Carl A K; Persson, Helena

    2016-10-01

    Antibody-based proteomics offers distinct advantages in the analysis of complex samples for discovery and validation of biomarkers associated with disease. However, its large-scale implementation requires tools and technologies that allow development of suitable antibody or antibody fragments in a high-throughput manner. To address this we designed and constructed two human synthetic antibody fragment (scFv) libraries denoted HelL-11 and HelL-13. By the use of phage display technology, in total 466 unique scFv antibodies specific for 114 different antigens were generated. The specificities of these antibodies were analyzed in a variety of immunochemical assays and a subset was further evaluated for functionality in protein microarray applications. This high-throughput approach demonstrates the ability to rapidly generate a wealth of reagents not only for proteome research, but potentially also for diagnostics and therapeutics. In addition, this work provides a great example on how a synthetic approach can be used to optimize library designs. By having precise control of the diversity introduced into the antigen-binding sites, synthetic libraries offer increased understanding of how different diversity contributes to antibody binding reactivity and stability, thereby providing the key to future library optimization. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  14. Visceral organ mass and hepatic protein synthetic capacity in fed and fasted rats

    International Nuclear Information System (INIS)

    Burrin, D.G.; Britton, R.A.; Ferrell, C.L.

    1986-01-01

    Forty-two male rats (avg wt. = 320 g) were used to assess the effect of severe nutrient restriction (72 h fast) on visceral organ mass and hepatic protein synthetic capacity as measured by in vitro incorporation of U- 14 -C-VALINE ( 14 C-VAL) into isolated hepatocytes. Organ weights expressed as a percent of empty body weight for fed vs. fasted rats were; liver (5.21 +/- .54 vs 3.82 +/- .46), kidney (.87 +/- 0.6 vs .89 +/- .05), stomach (.60 +/- .06 vs .61 +/- .06), intestines (3.70 +/- .44 vs 3.41 +/- .37). No differences were observed in in vitro oxygen consumption (15.7 +/- 3.1 vs 16.1 +/- 3.3, umole min -1 g -1 dry tissue) or 14 -C VAL incorporation (4.93 +/- 1.28 vs 4.31 +/- 1.48, dpm min -1 mg -1 dry tissue) for hepatocytes from fed vs. fasted rats. Analysis of perfused liver tissue indicated fed rats had higher protein (152.1 +/- 16.3 vs 136.6 +/- 29.6, mg/g tissue) and RNA (8.81 +/- 1.66 vs 5.97 +/- 1.87, mg/g tissue) with lower DNA (2.19 +/- .31 vs 3.19 +/- .54, mg/g tissue) compared to fasted rats. Protein-nucleic acid ratios suggest liver tissue from fed rats had a greater capacity for protein synthesis compared to fasted rats, however, this was not evident from in vitro hepatocyte 14 -C VAL incorporation estimates. These data indicate that severe nutrient restriction (72 h fast) affects visceral organ mass largely by reduced liver and gut size as well as decreased hepatic protein synthetic capacity

  15. Conversion of Synthetic Aβ to In Vivo Active Seeds and Amyloid Plaque Formation in a Hippocampal Slice Culture Model.

    Science.gov (United States)

    Novotny, Renata; Langer, Franziska; Mahler, Jasmin; Skodras, Angelos; Vlachos, Andreas; Wegenast-Braun, Bettina M; Kaeser, Stephan A; Neher, Jonas J; Eisele, Yvonne S; Pietrowski, Marie J; Nilsson, K Peter R; Deller, Thomas; Staufenbiel, Matthias; Heimrich, Bernd; Jucker, Mathias

    2016-05-04

    The aggregation of amyloid-β peptide (Aβ) in brain is an early event and hallmark of Alzheimer's disease (AD). We combined the advantages of in vitro and in vivo approaches to study cerebral β-amyloidosis by establishing a long-term hippocampal slice culture (HSC) model. While no Aβ deposition was noted in untreated HSCs of postnatal Aβ precursor protein transgenic (APP tg) mice, Aβ deposition emerged in HSCs when cultures were treated once with brain extract from aged APP tg mice and the culture medium was continuously supplemented with synthetic Aβ. Seeded Aβ deposition was also observed under the same conditions in HSCs derived from wild-type or App-null mice but in no comparable way when HSCs were fixed before cultivation. Both the nature of the brain extract and the synthetic Aβ species determined the conformational characteristics of HSC Aβ deposition. HSC Aβ deposits induced a microglia response, spine loss, and neuritic dystrophy but no obvious neuron loss. Remarkably, in contrast to in vitro aggregated synthetic Aβ, homogenates of Aβ deposits containing HSCs induced cerebral β-amyloidosis upon intracerebral inoculation into young APP tg mice. Our results demonstrate that a living cellular environment promotes the seeded conversion of synthetic Aβ into a potent in vivo seeding-active form. In this study, we report the seeded induction of Aβ aggregation and deposition in long-term hippocampal slice cultures. Remarkably, we find that the biological activities of the largely synthetic Aβ aggregates in the culture are very similar to those observed in vivo This observation is the first to show that potent in vivo seeding-active Aβ aggregates can be obtained by seeded conversion of synthetic Aβ in a living (wild-type) cellular environment. Copyright © 2016 the authors 0270-6474/16/365084-10$15.00/0.

  16. Structural analysis of protein-ligand interactions: the binding of endogenous compounds and of synthetic drugs.

    Science.gov (United States)

    Gallina, Anna M; Bork, Peer; Bordo, Domenico

    2014-02-01

    The large number of macromolecular structures deposited with the Protein Data Bank (PDB) describing complexes between proteins and either physiological compounds or synthetic drugs made it possible a systematic analysis of the interactions occurring between proteins and their ligands. In this work, the binding pockets of about 4000 PDB protein-ligand complexes were investigated and amino acid and interaction types were analyzed. The residues observed with lowest frequency in protein sequences, Trp, His, Met, Tyr, and Phe, turned out to be the most abundant in binding pockets. Significant differences between drug-like and physiological compounds were found. On average, physiological compounds establish with respect to drugs about twice as many hydrogen bonds with protein atoms, whereas drugs rely more on hydrophobic interactions to establish target selectivity. The large number of PDB structures describing homologous proteins in complex with the same ligand made it possible to analyze the conservation of binding pocket residues among homologous protein structures bound to the same ligand, showing that Gly, Glu, Arg, Asp, His, and Thr are more conserved than other amino acids. Also in the cases in which the same ligand is bound to unrelated proteins, the binding pockets showed significant conservation in the residue types. In this case, the probability of co-occurrence of the same amino acid type in the binding pockets could be up to thirteen times higher than that expected on a random basis. The trends identified in this study may provide an useful guideline in the process of drug design and lead optimization. Copyright © 2014 John Wiley & Sons, Ltd.

  17. Rational Design of Adjuvant for Skin Delivery: Conjugation of Synthetic β-Glucan Dectin-1 Agonist to Protein Antigen.

    Science.gov (United States)

    Donadei, Agnese; Gallorini, Simona; Berti, Francesco; O'Hagan, Derek T; Adamo, Roberto; Baudner, Barbara C

    2015-05-04

    The potential benefits of skin delivery of vaccines derive from the presence of a densely connected network of antigen presenting cells in the skin layer, most significantly represented by Langerhans cells and dermal dendritic cells. Targeting these cells by adjuvant conjugated to an antigen should result in enhanced immunogenicity of a vaccine. Since one of the most widely used adjuvants is an insoluble salt of aluminum (aluminum hydroxide) that cannot be used for skin delivery due to reactogenicity, we focused our attention on agonists of receptors present on skin dendritic cells, including the Dectin-1 receptor. β-(1-3)-glucans, which are the most abundant components of the fungal surface, are known to activate the innate immune response by interaction with the C-type lectin-like Dectin-1 receptor. In this work we identified by rational design a well-defined synthetic β-(1-3)-glucan hexasaccharide as a Dectin-1 agonist and chemically conjugated it to the genetically detoxified diphtheria toxin (CRM197) protein antigen, as a means to increase the binding to Dectin-1 receptor and to target to skin dendritic cells. We demonstrated that the in vitro activation of the receptor was significantly impacted by the presentation of the glucan on the protein carrier. In vivo results in mice showed that the conjugation of the synthetic β-(1-3)-glucan when delivered intradermally resulted in higher antibody titers in comparison to intramuscular (i.m.) immunization and was not different from subcutaneous (s.c.) delivery. These findings suggest that weak receptor binders can be turned into more potent agonists by the multivalent presentation of many ligands covalently conjugated to the protein core. Moreover, this approach is particularly valuable to increase the immunogenicity of antigens administered via skin delivery.

  18. Direct interaction of natural and synthetic catechins with signal transducer activator of transcription 1 affects both its phosphorylation and activity

    KAUST Repository

    Menegazzi, Marta; Mariotto, Sofia; Dal Bosco, Martina; Darra, Elena; Vaiana, Nadia; Shoji, Kazuo; Safwat, Abdel Azeim; Marechal, Jean Didier; Perahia, David; Suzuki, Hisanori; Romeo, Sergio

    2013-01-01

    Our previous studies showed that (-)-epigallocatechin-3-gallate (EGCG) inhibits signal transducer activator of transcription 1 (STAT1) activation. Since EGCG may be a promising lead compound for new anti-STAT1 drug design, 15 synthetic catechins

  19. A systematic investigation of production of synthetic prions from recombinant prion protein.

    Science.gov (United States)

    Schmidt, Christian; Fizet, Jeremie; Properzi, Francesca; Batchelor, Mark; Sandberg, Malin K; Edgeworth, Julie A; Afran, Louise; Ho, Sammy; Badhan, Anjna; Klier, Steffi; Linehan, Jacqueline M; Brandner, Sebastian; Hosszu, Laszlo L P; Tattum, M Howard; Jat, Parmjit; Clarke, Anthony R; Klöhn, Peter C; Wadsworth, Jonathan D F; Jackson, Graham S; Collinge, John

    2015-12-01

    According to the protein-only hypothesis, infectious mammalian prions, which exist as distinct strains with discrete biological properties, consist of multichain assemblies of misfolded cellular prion protein (PrP). A critical test would be to produce prion strains synthetically from defined components. Crucially, high-titre 'synthetic' prions could then be used to determine the structural basis of infectivity and strain diversity at the atomic level. While there have been multiple reports of production of prions from bacterially expressed recombinant PrP using various methods, systematic production of high-titre material in a form suitable for structural analysis remains a key goal. Here, we report a novel high-throughput strategy for exploring a matrix of conditions, additives and potential cofactors that might generate high-titre prions from recombinant mouse PrP, with screening for infectivity using a sensitive automated cell-based bioassay. Overall, approximately 20,000 unique conditions were examined. While some resulted in apparently infected cell cultures, this was transient and not reproducible. We also adapted published methods that reported production of synthetic prions from recombinant hamster PrP, but again did not find evidence of significant infectious titre when using recombinant mouse PrP as substrate. Collectively, our findings are consistent with the formation of prion infectivity from recombinant mouse PrP being a rare stochastic event and we conclude that systematic generation of prions from recombinant PrP may only become possible once the detailed structure of authentic ex vivo prions is solved. © 2015 The Authors.

  20. Protein covalent modification by biologically active quinones

    Directory of Open Access Journals (Sweden)

    MIROSLAV J. GASIC

    2004-11-01

    Full Text Available The avarone/avarol quinone/hydroquinone couple shows considerable antitumor activity. In this work, covalent modification of b-lactoglobulin by avarone and its derivatives as well as by the synthetic steroidal quinone 2,5(10-estradiene-1,4,17-trione and its derivatives were studied. The techniques for studying chemical modification of b-lactoglobulin by quinones were: UV/Vis spectrophotometry, SDS PAGE and isoelectrofocusing. SDS PAGE results suggest that polymerization of the protein occurs. It could be seen that the protein of 18 kD gives the bands of 20 kD, 36 kD, 40 kD, 45 kD, 64 kD and 128 kD depending on modification agent. The shift of the pI of the protein (5.4 upon modification toward lower values (from pI 5.0 to 5.3 indicated that lysine amino groups are the principal site of the reaction of b-lactoglobulin with the quinones.

  1. Strong supramolecular control over protein self-assembly using a polyamine decorated β-cyclodextrin as synthetic recognition element

    NARCIS (Netherlands)

    Uhlenheuer, D.A.; Milroy, L.G.; Neirynck, P.; Brunsveld, L.

    2011-01-01

    The supramolecular host molecule heptakis-[6-deoxy-6-(2-aminoethylsulfanyl)]-ß-cyclodextrin provides strong control over protein self-assembly in synthetic supramolecular protein constructs. Mono-functionalization of this modified ß-cyclodextrin with a cysteine residue allows for site-selective

  2. Chemical protein synthesis: Inventing synthetic methods to decipher how proteins work.

    Science.gov (United States)

    Kent, Stephen

    2017-09-15

    Total chemical synthesis of proteins has been rendered practical by the chemical ligation principle: chemoselective condensation of unprotected peptide segments equipped with unique, mutually reactive functional groups, enabled by formation of a non-native replacement for the peptide bond. Ligation chemistries are briefly described, including native chemical ligation - thioester-mediated, amide-forming reaction at Xaa-Cys sites - and its extensions. Case studies from the author's own works are used to illustrate the utility and applications of chemical protein synthesis. Selected recent developments in the field are briefly discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Labelled Thioamino Acids to Indicate the Synthetic Activity of the Rumen Bacteria in In-Vitro Experiments

    Energy Technology Data Exchange (ETDEWEB)

    Panic, B.; Jovanovic, M.; Cuperlovic, M.; Djordjevic, D. [Institute for the Application of Nuclear Energy in Agriculture, Veterinary Medicine and Forestry, Belgrade, Yugoslavia (Serbia)

    1968-07-01

    The synthetic activity of rumen bacteria has been studied in vitro through the investigation of cystine and methionine concentration and their specific activity. {sup 35}S-sulphate has been used as a radioactive tracer. Two diets, different in the level of nutrients - energy and protein - were added to the artificial tumen. The incubation with bacteria from the rumen content of the cows, fed for four weeks with the same diet, lasted 19 h. The diet with the higher level of protein and energy increased the cystine content (per 100 mg of N{sub 2}) by 23.3% and the methionine content by 39.4%. The concentration of radioactive cystine was increased at the same percentage rate by 25%, but radioactive methionine was much lower and increased only 6.4%. The difference between the specific activities of the investigated amino acids can be explained by the different catabolism rate and utilization of dietary cystine, and methionine by the rumen bacterial flora. Since the dietary methionine is catabolized slowly, it can, especially by the use of the diets with a high protein level, significantly decrease the specific activity of the radioactive methionine synthesized by rumen bacteria. Therefore, the incorporation of {sup 35}S into the cystine represents a more reliable indicator of the synthetic activity of the rumen bacteria. (author)

  4. Plant Proteins and Synthetic Amino Acids in the Nutrition of Non-Ruminants

    Energy Technology Data Exchange (ETDEWEB)

    Lewis, D. [Department of Applied Biochemistry and Nutrition, University Of Nottingham, Nottingham (United Kingdom)

    1968-07-01

    It is to be emphasized that in formulating diets for farm animals other than ruminants it is important to meet the requirements for individual essential amino acids and not merely to give regard to over-ail protein quality. The protein component serves to meet the needs for essential amino acids and also supplies material to synthesize those amino acids that are individually dispensable. In arranging for efficient formulation it is important to have available amino acid requirement standards to meet a particular production objective and data on the quantity of amino acids supplied by the various ingredients available. In considering the amino acid content of ingredients it is important to pay due regard to the problems of availability. Efforts to define amino acid requirements for the pig and chick have given somewhat variable results: it is possible to account for some of this variability. It is recognized that under certain circumstances non-amino nitrogen can be utilized by such species as the chick and the pig. The mechanisms involved are briefly considered. Some experimental work has shown that non-amino nitrogen can support growth, but it is difficult to establish a situation in which the non-essential amino acid levels are sufficiently low to take advantage of this fact. Extensive use of synthetic essential amino acids could change this situation. The case for the use of synthetic amino acids in the diets of farm animals is essentially an economic one. It is no longer necessary to demonstrate that free dietary amino acids can meet the needs of the animal. The only question is whether the needs of the animal are more effectively met by the addition of amino acids or more intact protein. The place of alternative protein sources to such attractive commodities as fish meal or soyabean meal must be considered in terms of amino acid supply. Whilst synthetic methionine and lysine are available there is a developing case for the use of such products as sunflower

  5. Immunochemical characterization of rhesus proteins with antibodies raised against synthetic peptides.

    Science.gov (United States)

    Hermand, P; Mouro, I; Huet, M; Bloy, C; Suyama, K; Goldstein, J; Cartron, J P; Bailly, P

    1993-07-15

    Rabbit polyclonal antibodies were raised against synthetic peptides corresponding to hydrophilic regions of the human Rhesus (Rh) IX cDNA-encoded polypeptide predicted to be extracellularly or intracellularly exposed in the topologic model of the Rh blood group protein. Four antibodies encompassing residues 33-45 (MPC1), 224-233 (MPC4), 390-404 (MPC6), and 408-416 (MPC8) were characterized and compared with a polyclonal anti-Rh protein obtained by immunization with purified Rh proteins. All antibodies had specificity for authentic Rh polypeptides and reacted on Western blot with Rh proteins immunoprecipitated with human monoclonal anti-RhD, -c, and -E. MPC1, but not the other antibodies, agglutinated all human erythrocytes except Rhnull and Rhmod cells, which either lack totally or are severely deficient in Rh proteins, respectively. Immunoblotting analysis with membrane proteins from common and rare variants showed that MPC1 and MPC8 reacted in Western blot with 32-Kd Rh polypeptides from all common red blood cells except those from Rhnull and Rhmod, indicating that peptide regions 33-45 and 408-416 may be common to several if not all Rh proteins, whatever the Rh blood group specificity. MPC4 reacted only with membrane preparations from cells carrying the E antigen, whereas MPC6 recognized preferentially the Rh proteins from E and Ee preparations, suggesting that the protein encoded by the RhIXb cDNA carries the E and/or e antigen(s). Immunoadsorption experiments using inside-out or right-side-out sealed vesicules from DccEE red blood cells as competing antigen showed that the MPC6 and MPC8 antibodies bound only to the cytoplasmic side of the erythrocyte membrane, thus providing evidence for the intracellular orientation of the C-terminal 27 residues of the Rh polypeptides. Attempts to transiently or stably express the Rh polypeptides. Attempts to transiently or stably express the Rh cDNA in eukaryotic cells were largely unsuccessful, suggesting that Rh antigen

  6. Antileishmanial activities of dihydrochalcones from piper elongatum and synthetic related compounds. Structural requirements for activity.

    Science.gov (United States)

    Hermoso, Alicia; Jiménez, Ignacio A; Mamani, Zulma A; Bazzocchi, Isabel L; Piñero, José E; Ravelo, Angel G; Valladares, Basilio

    2003-09-01

    Two dihydrochalcones (1 and 2) were isolated from Piper elongatum Vahl by activity-guided fractionation against extracellular promastigotes of Leishmania braziliensis in vitro. Their structures were elucidated by spectral analysis, including homonuclear and heteronuclear correlation NMR experiments. Derivatives 3-7 and 20 synthetic related compounds (8-27) were also assayed to establish the structural requirements for antileishmanial activity. Compounds 1-11 that proved to be more active that ketoconazol, used as positive control, were further assayed against promastigotes of Leishmania tropica and Leishmania infantum. Compounds 7 and 11, with a C(6)-C(3)-C(6) system, proved to be the most promising compounds, with IC(50) values of 2.98 and 3.65 microg/mL, respectively, and exhibited no toxic effect on macrophages (around 90% viability). Correlation between the molecular structures and antileishmanial activity is discussed in detail.

  7. Synthetic Training Data Generation for Activity Monitoring and Behavior Analysis

    Science.gov (United States)

    Monekosso, Dorothy; Remagnino, Paolo

    This paper describes a data generator that produces synthetic data to simulate observations from an array of environment monitoring sensors. The overall goal of our work is to monitor the well-being of one occupant in a home. Sensors are embedded in a smart home to unobtrusively record environmental parameters. Based on the sensor observations, behavior analysis and modeling are performed. However behavior analysis and modeling require large data sets to be collected over long periods of time to achieve the level of accuracy expected. A data generator - was developed based on initial data i.e. data collected over periods lasting weeks to facilitate concurrent data collection and development of algorithms. The data generator is based on statistical inference techniques. Variation is introduced into the data using perturbation models.

  8. Glycosylation of Recombinant Antigenic Proteins from Mycobacterium tuberculosis: In Silico Prediction of Protein Epitopes and Ex Vivo Biological Evaluation of New Semi-Synthetic Glycoconjugates.

    Science.gov (United States)

    Bavaro, Teodora; Tengattini, Sara; Piubelli, Luciano; Mangione, Francesca; Bernardini, Roberta; Monzillo, Vincenzina; Calarota, Sandra; Marone, Piero; Amicosante, Massimo; Pollegioni, Loredano; Temporini, Caterina; Terreni, Marco

    2017-06-29

    Tuberculosis is still one of the most deadly infectious diseases worldwide, and the use of conjugated antigens, obtained by combining antigenic oligosaccharides, such as the lipoarabinomannane (LAM), with antigenic proteins from Mycobacterium tuberculosis (MTB), has been proposed as a new strategy for developing efficient vaccines. In this work, we investigated the effect of the chemical glycosylation on two recombinant MTB proteins produced in E. coli with an additional seven-amino acid tag (recombinant Ag85B and TB10.4). Different semi-synthetic glycoconjugated derivatives were prepared, starting from mannose and two disaccharide analogs. The glycans were activated at the anomeric position with a thiocyanomethyl group, as required for protein glycosylation by selective reaction with lysines. The glycosylation sites and the ex vivo evaluation of the immunogenic activity of the different neo- glycoproteins were investigated. Glycosylation does not modify the immunological activity of the TB10.4 protein. Similarly, Ag85B maintains its B-cell activity after glycosylation while showing a significant reduction in the T-cell response. The results were correlated with the putative B- and T-cell epitopes, predicted using a combination of in silico systems. In the recombinant TB10.4, the unique lysine is not included in any T-cell epitope. Lys30 of Ag85B, identified as the main glycosylation site, proved to be the most important site involved in the formation of T-cell epitopes, reasonably explaining why its glycosylation strongly influenced the T-cell activity. Furthermore, additional lysines included in different epitopes (Lys103, -123 and -282) are also glycosylated. In contrast, B-cell epitopic lysines of Ag85B were found to be poorly glycosylated and, thus, the antibody interaction of Ag85B was only marginally affected after coupling with mono- or disaccharides.

  9. In situ detection of a heat-shock regulatory element binding protein using a soluble short synthetic enhancer sequence

    Energy Technology Data Exchange (ETDEWEB)

    Harel-Bellan, A; Brini, A T; Farrar, W L [National Cancer Institute, Frederick, MD (USA); Ferris, D K [Program Resources, Inc., Frederick, MD (USA); Robin, P [Institut Gustave Roussy, Villejuif (France)

    1989-06-12

    In various studies, enhancer binding proteins have been successfully absorbed out by competing sequences inserted into plasmids, resulting in the inhibition of the plasmid expression. Theoretically, such a result could be achieved using synthetic enhancer sequences not inserted into plasmids. In this study, a double stranded DNA sequence corresponding to the human heat shock regulatory element was chemically synthesized. By in vitro retardation assays, the synthetic sequence was shown to bind specifically a protein in extracts from the human T cell line Jurkat. When the synthetic enhancer was electroporated into Jurkat cells, not only the enhancer was shown to remain undegraded into the cells for up to 2 days, but also its was shown to bind intracellularly a protein. The binding was specific and was modulated upon heat shock. Furthermore, the binding protein was shown to be of the expected molecular weight by UV crosslinking. However, when the synthetic enhancer element was co-electroporated with an HSP 70-CAT reporter construct, the expression of the reporter plasmid was consistently enhanced in the presence of the exogenous synthetic enhancer.

  10. A new strategy to deliver synthetic protein drugs: self-reproducible biologics using minicircles.

    Science.gov (United States)

    Yi, Hyoju; Kim, Youngkyun; Kim, Juryun; Jung, Hyerin; Rim, Yeri Alice; Jung, Seung Min; Park, Sung-Hwan; Ju, Ji Hyeon

    2014-08-05

    Biologics are the most successful drugs used in anticytokine therapy. However, they remain partially unsuccessful because of the elevated cost of their synthesis and purification. Development of novel biologics has also been hampered by the high cost. Biologics are made of protein components; thus, theoretically, they can be produced in vivo. Here we tried to invent a novel strategy to allow the production of synthetic drugs in vivo by the host itself. The recombinant minicircles encoding etanercept or tocilizumab, which are synthesized currently by pharmaceutical companies, were injected intravenously into animal models. Self-reproduced etanercept and tocilizumab were detected in the serum of mice. Moreover, arthritis subsided in mice that were injected with minicircle vectors carrying biologics. Self-reproducible biologics need neither factory facilities for drug production nor clinical processes, such as frequent drug injection. Although this novel strategy is in its very early conceptual stage, it seems to represent a potential alternative method for the delivery of biologics.

  11. Recoding aminoacyl-tRNA synthetases for synthetic biology by rational protein-RNA engineering.

    Science.gov (United States)

    Hadd, Andrew; Perona, John J

    2014-12-19

    We have taken a rational approach to redesigning the amino acid binding and aminoacyl-tRNA pairing specificities of bacterial glutaminyl-tRNA synthetase. The four-stage engineering incorporates generalizable design principles and improves the pairing efficiency of noncognate glutamate with tRNA(Gln) by over 10(5)-fold compared to the wild-type enzyme. Better optimized designs of the protein-RNA complex include substantial reengineering of the globular core region of the tRNA, demonstrating a role for specific tRNA nucleotides in specifying the identity of the genetically encoded amino acid. Principles emerging from this engineering effort open new prospects for combining rational and genetic selection approaches to design novel aminoacyl-tRNA synthetases that ligate noncanonical amino acids onto tRNAs. This will facilitate reconstruction of the cellular translation apparatus for applications in synthetic biology.

  12. Effect of chemical composition on the flocculation dynamics of latex-based synthetic activated sludge

    International Nuclear Information System (INIS)

    Tan Phong Nguyen; Hankins, Nicholas P.; Hilal, Nidal

    2007-01-01

    This study investigates the effect of calcium, alginate, fibrous cellulose, and pH on the flocculation dynamics and final properties of synthetic activated sludges. A laboratory-scale batch reactor, fed with standard synthetic sludges was used. The effects of varying calcium concentration (5-25 mM), alginate concentration (25-125 mg/L), fibrous cellulose concentration (0.2-0.8 g/L) and pH (3-9) on the sludge characteristics were studied by varying one parameter whilst keeping the others constant. The results from experiments indicated that the calcium, alginate, fibrous cellulose, and pH had the critical effect on the aggregation rate, flocs size, and made the improvement of the final properties of sludge. Dynamic measurements have established the optimum conditions for floc formation and can accurately reflect the state of formation of the synthetic activated sludge flocs. These correlate well with measurements of settleability and turbidity of the synthetic activated sludge. The results of this study support the bonding theory and indicate that formation of cations-polymer complexes and polymer gelation are important means of flocculation. The development of synthetic activated sludges is suggested also to be a possible surrogate for studying the final properties of activated sludge

  13. Synthetic multielement standards used for instrumental neutron activation analysis as rock imitations

    International Nuclear Information System (INIS)

    Leypunskaya, D.I.; Drynkin, V.I.; Belenky, B.V.; Kolomijtsev, M.A.; Dundera, V.Yu.; Pachulia, N.V.

    1975-01-01

    Complex (multielemental) standards representing microelement composition of standard rocks such as trap ST-1 (USSR), gabbrodiorite SGD-1 (USSR), albitized granite SG-1 (USSR), basalt BCR-1 (USA) and granodiorite GSP-1 (USA) have been synthesized. It has been shown that the concentration of each microelement in the synthetic standards can be given with a high precision. Comparative investigation has been carried out of the synthetic imitations and the above natural standard rocks. It has been found that the result of the instrumental neutron activation analysis using the synthetic standards is as good as in the case when natural standard rocks are used. The results obtained have been also used for substantiation of the versatility of the method used for standard preparation, i.e. a generalization has been made of a possibility of using this method for the preparation of synthetic standards representing the microelement composition of any natural rocks with various compositions and concentrations of microelements. (T.G.)

  14. Evaluation of dermal wound healing activity of synthetic peptide SVVYGLR.

    Science.gov (United States)

    Uchinaka, Ayako; Kawaguchi, Naomasa; Ban, Tsuyoshi; Hamada, Yoshinosuke; Mori, Seiji; Maeno, Yoshitaka; Sawa, Yoshiki; Nagata, Kohzo; Yamamoto, Hirofumi

    2017-09-23

    SVVYGLR peptide (SV peptide) is a 7-amino-acid sequence with angiogenic properties that is derived from osteopontin in the extracellular matrix and promotes differentiation of fibroblasts to myofibroblast-like cells and the production of collagen type Ⅲ by cardiac fibroblasts. However, the effects of SV peptide on dermal cells and tissue are unknown. In this study, we evaluated the effects of this peptide in a rat model of dermal wound healing. The synthetic SV peptide was added to dermal fibroblasts or keratinocytes, and their cellular motility was evaluated. In an in vivo wound healing exeriment, male rats aged 8 weeks were randomly assigned to the SV peptide treatment, non-treated control, or phosphate-buffered saline (PBS) groups. Wound healing was assessed by its repair rate and histological features. Scratch assay and cell migration assays using the Chemotaxicell method showed that SV peptide significantly promoted the cell migration in both fibroblasts and keratinocytes. In contrast the proliferation potency of these cells was not affected by SV peptide. In the rat model, wound healing progressed faster in the SV peptide-treated group than in the control and PBS groups. The histopathological analyses showed that the SV peptide treatment stimulated the migration of fibroblasts to the wound area and increased the number of myofibroblasts. Immunohistochemical staining showed a marked increase of von Willebland factor-positive neomicrovessels in the SV peptide-treated group. In conclusion, SV peptide has a beneficial function to promote wound healing by stimulating granulation via stimulating angiogenesis, cell migration, and the myofibroblastic differentiation of fibroblasts. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Antimicrobial activity predictors benchmarking analysis using shuffled and designed synthetic peptides.

    Science.gov (United States)

    Porto, William F; Pires, Állan S; Franco, Octavio L

    2017-08-07

    The antimicrobial activity prediction tools aim to help the novel antimicrobial peptides (AMP) sequences discovery, utilizing machine learning methods. Such approaches have gained increasing importance in the generation of novel synthetic peptides by means of rational design techniques. This study focused on predictive ability of such approaches to determine the antimicrobial sequence activities, which were previously characterized at the protein level by in vitro studies. Using four web servers and one standalone software, we evaluated 78 sequences generated by the so-called linguistic model, being 40 designed and 38 shuffled sequences, with ∼60 and ∼25% of identity to AMPs, respectively. The ab initio molecular modelling of such sequences indicated that the structure does not affect the predictions, as both sets present similar structures. Overall, the systems failed on predicting shuffled versions of designed peptides, as they are identical in AMPs composition, which implies in accuracies below 30%. The prediction accuracy is negatively affected by the low specificity of all systems here evaluated, as they, on the other hand, reached 100% of sensitivity. Our results suggest that complementary approaches with high specificity, not necessarily high accuracy, should be developed to be used together with the current systems, overcoming their limitations. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Synthetic analogs of anoplin show improved antimicrobial activities

    DEFF Research Database (Denmark)

    Munk, Jens; Uggerhøj, Lars Erik; Poulsen, Tanja Juul

    2013-01-01

    We present the antimicrobial and hemolytic activities of the decapeptide anoplin and 19 analogs thereof tested against methicillin-resistant Staphylococcus aureus ATCC 33591 (MRSA), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), vancomycin-resistant Enterococcus faecium (ATCC...... that increasing the charge and/or hydrophobicity improves antimicrobial activity and increases hemolytic activity. For each strain tested, we identify at least six anoplin analogs with an improved therapeutic index compared with anoplin, the only exception being Enterococcus faecium, against which only few...

  17. Antifungal activity of natural and synthetic amides from Piper species

    Energy Technology Data Exchange (ETDEWEB)

    Marques, Joaquim V.; Oliveira, Alberto de; Kato, Massuo J., E-mail: majokato@iq.usp.b [Universidade de Sao Paulo (IQ/USP), SP (Brazil). Inst. de Quimica; Raggi, Ludmila; Young, Maria C. [Instituto de Botanica, Sao Paulo, SP (Brazil). Secao de Fisiologia e Bioquimica de Plantas

    2010-07-01

    The antifungal leaves extract from Piper scutifolium was submitted to bioactivity-guided chromatographic separation against Cladosporium cladosporioides and C. sphaerospermum yielding piperine, piperlonguminine and corcovadine as the active principles which displayed a detection limit of 1 {mu}g. Structure-activity relationships were investigated with the preparation of twelve analogs having differences in the number of unsaturations, aromatic ring substituents and in the amide moiety. Analogs having a single double-bond and no substituent in the aromatic ring displayed higher activity, while N,N,-diethyl analogs displayed higher dose-dependent activity. (author)

  18. Synthetic protein scaffolds based on peptide motifs and cognate adaptor domains for improving metabolic productivity

    Directory of Open Access Journals (Sweden)

    Anselm H.C. Horn

    2015-11-01

    Full Text Available The efficiency of many cellular processes relies on the defined interaction among different proteins within the same metabolic or signaling pathway. Consequently, a spatial colocalization of functionally interacting proteins has frequently emerged during evolution. This concept has been adapted within the synthetic biology community for the purpose of creating artificial scaffolds. A recent advancement of this concept is the use of peptide motifs and their cognate adaptor domains. SH2, SH3, GBD, and PDZ domains have been used most often in research studies to date. The approach has been successfully applied to the synthesis of a variety of target molecules including catechin, D-glucaric acid, H2, hydrochinone, resveratrol, butyrate, gamma-aminobutyric acid, and mevalonate. Increased production levels of up to 77-fold have been observed compared to non-scaffolded systems. A recent extension of this concept is the creation of a covalent linkage between peptide motifs and adaptor domains, which leads to a more stable association of the scaffolded systems and thus bears the potential to further enhance metabolic productivity.

  19. Natural Cinnamic Acids, Synthetic Derivatives and Hybrids with Antimicrobial Activity

    Directory of Open Access Journals (Sweden)

    Juan David Guzman

    2014-11-01

    Full Text Available Antimicrobial natural preparations involving cinnamon, storax and propolis have been long used topically for treating infections. Cinnamic acids and related molecules are partly responsible for the therapeutic effects observed in these preparations. Most of the cinnamic acids, their esters, amides, aldehydes and alcohols, show significant growth inhibition against one or several bacterial and fungal species. Of particular interest is the potent antitubercular activity observed for some of these cinnamic derivatives, which may be amenable as future drugs for treating tuberculosis. This review intends to summarize the literature data on the antimicrobial activity of the natural cinnamic acids and related derivatives. In addition, selected hybrids between cinnamic acids and biologically active scaffolds with antimicrobial activity were also included. A comprehensive literature search was performed collating the minimum inhibitory concentration (MIC of each cinnamic acid or derivative against the reported microorganisms. The MIC data allows the relative comparison between series of molecules and the derivation of structure-activity relationships.

  20. CELL-SURFACE DISPLAY OF SYNTHETIC PHYTOCHELATINS USING ICE NUCLEATION PROTEIN FOR ENHANCED HEAVY-METAL BIOACCUMULATION. (R827227)

    Science.gov (United States)

    Synthetic phytochelatins (ECs) composed of (Glu–Cys)nGly are protein analogs of phytochelatin that exhibit improved metal-binding capacity over metallothioneins (MTs). Expression of EC20 on the surface of E. coli using the Lpp-OmpA anchor resulted in i...

  1. Fast and easy protocol for the purification of recombinant S-layer protein for synthetic biology applications

    KAUST Repository

    Norville, Julie E.; Kelly, Deborah F.; Knight, Thomas F.; Belcher, Angela M.; Walz, Thomas

    2011-01-01

    A goal of synthetic biology is to make biological systems easier to engineer. One of the aims is to design, with nanometer-scale precision, biomaterials with well-defined properties. The surface-layer protein SbpA forms 2D arrays naturally

  2. Selection of glutamate-rich protein long synthetic peptides for vaccine development: antigenicity and relationship with clinical protection and immunogenicity

    DEFF Research Database (Denmark)

    Theisen, M; Dodoo, D; Toure-Balde, A

    2001-01-01

    Antibodies against three long synthetic peptides (LSPs) derived from the glutamate-rich protein (GLURP) of Plasmodium falciparum were analyzed in three cohorts from Liberia, Ghana, and Senegal. Two overlapping LSPs, LR67 and LR68, are derived from the relatively conserved N-terminal nonrepeat...

  3. Measurement of Muscle Protein Fractional Synthetic Rate by Capillary Gas Chromatography/Combustion Isotope Ratio Mass Spectrometry

    OpenAIRE

    Yarasheski, Kevin E.; Smith, Kenneth; Rennie, Michael J.; Bier, Dennis M.

    1992-01-01

    The measurement of skeletal muscle protein fractional synthetic rate using an infusion of (1-13C)leucine and measuring the isotopic abundance of the tracer in skeletal muscle protein by preparative gas chromatography (GC)/ninhydrin isotope ratio mass spectrometry (IRMS) is laborious and subject to errors owing to contamination by 12C. The purpose of this study was to compare muscle (13C)leucine enrichment measured with the conventional preparative GC/ninhydrin IRMS approach to a new, continuo...

  4. Synthetic Approaches and Biological Activities of 4-Hydroxycoumarin Derivatives

    Directory of Open Access Journals (Sweden)

    Oee-Sook Park

    2009-11-01

    Full Text Available The main purpose of this review is to summarize recent chemical syntheses and structural modifications of 4-hydroxycoumarin and its derivatives, of interest due to their characteristic conjugated molecular architecture and biological activities.

  5. A synthetic peptide with the putative iron binding motif of amyloid precursor protein (APP does not catalytically oxidize iron.

    Directory of Open Access Journals (Sweden)

    Kourosh Honarmand Ebrahimi

    Full Text Available The β-amyloid precursor protein (APP, which is a key player in Alzheimer's disease, was recently reported to possess an Fe(II binding site within its E2 domain which exhibits ferroxidase activity [Duce et al. 2010, Cell 142: 857]. The putative ligands of this site were compared to those in the ferroxidase site of ferritin. The activity was indirectly measured using transferrin, which scavenges the Fe(III product of the reaction. A 22-residue synthetic peptide, named FD1, with the putative ferroxidase site of APP, and the E2 domain of APP were each reported to exhibit 40% of the ferroxidase activity of APP and of ceruloplasmin. It was also claimed that the ferroxidase activity of APP is inhibited by Zn(II just as in ferritin. We measured the ferroxidase activity indirectly (i by the incorporation of the Fe(III product of the ferroxidase reaction into transferrin and directly (ii by monitoring consumption of the substrate molecular oxygen. The results with the FD1 peptide were compared to the established ferroxidase activities of human H-chain ferritin and of ceruloplasmin. For FD1 we observed no activity above the background of non-enzymatic Fe(II oxidation by molecular oxygen. Zn(II binds to transferrin and diminishes its Fe(III incorporation capacity and rate but it does not specifically bind to a putative ferroxidase site of FD1. Based on these results, and on comparison of the putative ligands of the ferroxidase site of APP with those of ferritin, we conclude that the previously reported results for ferroxidase activity of FD1 and - by implication - of APP should be re-evaluated.

  6. Low-Cytotoxic Synthetic Bromorutaecarpine Exhibits Anti-Inflammation and Activation of Transient Receptor Potential Vanilloid Type 1 Activities

    Directory of Open Access Journals (Sweden)

    Chi-Ming Lee

    2013-01-01

    Full Text Available Rutaecarpine (RUT, the major bioactive ingredient isolated from the Chinese herb Evodia rutaecarpa, possesses a wide spectrum of biological activities, including anti-inflammation and preventing cardiovascular diseases. However, its high cytotoxicity hampers pharmaceutical development. We designed and synthesized a derivative of RUT, bromo-dimethoxyrutaecarpine (Br-RUT, which showed no cytotoxicity at 20 μM. Br-RUT suppressed nitric oxide (NO production and tumor necrosis factor-α release in concentration-dependent (0~20 μM manners in lipopolysaccharide (LPS-treated RAW 264.7 macrophages; protein levels of inducible NO synthase (iNOS and cyclooxygenase-2 induced by LPS were downregulated. Br-RUT inhibited cell migration and invasion of ovarian carcinoma A2780 cells with 0~48 h of treatment. Furthermore, Br-RUT enhanced the expression of transient receptor potential vanilloid type 1 and activated endothelial NOS in human aortic endothelial cells. These results suggest that the synthetic Br-RUT possesses very low cytotoxicity but retains its activities against inflammation and vasodilation that could be beneficial for cardiovascular disease therapeutics.

  7. Synthetic histatin analogues with broad-spectrum antimicrobial activity.

    OpenAIRE

    Helmerhorst, E J; Van't Hof, W; Veerman, E C; Simoons-Smit, I; Nieuw Amerongen, A V

    1997-01-01

    Histatins are salivary histidine-rich cationic peptides, ranging from 7 to 38 amino acid residues in length, that exert a potent killing effect in vitro on Candida albicans. Starting from the C-terminal fungicidal domain of histatin 5 (residues 11-24, called dh-5) a number of substitution analogues were chemically synthesized to study the effect of amphipathicity of the peptide in helix conformation on candidacidal activity. Single substitutions in dh-5 at several positions did not have any e...

  8. On different activities of two synthetic coumarin derivatives

    Czech Academy of Sciences Publication Activity Database

    Drábiková, K.; Jančinová, V.; Perečko, T.; Nosáľ, R.; Ambrožová, Gabriela; Lojek, Antonín; Šmidrkal, J.; Harmatha, Juraj

    2010-01-01

    Roč. 3, č. 3 (2010), A41-A41 ISSN 1337-6853. [Toxcon 2010, Borderless Toxicology. 15th Interdisciplinary Toxicological Conference & Advanced Toxicological Course. 06.09.-10.09.2010, Stará Lesná - Hotel Academia] R&D Projects: GA ČR(CZ) GA203/07/1227 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50040507 Keywords : coumarins * activities * antiinflammatory * antioxidant Subject RIV: CC - Organic Chemistry

  9. Synthetic activity of rat blood lymphocytes under acute and continuous gamma-irradiation - fluorescent microspectral study

    International Nuclear Information System (INIS)

    Karnaukhova, N.A.; Sergiyevich, L.A.; Aksenova, G.Y.; Karnaukhov, V.N.

    1999-01-01

    The effects of different doses of acute and continuous gamma-irradiation on the synthetic activity of rat blood lymphocytes stained with acridine orange were studied by fluorescent microspectrometry. Male rats were exposed to acute gamma-irradiation with doses of 7.5, 4 and 3 Gy, or to continuous irradiation with dose rates of 14.4, 2.1, 1.1 and 0.43 cGy/day, respectively. The changes of the synthetic activity of blood lymphocytes occurred in three main stages after acute gamma-irradiation and in four stages under continuous irradiation. The stages reflect the processes of depression and activation of the immune system under irradiation. Essential differences between the acute and continuous effects were observed in the first stage. After acute gamma-irradiation, the synthetic activity decreased sharply, indicating the predominant contribution of the damaging effect of irradiation, whereas under continuous irradiation, as a result of the stimulatory effect of low-dose irradiation, the synthetic activity increased during the first stage. (orig.)

  10. Direct interaction of natural and synthetic catechins with signal transducer activator of transcription 1 affects both its phosphorylation and activity

    KAUST Repository

    Menegazzi, Marta

    2013-12-10

    Our previous studies showed that (-)-epigallocatechin-3-gallate (EGCG) inhibits signal transducer activator of transcription 1 (STAT1) activation. Since EGCG may be a promising lead compound for new anti-STAT1 drug design, 15 synthetic catechins, characterized by the (-)-gallocatechin-3-gallate stereochemistry, were studied in the human mammary MDA-MB-231 cell line to identify the minimal structural features that preserve the anti-STAT1 activity. We demonstrate that the presence of three hydroxyl groups of B ring and one hydroxyl group in D ring is essential to preserve their inhibitory action. Moreover, a possible molecular target of these compounds in the STAT1 pathway was investigated. Our results demonstrate a direct interaction between STAT1 protein and catechins displaying anti-STAT1 activity. In particular, surface plasmon resonance (SPR) analysis and molecular modeling indicate the presence of two putative binding sites (a and b) with different affinity. Based on docking data, site-directed mutagenesis was performed, and interaction of the most active catechins with STAT1 was studied with SPR to test whether Gln518 on site a and His568 on site b could be important for the catechin-STAT1 interaction. Data indicate that site b has higher affinity for catechins than site a as the highest affinity constant disappears in the H568ASTAT1 mutant. Furthermore, Janus kinase 2 (JAK2) kinase assay data suggest that the contemporary presence in vitro of STAT1 and catechins inhibits JAK2-elicited STAT1 phosphorylation. The very tight catechin-STAT1 interaction prevents STAT1 phosphorylation and represents a novel, specific and efficient molecular mechanism for the inhibition of STAT1 activation. © Copyright 2014 Federation of European Biochemical Societies. All rights reserved.

  11. Study of different coupling agents in the conjugation of a V3-based synthetic MAP to carrier proteins.

    Science.gov (United States)

    Cruz, L J; Iglesias, E; Aguilar, J C; Quintana, D; Garay, H E; Duarte, C; Reyes, O

    2001-09-01

    The conjugation of synthetic peptides to carrier proteins is a widely used method for immunological studies. Different coupling agents have been described to form the conjugate with carrier proteins. In this paper, we demonstrate that the antibody response toward V3-based synthetic MAPs derived from HIV-1, JY1 isolate, conjugated to two different carrier proteins using either m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) or beta-maleimidopropionic acid N-hydroxysuccinimide ester (MPS), or succinic anhydride (SA) show different behaviors. An excellent anti-JY1 response without a strong response to the coupling agent is observed in the case of succinic anhydride spacer. In contrast, MBS produces total abrogation of the antibody response with a high response toward the coupling agent.

  12. Engineering Synthetic Proteins to Generate Ca2+ Signals in Mammalian Cells.

    Science.gov (United States)

    Qudrat, Anam; Truong, Kevin

    2017-03-17

    The versatility of Ca 2+ signals allows it to regulate diverse cellular processes such as migration, apoptosis, motility and exocytosis. In some receptors (e.g., VEGFR2), Ca 2+ signals are generated upon binding their ligand(s) (e.g., VEGF-A). Here, we employed a design strategy to engineer proteins that generate a Ca 2+ signal upon binding various extracellular stimuli by creating fusions of protein domains that oligomerize to the transmembrane domain and the cytoplasmic tail of the VEGFR2. To test the strategy, we created chimeric proteins that generate Ca 2+ signals upon stimulation with various extracellular stimuli (e.g., rapamycin, EDTA or extracellular free Ca 2+ ). By coupling these chimeric proteins that generate Ca 2+ signals with proteins that respond to Ca 2+ signals, we rewired, for example, dynamic cellular blebbing to increases in extracellular free Ca 2+ . Thus, using this design strategy, it is possible to engineer proteins to generate a Ca 2+ signal to rewire a wide range of extracellular stimuli to a wide range of Ca 2+ -activated processes.

  13. Protein nanocoatings on synthetic polymeric nanofibrous membranes designed as carriers for skin cells.

    Science.gov (United States)

    Bacakova, Marketa; Pajorova, Julia; Stranska, Denisa; Hadraba, Daniel; Lopot, Frantisek; Riedel, Tomas; Brynda, Eduard; Zaloudkova, Margit; Bacakova, Lucie

    2017-01-01

    Protein-coated resorbable synthetic polymeric nanofibrous membranes are promising for the fabrication of advanced skin substitutes. We fabricated electrospun polylactic acid and poly(lactide- co -glycolic acid) nanofibrous membranes and coated them with fibrin or collagen I. Fibronectin was attached to a fibrin or collagen nanocoating, in order further to enhance the cell adhesion and spreading. Fibrin regularly formed a coating around individual nanofibers in the membranes, and also formed a thin noncontinuous nanofibrous mesh on top of the membranes. Collagen also coated most of the fibers of the membrane and randomly created a soft gel on the membrane surface. Fibronectin predominantly adsorbed onto a thin fibrin mesh or a collagen gel, and formed a thin nanofibrous structure. Fibrin nanocoating greatly improved the attachment, spreading, and proliferation of human dermal fibroblasts, whereas collagen nanocoating had a positive influence on the behavior of human HaCaT keratinocytes. In addition, fibrin stimulated the fibroblasts to synthesize fibronectin and to deposit it as an extracellular matrix. Fibrin coating also showed a tendency to improve the ultimate tensile strength of the nanofibrous membranes. Fibronectin attached to fibrin or to a collagen coating further enhanced the adhesion, spreading, and proliferation of both cell types.

  14. Structure and Biological Activity of Pathogen-like Synthetic Nanomedicines

    Science.gov (United States)

    Lőrincz, Orsolya; Tőke, Enikő R.; Somogyi, Eszter; Horkay, Ferenc; Chandran, Preethi; Douglas, Jack F.; Szebeni, János; Lisziewicz, Julianna

    2011-01-01

    Here we characterize the structure, stability and intracellular mode-of-action of DermaVir nanomedicine that is under clinical development for the treatment of HIV/AIDS. This nanomedicine is comprised of pathogen-like pDNA/PEIm nanoparticles (NPs) having the structure and function resembling spherical viruses that naturally evolved to deliver nucleic acids to the cells. Atomic force microscopy demonstrated spherical 100–200nm NPs with a smooth polymer surface protecting the pDNA in the core. Optical-absorption determined both the NP structural stability and biological activity relevant to their ability to escape from the endosome and release the pDNA at the nucleus. Salt, pH and temperature influence the nanomedicine shelf-life and intracellular stability. This approach facilitates the development of diverse polyplex nanomedicines where the delivered pDNA-expressed antigens induce immune responses to kill infected cells. PMID:21839051

  15. Fluorogen-activating proteins: beyond classical fluorescent proteins

    Directory of Open Access Journals (Sweden)

    Shengnan Xu

    2018-05-01

    Full Text Available Fluorescence imaging is a powerful technique for the real-time noninvasive monitoring of protein dynamics. Recently, fluorogen activating proteins (FAPs/fluorogen probes for protein imaging were developed. Unlike the traditional fluorescent proteins (FPs, FAPs do not fluoresce unless bound to their specific small-molecule fluorogens. When using FAPs/fluorogen probes, a washing step is not required for the removal of free probes from the cells, thus allowing rapid and specific detection of proteins in living cells with high signal-to-noise ratio. Furthermore, with different fluorogens, living cell multi-color proteins labeling system was developed. In this review, we describe about the discovery of FAPs, the design strategy of FAP fluorogens, the application of the FAP technology and the advances of FAP technology in protein labeling systems. KEY WORDS: Fluorogen activating proteins, Fluorogens, Genetically encoded sensors, Fluorescence imaging, Molecular imaging

  16. Controlling T-Cell Activation with Synthetic Dendritic Cells Using the Multivalency Effect

    NARCIS (Netherlands)

    Hammink, R.; Mandal, S.; Eggermont, L.J.; Nooteboom, M.; Willems, P.H.G.M.; Tel, J.; Rowan, A.E.; Figdor, C.G.; Blank, K.G.

    2017-01-01

    Artificial antigen-presenting cells (aAPCs) have recently gained a lot of attention. They efficiently activate T cells and serve as powerful replacements for dendritic cells in cancer immunotherapy. Focusing on a specific class of polymer-based aAPCs, so-called synthetic dendritic cells (sDCs), we

  17. Active aeroelastic control aspects of an aircraft wing by using synthetic jet actuators : Modeling, simulations, experiments

    NARCIS (Netherlands)

    Donnell, K.O.; Schober, S.; Stolk, M.; Marzocca, P.; De Breuker, R.; Abdalla, M.; Nicolini, E.; Gürdal, Z.

    2007-01-01

    This paper discusses modeling, simulations and experimental aspects of active aeroelastic control on aircraft wings by using Synthetic Jet Actuators (SJAs). SJAs, a particular class of zero-net mass-flux actuators, have shown very promising results in numerous aeronautical applications, such as

  18. Novel Protein-Protein Inhibitor Based Approach to Control Plant Ethylene Responses: Synthetic Peptides for Ripening Control

    Directory of Open Access Journals (Sweden)

    Mareike Kessenbrock

    2017-09-01

    Full Text Available Ethylene signaling is decisive for many plant developmental processes. Among these, control of senescence, abscission and fruit ripening are of fundamental relevance for global agriculture. Consequently, detailed knowledge of the signaling network along with the molecular processes of signal perception and transfer are expected to have high impact on future food production and agriculture. Recent advances in ethylene research have demonstrated that signaling of the plant hormone critically depends on the interaction of the ethylene receptor family with the NRAMP-like membrane protein ETHYLENE INSENSITIVE 2 (EIN2 at the ER membrane, phosphorylation-dependent proteolytic processing of ER-localized EIN2 and subsequent translocation of the cleaved EIN2 C-terminal polypeptide (EIN2-CEND to the nucleus. EIN2 nuclear transport, but also interaction with the receptors sensing the ethylene signal, both, depend on a nuclear localization signal (NLS located at the EIN2 C-terminus. Loss of the tight interaction between receptors and EIN2 affects ethylene signaling and impairs plant ethylene responses. Synthetic peptides derived from the NLS sequence interfere with the EIN2–receptor interaction and have utility in controlling plant ethylene responses such as ripening. Here, we report that a synthetic peptide (NOP-1 corresponding to the NLS motif of Arabidopsis EIN2 (aa 1262–1269 efficiently binds to tomato ethylene receptors LeETR4 and NR and delays ripening in the post-harvest phase when applied to the surface of sampled green fruits pre-harvest. In particular, degradation of chlorophylls was delayed by several days, as monitored by optical sensors and confirmed by analytical methods. Similarly, accumulation of β-carotene and lycopene in the fruit pulp after NOP-1 application was delayed, without having impact on the total pigment concentration in the completely ripe fruits. Likewise, the peptide had no negative effects on fruit quality. Our molecular

  19. Marine bioactive compounds: stereospecific anti-inflammatory activity of natural and synthetic cordiachromene A.

    Science.gov (United States)

    Benslimane, A F; Pouchus, Y F; Verbist, J F; Petit, J Y; Khettab, E N; Welin, L; Brion, J D

    1992-01-01

    A new synthesis is proposed for cordiachromene A (CCA), a bioactive component of the ascidian Aplidium antillense Gravier, using a method producing a racemic mixture. The anti-inflammatory activities of a natural extract and a chemically synthetic form of CCA were assessed in vivo by carrageenan-induced rat-paw edema. The activity of synthetic CCA was confirmed by a test on kaolin-induced granuloma in the rat. Strong activities were measured for both CCA, but comparison of results of the first test suggests that only the natural optically active isomer has an anti-inflammatory effect. CCA is similar to indomethacin in its effect on carrageenan-induced rat-paw edema and ten times as active as phenylbutazone.

  20. Membrane Recruitment of the Non-receptor Protein GIV/Girdin (Gα-interacting, Vesicle-associated Protein/Girdin) Is Sufficient for Activating Heterotrimeric G Protein Signaling.

    Science.gov (United States)

    Parag-Sharma, Kshitij; Leyme, Anthony; DiGiacomo, Vincent; Marivin, Arthur; Broselid, Stefan; Garcia-Marcos, Mikel

    2016-12-30

    GIV (aka Girdin) is a guanine nucleotide exchange factor that activates heterotrimeric G protein signaling downstream of RTKs and integrins, thereby serving as a platform for signaling cascade cross-talk. GIV is recruited to the cytoplasmic tail of receptors upon stimulation, but the mechanism of activation of its G protein regulatory function is not well understood. Here we used assays in humanized yeast models and G protein activity biosensors in mammalian cells to investigate the role of GIV subcellular compartmentalization in regulating its ability to promote G protein signaling. We found that in unstimulated cells GIV does not co-fractionate with its substrate G protein Gα i3 on cell membranes and that constitutive membrane anchoring of GIV in yeast cells or rapid membrane translocation in mammalian cells via chemically induced dimerization leads to robust G protein activation. We show that membrane recruitment of the GIV "Gα binding and activating" motif alone is sufficient for G protein activation and that it does not require phosphomodification. Furthermore, we engineered a synthetic protein to show that recruitment of the GIV "Gα binding and activating" motif to membranes via association with active RTKs, instead of via chemically induced dimerization, is also sufficient for G protein activation. These results reveal that recruitment of GIV to membranes in close proximity to its substrate G protein is a major mechanism responsible for the activation of its G protein regulatory function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Potent Skin Cancer Chemopreventing Activity of Some Novel Semi-synthetic Cembranoids from Marine Sources

    OpenAIRE

    Fahmy, Hesham; Zjawiony, Jordan K.; Konoshima, Takao; Tokuda, Harukuni; Khan, Shabana; Khalifa, Sherief

    2006-01-01

    Abstract: In the course of our continuing research in development and evaluation of novel skin cancer chemopreventive agents from marine sources, five semi-synthetic cembranoids derived from the marine natural product sarcophine, isolated from the soft coral Sarcophyton glaucum, were synthesized and shown to exhibit a remarkable chemopreventive activity in the in-vitro Epstein Barr Virus Early Antigen (EBV-EA) activation assay. These compounds were assayed in vivo using the two-stage carcinog...

  2. Reducing crude protein content with supplementation of synthetic lysine and threonine in barley - rapeseed meal - pea diets for growing pigs

    Directory of Open Access Journals (Sweden)

    Jarmo Valaja

    1993-03-01

    Full Text Available This study was conducted to determine the possibility to use synthetic amino acids to lower the nitrogen output from pig production. A performance experiment was carried out with 120triplet-fed growing pigs whose dietary crude protein was reduced from 179 g/feed unit (FU= 0.7 kg starch equivalent to 160, 140 and 122 g/FU, respectively. The diets were supplemented with synthetic lysine and threonine to keep the level of these amino acids constant. Dietary protein reduction did not affect the growth performance or feed conversion ratio of the pigs, but it did linearly increase the portion of fat to lean in the carcass. Significant linear effect was found in back fat (p

  3. Effective sampling range of a synthetic protein-based attractant for Ceratitis capitata (Diptera: Tephritidae).

    Science.gov (United States)

    Epsky, Nancy D; Espinoza, Hernán R; Kendra, Paul E; Abernathy, Robert; Midgarden, David; Heath, Robert R

    2010-10-01

    Studies were conducted in Honduras to determine effective sampling range of a female-targeted protein-based synthetic attractant for the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). Multilure traps were baited with ammonium acetate, putrescine, and trimethylamine lures (three-component attractant) and sampled over eight consecutive weeks. Field design consisted of 38 traps (over 0.5 ha) placed in a combination of standard and high-density grids to facilitate geostatistical analysis, and tests were conducted in coffee (Coffea arabica L.),mango (Mangifera indica L.),and orthanique (Citrus sinensis X Citrus reticulata). Effective sampling range, as determined from the range parameter obtained from experimental variograms that fit a spherical model, was approximately 30 m for flies captured in tests in coffee or mango and approximately 40 m for flies captured in orthanique. For comparison, a release-recapture study was conducted in mango using wild (field-collected) mixed sex C. capitata and an array of 20 baited traps spaced 10-50 m from the release point. Contour analysis was used to document spatial distribution of fly recaptures and to estimate effective sampling range, defined by the area that encompassed 90% of the recaptures. With this approach, effective range of the three-component attractant was estimated to be approximately 28 m, similar to results obtained from variogram analysis. Contour maps indicated that wind direction had a strong influence on sampling range, which was approximately 15 m greater upwind compared with downwind from the release point. Geostatistical analysis of field-captured insects in appropriately designed trapping grids may provide a supplement or alternative to release-recapture studies to estimate sampling ranges for semiochemical-based trapping systems.

  4. IL-6/IL-12 Cytokine Receptor Shuffling of Extra- and Intracellular Domains Reveals Canonical STAT Activation via Synthetic IL-35 and IL-39 Signaling.

    Science.gov (United States)

    Floss, D M; Schönberg, M; Franke, M; Horstmeier, F C; Engelowski, E; Schneider, A; Rosenfeldt, E M; Scheller, J

    2017-11-09

    IL-35 and IL-39 are recently discovered shared members of the IL-6- and IL-12-type cytokine family with immune-suppressive capacity. IL-35 has been reported to induce the formation of four different receptor complexes: gp130:IL-12β2, gp130:gp130, IL-12β2:IL-12β2, and IL-12β2:WSX-1. IL-39 was proposed to form a gp130:IL-23R receptor complex. IL-35, but not IL-39, has been reported to activate non-conventional STAT signaling, depending on the receptor complex and target cell. Analyses of IL-35 and IL-39 are, however, hampered by the lack of biologically active recombinant IL-35 and IL-39 proteins. Therefore, we engineered chimeric cytokine receptors to accomplish synthetic IL-35 and IL- 39 signaling by shuffling the extra- and intracellular domains of IL-6/IL-12-type cytokine receptors, resulting in biological activity for all previously described IL-35 receptor complexes. Moreover, we found that the proposed IL-39 receptor complex is biologically active and discovered two additional biologically active synthetic receptor combinations, gp130/IL-12Rβ1 and IL-23R/IL-12Rβ2. Surprisingly, synthetic IL-35 activation led to more canonical STAT signaling of all receptor complexes. In summary, our receptor shuffling approach highlights an interchangeable, modular domain structure among IL-6- and IL-12-type cytokine receptors and enabled synthetic IL-35 and IL-39 signaling.

  5. Synthetic Polymer with a Structure-Driven Hepatic Deposition and Curative Pharmacological Activity in Hepatic Cells

    DEFF Research Database (Denmark)

    Riber, Camilla Frich; Halling Folkmar Andersen, Anna; Anegaard Rolskov, Lærke

    2017-01-01

    Synthetic polymers make strong contributions as tools for delivery of biological drugs and chemotherapeutics. The most praised characteristic of polymers in these applications is complete lack of pharmacological function such as to minimize the side effects within the human body. In contrast......, synthetic polymers with curative pharmacological activity are truly rare. Moreover, such activity is typically nonspecific rather than structure-defined. In this work, we present the discovery of poly(ethylacrylic acid) (PEAA) as a polymer with a suit of structure-defined, unexpected, pharmacological......, and pharmacokinetic properties not observed in close structural analogues. Specifically, PEAA reveals capacity to bind to albumin with ensuing natural hepatic deposition in vivo and exhibits concurrent inhibitory activity against the hepatitis C virus and inflammation in hepatic cells. Our findings provide a view...

  6. In vitro biological characterization of a novel, synthetic diaryl pyrazole resorcinol class of heat shock protein 90 inhibitors.

    Science.gov (United States)

    Sharp, Swee Y; Boxall, Kathy; Rowlands, Martin; Prodromou, Chrisostomos; Roe, S Mark; Maloney, Alison; Powers, Marissa; Clarke, Paul A; Box, Gary; Sanderson, Sharon; Patterson, Lisa; Matthews, Thomas P; Cheung, Kwai-Ming J; Ball, Karen; Hayes, Angela; Raynaud, Florence; Marais, Richard; Pearl, Laurence; Eccles, Sue; Aherne, Wynne; McDonald, Edward; Workman, Paul

    2007-03-01

    The molecular chaperone heat shock protein 90 (HSP90) has emerged as an exciting molecular target. Derivatives of the natural product geldanamycin, such as 17-allylamino-17-demethoxy-geldanamycin (17-AAG), were the first HSP90 ATPase inhibitors to enter clinical trial. Synthetic small-molecule HSP90 inhibitors have potential advantages. Here, we describe the biological properties of the lead compound of a new class of 3,4-diaryl pyrazole resorcinol HSP90 inhibitor (CCT018159), which we identified by high-throughput screening. CCT018159 inhibited human HSP90beta with comparable potency to 17-AAG and with similar ATP-competitive kinetics. X-ray crystallographic structures of the NH(2)-terminal domain of yeast Hsp90 complexed with CCT018159 or its analogues showed binding properties similar to radicicol. The mean cellular GI(50) value of CCT018159 across a panel of human cancer cell lines, including melanoma, was 5.3 mumol/L. Unlike 17-AAG, the in vitro antitumor activity of the pyrazole resorcinol analogues is independent of NQO1/DT-diaphorase and P-glycoprotein expression. The molecular signature of HSP90 inhibition, comprising increased expression of HSP72 protein and depletion of ERBB2, CDK4, C-RAF, and mutant B-RAF, was shown by Western blotting and quantified by time-resolved fluorescent-Cellisa in human cancer cell lines treated with CCT018159. CCT018159 caused cell cytostasis associated with a G(1) arrest and induced apoptosis. CCT018159 also inhibited key endothelial and tumor cell functions implicated in invasion and angiogenesis. Overall, we have shown that diaryl pyrazole resorcinols exhibited similar cellular properties to 17-AAG with potential advantages (e.g., aqueous solubility, independence from NQO1 and P-glycoprotein). These compounds form the basis for further structure-based optimization to identify more potent inhibitors suitable for clinical development.

  7. Radioreceptor assay for evaluation of the plasma glucocorticoid activity of natural and synthetic steroids in man

    International Nuclear Information System (INIS)

    Ballard, P.L.; Carter, J.P.; Graham, B.S.; Baxter, J.D.

    1975-01-01

    An assay for plasma glucocorticoid activity has been developed using specific glucocorticoid receptors. Unlike other assays for cortisol and certain synthetic corticosteroids, this radioreceptor assay measures the glucocorticoid activity of all natural and synthetic steroids. Steroids extracted from as little as 0.05 ml of plasma are incubated with 3 H-dexamethasone and cytosol receptors from cultured rat hepatoma cells. From 0.5 to 50 ng of cortisol are accurately detected. Glucocorticoid activities of adult plasmas determined by the assay correlate closely with corticoid levels obtained in the CBG-isotope and fluorometric assays. Other steroids are measured in proportion to both concentration and potency as glucocorticoids. Relative activities include: cortisol 100, dexamethasone 940, prednisolone 230, prednisone 3, estradiol 1 and androstenedione 1. A similar ranking of steroids was found using receptors from a human source (fetal lung). The assay has been useful in detecting glucocorticoid activity in unidentified medications and in measuring plasma glucocorticoid levels after administration of synthetic corticosteroids. (auth)

  8. Suppressive effect on polyclonal B-cell activation of a synthetic peptide homologous to a transmembrane component of oncogenic retroviruses

    Energy Technology Data Exchange (ETDEWEB)

    Mitani, M.; Cianciolo, G.J.; Snyderman, R.; Yasuda, M.; Good, R.A.; Day, N.K.

    1987-01-01

    Purified feline leukemia virus, UV light-inactivated feline leukemia virus, and a synthetic peptide (CKS-17) homologous to a well-conserved region of the transmembrane components of several human and animal retroviruses were each studied for their effect on IgG production by feline peripheral blood lymphocytes. Using a reverse hemolytic plaque assay, both the viable virus and the UV-inactivated feline leukemia virus, but not the CKS-17, activated B lymphocytes to secrete IgG. When staphylococcal protein A, a polyclonal B-cell activator, was used to stimulate IgG synthesis by feline lymphocytes, the viable virus, the UV-inactivated virus, and the CKS-17 peptide each strongly suppressed IgG secretion without compromising viability of the lymphocytes. These finding suggest that the immunosuppressive influences of feline leukemia virus on immunoglobulin synthesis may reside in a conserved portion of the envelope glycoprotein that includes the region homologous to CKS-17.

  9. Suppressive effect on polyclonal B-cell activation of a synthetic peptide homologous to a transmembrane component of oncogenic retroviruses

    International Nuclear Information System (INIS)

    Mitani, M.; Cianciolo, G.J.; Snyderman, R.; Yasuda, M.; Good, R.A.; Day, N.K.

    1987-01-01

    Purified feline leukemia virus, UV light-inactivated feline leukemia virus, and a synthetic peptide (CKS-17) homologous to a well-conserved region of the transmembrane components of several human and animal retroviruses were each studied for their effect on IgG production by feline peripheral blood lymphocytes. Using a reverse hemolytic plaque assay, both the viable virus and the UV-inactivated feline leukemia virus, but not the CKS-17, activated B lymphocytes to secrete IgG. When staphylococcal protein A, a polyclonal B-cell activator, was used to stimulate IgG synthesis by feline lymphocytes, the viable virus, the UV-inactivated virus, and the CKS-17 peptide each strongly suppressed IgG secretion without compromising viability of the lymphocytes. These finding suggest that the immunosuppressive influences of feline leukemia virus on immunoglobulin synthesis may reside in a conserved portion of the envelope glycoprotein that includes the region homologous to CKS-17

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

    Indian Academy of Sciences (India)

    2012-01-19

    Jan 19, 2012 ... CD44, an adhesion molecule, has been reported to be a binding site for ... receptors in mediating mitogen-activated protein kinase activation. ... surface expression and tumour necrosis factor-alpha levels, ... Abbreviations used: Abs, antibodies; ANOVA, analysis of variance; AP-1, activator protein -1; BCG, ...

  11. Antileishmanial Activity of the Hydroalcoholic Extract of Miconia langsdorffii, Isolated Compounds, and Semi-Synthetic Derivatives

    Directory of Open Access Journals (Sweden)

    Wilson R. Cunha

    2011-02-01

    Full Text Available The in vitro activity of the crude hydroalcoholic extract of the aerial parts of Miconia langsdorffii Cogn. was evaluated against the promastigote forms of L. amazonensis, the causative agent of cutaneous leishmaniasis in humans. The bioassay-guided fractionation of this extract led to identification of the triterpenes ursolic acid and oleanolic acid as the major compounds in the fraction that displayed the highest activity. Several ursolic acid semi-synthetic derivatives were prepared, to find out whether more active compounds could be obtained. Among these ursolic acid-derived substances, the C-28 methyl ester derivative exhibited the best antileishmanial activity.

  12. Pre-ERCP infusion of semapimod, a mitogen-activated protein kinases inhibitor, lowers post-ERCP hyperamylasemia but not pancreatitis incidence

    NARCIS (Netherlands)

    van Westerloo, David J.; Rauws, Erik A.; Hommes, Daan; de Vos, Alex F.; van der Poll, Tom; Powers, Barbara L.; Fockens, Paul; Dijkgraaf, Marcel G. W.; Bruno, Marco J.

    2008-01-01

    BACKGROUND: Acute pancreatitis and hyperamylasemia are frequent complications of an ERCP. Semapimod is a synthetic guanylhydrazone that inhibits the mitogen-activated protein kinase (MAPK) pathway, macrophage activation, and the production of several inflammatory cytokines. OBJECTIVE: This study

  13. The preparation of synthetic standards for use in instrumental neutron-activation analysis

    International Nuclear Information System (INIS)

    Eddy, B.T.; Watterson, J.I.W.; Erasmus, C.S.

    1979-01-01

    An account is given of the formulation and preparation of synthetic standards suitable for the routine analysis of minerals, ores, and ore concentrates by instrumental neutron activation. Fifteen standards were prepared, each containing from one to seven elements. The standards contain forty-four elements that produce isotopes with half-lives longer than 12 hours. An evaluation of the accuracy and precision of the method of preparation is given

  14. Synthetic ligands of the elastin receptor induce elastogenesis in human dermal fibroblasts via activation of their IGF-1 receptors.

    Science.gov (United States)

    Qa'aty, Nour; Vincent, Matthew; Wang, Yanting; Wang, Andrew; Mitts, Thomas F; Hinek, Aleksander

    2015-12-01

    We have previously reported that a mixture of peptides obtained after chemical or enzymatic degradation of bovine elastin, induced new elastogenesis in human skin. Now, we investigated the elastogenic potential of synthetic peptides mimicking the elastin-derived, VGVAPG sequence, IGVAPG sequence that we found in the rice bran, and a similar peptide, VGVTAG that we identified in the IGF-1-binding protein-1 (IGFBP-1). We now demonstrate that treatment with each of these xGVxxG peptides (recognizable by the anti-elastin antibody), up-regulated the levels of elastin-encoding mRNA, tropoelastin protein, and the deposition of new elastic fibers in cultures of human dermal fibroblasts and in cultured explants of human skin. Importantly, we found that such induction of new elastogenesis may involve two parallel signaling pathways triggered after activation of IGF-1 receptor. In the first one, the xGVxxG peptides interact with the cell surface elastin receptor, thereby causing the downstream activation of the c-Src kinase and a consequent cross-activation of the adjacent IGF-1R, even in the absence of its principal ligand. In the second pathway their hydrophobic association with the N-terminal domain (VGVTAG) of the serum-derived IGFBP-1 induces conformational changes of this IGF-1 chaperone allowing for the release of its cargo and a consequent ligand-specific phosphorylation of IGF-1R. We present a novel, clinically relevant mechanism in which products of partial degradation of dermal elastin may stimulate production of new elastic fibers by dermal fibroblasts. Our findings particularly encourage the use of biologically safe synthetic xGVxxG peptides for regeneration of the injured or aged human skin. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  15. Chitin and stress induced protein kinase activation

    DEFF Research Database (Denmark)

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

    2017-01-01

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

  16. Selecting highly structure-specific antibodies using structured synthetic mimics of the cystine knot protein sclerostin

    NARCIS (Netherlands)

    Back, J.W.; Frisch, C.; Van Pee, K.; Boschert, V.; van Vught, R.; Puijk, W.; Mueller, T. D.; Knappik, A.; Timmerman, P.

    2012-01-01

    Antibodies directed against specific regions of a protein have traditionally been raised against full proteins, protein domains or simple unstructured peptides, containing contiguous stretches of primary sequence. We have used a new approach of selecting antibodies against restrained peptides

  17. Generation and analyses of human synthetic antibody libraries and their application for protein microarrays

    DEFF Research Database (Denmark)

    Säll, Anna; Walle, Maria; Wingren, Christer

    2016-01-01

    in a high-throughput manner. To address this we designed and constructed two human synthetic antibody fragment (scFv) libraries denoted HelL-11 and HelL-13. By the use of phage display technology, in total 466 unique scFv antibodies specific for 114 different antigens were generated. The specificities......Antibody-based proteomics offers distinct advantages in the analysis of complex samples for discovery and validation of biomarkers associated with disease. However, its large-scale implementation requires tools and technologies that allow development of suitable antibody or antibody fragments...... for diagnostics and therapeutics. In addition, this work provides a great example on how a synthetic approach can be used to optimize library designs. By having precise control of the diversity introduced into the antigen-binding sites, synthetic libraries offer increased understanding of how different diversity...

  18. Activation of peroxisome proliferator-activated receptors (PPARs) by their ligands and protein kinase A activators

    Science.gov (United States)

    Lazennec, Gwendal; Canaple, Laurence; Saugy, Damien; Wahli, Walter

    2000-01-01

    The nuclear peroxisome proliferator-activated receptors (PPARs) α, β and γ activate the transcription of multiple genes involved in lipid metabolism. Several natural and synthetic ligands have been identified for each PPAR isotype but little is known about the phosphorylation state of these receptors. We show here that activators of protein kinase A (PKA) can enhance mouse PPAR activity in the absence and the presence of exogenous ligands in transient transfection experiments. The activation function 1 (AF-1) of PPARs was dispensable for transcriptional enhancement, whereas the activation function 2 (AF-2) was required for this effect. We also show that several domains of PPAR can be phosphorylated by PKA in vitro. Moreover, gel experiments suggest that PKA stabilizes binding of the liganded PPAR to DNA. PKA inhibitors decreased not only the kinase dependent induction of PPARs but also their ligand-dependent induction, suggesting that the ligands may also mobilize the PKA pathway to lead to maximal transcriptional induction by PPARs. Moreover, comparing PPARα KO with PPARα wild-type mice, we show that the expression of the ACO gene can be regulated by PKA-activated PPARα in liver. These data demonstrate that the PKA pathway is an important modulator of PPAR activity and we propose a model associating this pathway in the control of fatty acid β-oxidation under conditions of fasting, stress and exercise. PMID:11117527

  19. Enhanced fuel efficiency on tractor-trailers using synthetic jet-based active flow control

    Science.gov (United States)

    Amitay, Michael; Menicovich, David; Gallardo, Daniele

    2016-04-01

    The application of piezo-electrically-driven synthetic-jet-based active flow control to reduce drag on tractor-trailers was explored experimentally in wind tunnel testing as well as full-scale road tests. Aerodynamic drag accounts for more than 50% of the usable energy at highway speeds, a problem that applies primarily to trailer trucks. Therefore, a reduction in aerodynamic drag results in large saving of fuel and reduction in CO2 emissions. The active flow control technique that is being used relies on a modular system comprised of distributed, small, highly efficient actuators. These actuators, called synthetic jets, are jets that are synthesized at the edge of an orifice by a periodic motion of a piezoelectric diaphragm(s) mounted on one (or more) walls of a sealed cavity. The synthetic jet is zero net mass flux (ZNMF), but it allows momentum transfer to flow. It is typically driven near diaphragm and/or cavity resonance, and therefore, small electric input [O(10W)] is required. Another advantage of this actuator is that no plumbing is required. The system doesn't require changes to the body of the truck, can be easily reconfigured to various types of vehicles, and consumes small amounts of electrical power from the existing electrical system of the truck. Preliminary wind tunnel results showed up to 18% reduction in fuel consumption, whereas road tests also showed very promising results.

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

    NARCIS (Netherlands)

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

    1998-01-01

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

  1. [Testing the pharmacological activity of some synthetic cannabinoids in mice (author's transl)].

    Science.gov (United States)

    Ganz, A J; Waser, P G

    1980-01-01

    A series of synthetic cannabinoids were tested in mice for analgesic, anticonvulsant, sedative and reserpine antagonistic properties as well as for influence on body temperature and on motor coordination and compared with the natural delta 9-tetrahydrocannabinol (delta 9-THC), delta 8-tetrahydrocannabinol (delta 8-THC) and cannabidiol (CBD). All cannabinoids were injected s.c. or i.p. in mice as solutions in olive oil. The synthetic cannabinoids, with the exception of the lipophilic ones, were less active than the natural delta 9-THC. 1',1'-dimethyl-delta 8-tetrahydrocannabinol (DM-delta 8-THC) has an analgesic ED 50 of 16 mg/kg s.c. (writhing test) and is three times more active than delta 9-THC, but also eight times less active than morphine. The lipophilic derivatives of delta 8-THC prolonged pentobarbitone narcosis and diminished locomotor activity in mice. Anticonvulsant activities could never be detected; all cannabinoids slightly diminished body temperature and antagonized weakly the hypothermia induced by reserpine. The trained capacity of remaining on the rotating rod was severely shortened for a long time after application of all cannabinoids but mainly by the lipophilic ones. The influence of derivation on the activity of delta 9-THC is discussed.

  2. Synthetic biology approaches for protein production optimization in bacterial cell factories

    DEFF Research Database (Denmark)

    Rennig, Maja; Andersen, Mikael Rørdam

    devices and their fusion to antibiotic selection markers enables subsequent selection of high-expressing constructs. The approach is a simple and inexpensive alternative to advanced screening techniques. In addition, a second synthetic biology approach provides the means for fast and efficient plasmid...

  3. A study of the thermal activation of synthetic zeolites (molecular sieve) for gas-solid chromatography

    International Nuclear Information System (INIS)

    Walker, J.A.J.

    1978-10-01

    The thermal activation of synthetic zeolites from two sources has been investigated with reference to the adsorption chromatography of inorganic gases. It was found that the heats of adsorption for oxygen and carbon monoxide increased with activation temperatures. Limits of detection for oxygen in argon and conversely argon in oxygen were determined as well as the chromatographic stability of the activated zeolite. The practical implications and importance of the results are discussed and the application to the analysis of fast reactor blanket gas is mentioned. An explanation is proposed for the adsorption behaviour of these activated materials, based on an electrostatic mechanism, and this has suggested a reason for the separation characteristics of oxygen and argon on polar zeolites. Further work is identified including the investigation of energy states of the oxygen molecule adsorbed on activated zeolite by means of ultra-violet photoelectron spectroscopy. (author)

  4. A study of the thermal activation of synthetic zeolites (molecular sieve) for gas-solid chromatography

    International Nuclear Information System (INIS)

    Walker, J.A.J.

    1978-10-01

    The thermal activation of synthetic zeolites from two sources has been investigated with reference to the adsorption chromatography of inorganic gases. It was found that the heats of adsorption for oxygen and carbon monoxide increased with activation temperature. Limits of detection for oxygen in argon and conversely argon in oxygen were determined as well as the chromatographic stability of the activated zeolite. The practical implications and importance of the results are discussed and the application to the analysis of fast reactor blanket gas is mentioned. An explanation is proposed for the adsorption behaviour of these activated materials, based on an electrostatic mechanism, and this has suggested a reason for the separation characteristics of oxygen and argon on polar zeolites. Further work is identified including the investigation of energy states of the oxygen molecule adsorbed on activated zeolite by means of ultra-violet photoelectron spectroscopy. (author)

  5. iNOS Activity Modulates Inflammation, Angiogenesis, and Tissue Fibrosis in Polyether-Polyurethane Synthetic Implants.

    Science.gov (United States)

    Cassini-Vieira, Puebla; Araújo, Fernanda Assis; da Costa Dias, Filipi Leles; Russo, Remo Castro; Andrade, Silvia Passos; Teixeira, Mauro Martins; Barcelos, Luciola Silva

    2015-01-01

    There is considerable interest in implantation techniques and scaffolds for tissue engineering and, for safety and biocompatibility reasons, inflammation, angiogenesis, and fibrosis need to be determined. The contribution of inducible nitric oxide synthase (iNOS) in the regulation of the foreign body reaction induced by subcutaneous implantation of a synthetic matrix was never investigated. Here, we examined the role of iNOS in angiogenesis, inflammation, and collagen deposition induced by polyether-polyurethane synthetic implants, using mice with targeted disruption of the iNOS gene (iNOS(-/-)) and wild-type (WT) mice. The hemoglobin content and number of vessels were decreased in the implants of iNOS(-/-) mice compared to WT mice 14 days after implantation. VEGF levels were also reduced in the implants of iNOS(-/-) mice. In contrast, the iNOS(-/-) implants exhibited an increased neutrophil and macrophage infiltration. However, no alterations were observed in levels of CXCL1 and CCL2, chemokines related to neutrophil and macrophage migration, respectively. Furthermore, the implants of iNOS(-/-) mice showed boosted collagen deposition. These data suggest that iNOS activity controls inflammation, angiogenesis, and fibrogenesis in polyether-polyurethane synthetic implants and that lack of iNOS expression increases foreign body reaction to implants in mice.

  6. iNOS Activity Modulates Inflammation, Angiogenesis, and Tissue Fibrosis in Polyether-Polyurethane Synthetic Implants

    Science.gov (United States)

    Cassini-Vieira, Puebla; Araújo, Fernanda Assis; da Costa Dias, Filipi Leles; Russo, Remo Castro; Andrade, Silvia Passos; Teixeira, Mauro Martins; Barcelos, Luciola Silva

    2015-01-01

    There is considerable interest in implantation techniques and scaffolds for tissue engineering and, for safety and biocompatibility reasons, inflammation, angiogenesis, and fibrosis need to be determined. The contribution of inducible nitric oxide synthase (iNOS) in the regulation of the foreign body reaction induced by subcutaneous implantation of a synthetic matrix was never investigated. Here, we examined the role of iNOS in angiogenesis, inflammation, and collagen deposition induced by polyether-polyurethane synthetic implants, using mice with targeted disruption of the iNOS gene (iNOS−/−) and wild-type (WT) mice. The hemoglobin content and number of vessels were decreased in the implants of iNOS−/− mice compared to WT mice 14 days after implantation. VEGF levels were also reduced in the implants of iNOS−/− mice. In contrast, the iNOS−/− implants exhibited an increased neutrophil and macrophage infiltration. However, no alterations were observed in levels of CXCL1 and CCL2, chemokines related to neutrophil and macrophage migration, respectively. Furthermore, the implants of iNOS−/− mice showed boosted collagen deposition. These data suggest that iNOS activity controls inflammation, angiogenesis, and fibrogenesis in polyether-polyurethane synthetic implants and that lack of iNOS expression increases foreign body reaction to implants in mice. PMID:26106257

  7. Geographic Variability and Anti-Staphylococcal Activity of the Chrysophaentins and Their Synthetic Fragments

    Directory of Open Access Journals (Sweden)

    Jared T. Hammill

    2012-05-01

    Full Text Available Drug-resistant Staphylococcus aureus is a continuing public health concern, both in the hospital and community settings. Antibacterial compounds that possess novel structural scaffolds and are effective against multiple S. aureus strains, including current drug-resistant ones, are needed. Previously, we have described the chrysophaentins, a family of bisdiarylbutene macrocycles from the chrysophyte alga Chrysophaeum taylori that inhibit the growth of S. aureus and methicillin-resistant S. aureus (MRSA. In this study we have analyzed the geographic variability of chrysophaentin production in C. taylori located at different sites on the island of St. John, U.S. Virgin Islands, and identified two new linear chrysophaentin analogs, E2 and E3. In addition, we have expanded the structure activity relationship through synthesis of fragments comprising conserved portions of the chrysophaentins, and determined the antimicrobial activity of natural chrysophaentins and their synthetic analogs against five diverse S. aureus strains. We find that the chrysophaentins show similar activity against all S. aureus strains, regardless of their drug sensitivity profiles. The synthetic chrysophaentin fragments indeed mimic the natural compounds in their spectrum of antibacterial activity, and therefore represent logical starting points for future medicinal chemistry studies of the natural products and their analogs.

  8. Optochemical Control of Protein Localization and Activity within Cell-like Compartments.

    Science.gov (United States)

    Caldwell, Reese M; Bermudez, Jessica G; Thai, David; Aonbangkhen, Chanat; Schuster, Benjamin S; Courtney, Taylor; Deiters, Alexander; Hammer, Daniel A; Chenoweth, David M; Good, Matthew C

    2018-05-08

    We report inducible dimerization strategies for controlling protein positioning, enzymatic activity, and organelle assembly inside synthetic cell-like compartments upon photostimulation. Using a photocaged TMP-Haloligand compound, we demonstrate small molecule and light-induced dimerization of DHFR and Haloenzyme to localize proteins to a compartment boundary and reconstitute tripartite sfGFP assembly. Using photocaged rapamycin and fragments of split TEV protease fused to FRB and FKBP, we establish optical triggering of protease activity inside cell-size compartments. We apply light-inducible protease activation to initiate assembly of membraneless organelles, demonstrating the applicability of these tools for characterizing cell biological processes in vitro. This modular toolkit, which affords spatial and temporal control of protein function in a minimal cell-like system, represents a critical step toward the reconstitution of a tunable synthetic cell, built from the bottom up.

  9. Separation of antimony from synthetic cloth. Application in forensic science using neutron activation analysis

    International Nuclear Information System (INIS)

    Bhadkambekar, C.A.; Swain, K.K.; Kayasth, S.R.; Mukherjee, T.

    2005-01-01

    A simple ion-exchange separation procedure was developed for selective removal of antimony from synthetic cloth to facilitate determination of several trace elements frequently used to identify gunshot residues by neutron activation analysis. Radiotracers of Sb, Ba, Cu, Co, As, Zn, Hg and Ag were employed to optimize the developed procedure. The method involves the quantitative retention of the above elements, except of Sb, from 0.2M ammonium carbonate solution using Chelex 100 resin and subsequent quantitative elution of the elements of interest with 2M nitric acid for gamma-ray spectrometry. The procedure was tested by simulated gunshot residues. (author)

  10. Lipid-lowering Activity of Natural and Semi-Synthetic Sterols and Stanols.

    Science.gov (United States)

    Taha, Dhiaa A; Wasan, Ellen K; Wasan, Kishor M; Gershkovich, Pavel

    2015-01-01

    Consumption of plant sterols/ stanols has long been demonstrated to reduce plasma cholesterol levels. The objective of this review is to demonstrate the lipid-lowering activity and anti-atherogenic effects of natural and semi-synthetic plant sterols/ stanols based on evidence from cell-culture studies, animal studies and clinical trials. Additionally, this review highlights certain molecular mechanisms by which plant sterols/ stanols lower plasma cholesterol levels with a special emphasis on factors that affect the cholesterol-lowering activity of plant sterols/stanols. The crystalline nature and the poor oil solubility of these natural products could be important factors that limit their cholesterol-lowering efficiency. Several attempts have been made to improve the cholesterol-lowering activity by enhancing the bioavailability of crystalline sterols and stanols. Approaches involved reduction of the crystal size and/or esterification with fatty acids from vegetable or fish oils. However, the most promising approach in this context is the chemical modification of plant sterols /stanols into water soluble disodium ascorbyl phytostanyl phosphates analogue by esterification with ascorbic acid. This novel semi-synthetic stanol derivative has improved efficacy over natural plant sterols/ stanols and can provide additional benefits by combining the cholesterol-lowering properties of plant stanols with the antioxidant potential of ascorbic acid. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

  11. A synthetic cadmium metallothionein gene (PMCd1syn) of Paramecium species: expression, purification and characteristics of metallothionein protein.

    Science.gov (United States)

    Dar, Saira; Shuja, Rukhsana N; Shakoori, Abdul Rauf

    2013-02-01

    Metallothioneins (MTs) are metal binding proteins that are rich in cysteine residues constituting 10-30 % of the total protein, and in which the thiol groups bind to the metal ions. The increasing amount of metal ions in the medium have shown increased production of MTs by different organisms such as bacteria, protozoa and mammals like humans. PMCd1 is the first gene ever discovered in Paramecium, a ciliated protozoan, that could produce this MT in response to cadmium. In this study the PMCd1syn gene has been cloned in pET41a expression vector and expressed in an Escherichia coli BL21-codonplus strain for the first time. Since the gene PMCd1 amplified from Paramecium contained 10 codons, which could act as stop codons during expression in E. coli, this gene of 612 bps was synthesized to substitute these (stop) codons for the Paramecium sp. specific amino acids. For stability of the expressed protein, glutathione-S-transferase gene was fused with PMCd1syn gene and coexpressed. The cells expressing PMCd1syn demonstrated increased accumulation of cadmium. This is the first report of cadmium MT protein expressed from Paramecium species, particularly from synthetic MT gene (PMCd1syn). This fusion protein, the molecular weight of which has been confirmed to be 53.03 kDa with MALDI analysis, is rich in cysteine residues, and has been shown for the first time in this ciliate to bind to and sequester Cd(2+)-ions.

  12. Radical-scavenging Activity of Natural Methoxyphenols vs. Synthetic Ones using the Induction Period Method

    Directory of Open Access Journals (Sweden)

    Seiichiro Fujisawa

    2007-02-01

    Full Text Available The radical-scavenging activities of the synthetic antioxidants 2-allyl-4-X-phenol (X=NO2, Cl, Br, OCH3, COCH3, CH3, t-(CH33, C6H5 and 2,4-dimethoxyphenol, and the natural antioxidants eugenol and isoeugenol, were investigated using differential scanning calorimetry (DSC by measuring their anti-1,1-diphenyl-2-picrylhydrazyl (DPPH radical activity and the induction period for polymerization of methyl methacrylate (MMA initiated by thermal decomposition of 2,2'-azobisisobutyronitrile (AIBN and benzoyl peroxide (BPO. 2-Allyl-4-methoxyphenol and 2,4-dimethoxy-phenol scavenged not only oxygen-centered radicals (PhCOO. derived from BPO, but also carbon-centered radicals (R. derived from the AIBN and DPPH radical much more efficiently, in comparison with eugenol and isoeugenol. 2-Allyl-4-methoxyphenol may be useful for its lower prooxidative activity.

  13. Protein-polymer nano-machines. Towards synthetic control of biological processes

    Directory of Open Access Journals (Sweden)

    Alexander Cameron

    2004-09-01

    Full Text Available Abstract The exploitation of nature's machinery at length scales below the dimensions of a cell is an exciting challenge for biologists, chemists and physicists, while advances in our understanding of these biological motifs are now providing an opportunity to develop real single molecule devices for technological applications. Single molecule studies are already well advanced and biological molecular motors are being used to guide the design of nano-scale machines. However, controlling the specific functions of these devices in biological systems under changing conditions is difficult. In this review we describe the principles underlying the development of a molecular motor with numerous potential applications in nanotechnology and the use of specific synthetic polymers as prototypic molecular switches for control of the motor function. The molecular motor is a derivative of a TypeI Restriction-Modification (R-M enzyme and the synthetic polymer is drawn from the class of materials that exhibit a temperature-dependent phase transition. The potential exploitation of single molecules as functional devices has been heralded as the dawn of new era in biotechnology and medicine. It is not surprising, therefore, that the efforts of numerous multidisciplinary teams 12. have been focused in attempts to develop these systems. as machines capable of functioning at the low sub-micron and nanometre length-scales 3. However, one of the obstacles for the practical application of single molecule devices is the lack of functional control methods in biological media, under changing conditions. In this review we describe the conceptual basis for a molecular motor (a derivative of a TypeI Restriction-Modification enzyme with numerous potential applications in nanotechnology and the use of specific synthetic polymers as prototypic molecular switches for controlling the motor function 4.

  14. Synthetic Polymer Affinity Ligand for Bacillus thuringiensis ( Bt) Cry1Ab/Ac Protein: The Use of Biomimicry Based on the Bt Protein-Insect Receptor Binding Mechanism.

    Science.gov (United States)

    Liu, Mingming; Huang, Rong; Weisman, Adam; Yu, Xiaoyang; Lee, Shih-Hui; Chen, Yalu; Huang, Chao; Hu, Senhua; Chen, Xiuhua; Tan, Wenfeng; Liu, Fan; Chen, Hao; Shea, Kenneth J

    2018-05-24

    We report a novel strategy for creating abiotic Bacillus thuringiensis ( Bt) protein affinity ligands by biomimicry of the recognition process that takes place between Bt Cry1Ab/Ac proteins and insect receptor cadherin-like Bt-R 1 proteins. Guided by this strategy, a library of synthetic polymer nanoparticles (NPs) was prepared and screened for binding to three epitopes 280 FRGSAQGIEGS 290 , 368 RRPFNIGINNQQ 379 and 436 FRSGFSNSSVSIIR 449 located in loop α8, loop 2 and loop 3 of domain II of Bt Cry1Ab/Ac proteins. A negatively charged and hydrophilic nanoparticle (NP12) was found to have high affinity to one of the epitopes, 368 RRPFNIGINNQQ 379 . This same NP also had specific binding ability to both Bt Cry1Ab and Bt Cry1Ac, proteins that share the same epitope, but very low affinity to Bt Cry2A, Bt Cry1C and Bt Cry1F closely related proteins that lack epitope homology. To locate possible NP- Bt Cry1Ab/Ac interaction sites, NP12 was used as a competitive inhibitor to block the binding of 865 NITIHITDTNNK 876 , a specific recognition site in insect receptor Bt-R 1 , to 368 RRPFNIGINNQQ 379 . The inhibition by NP12 reached as high as 84%, indicating that NP12 binds to Bt Cry1Ab/Ac proteins mainly via 368 RRPFNIGINNQQ 379 . This epitope region was then utilized as a "target" or "bait" for the separation and concentration of Bt Cry1Ac protein from the extract of transgenic Bt cotton leaves by NP12. This strategy, based on the antigen-receptor recognition mechanism, can be extended to other biotoxins and pathogen proteins when designing biomimic alternatives to natural protein affinity ligands.

  15. A synthetic peptide derived from the animo acid sequence of canine parvovirus structural proteins which defines a B cell epitope and elicits antiviral antibody in BALB c mice.

    NARCIS (Netherlands)

    G.F. Rimmelzwaan (Guus); J. Carlson; F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Albert)

    1990-01-01

    textabstractSynthetic peptides, recombinant fusion proteins and mouse monoclonal antibodies were used to delineate a B cell epitope of the VP'2 structural protein of canine parvovirus (CPV). Although this epitope is not preferentially recognized in the normal antibody response to CPV, virus-specific

  16. Structure-activity-based design of a synthetic malaria peptide eliciting sporozoite inhibitory antibodies in a virosomal formulation.

    NARCIS (Netherlands)

    Okitsu, S.L.; Kienzl, U.; Moehle, K.; Silvie, O.; Peduzzi, E.; Mueller, M.S.; Sauerwein, R.W.; Matile, H.; Zurbriggen, R.; Mazier, D.; Robinson, J.A.; Pluschke, G.

    2007-01-01

    The circumsporozoite protein (CSP) of Plasmodium falciparum is a leading candidate antigen for inclusion in a malaria subunit vaccine. We describe here the design of a conformationally constrained synthetic peptide, designated UK-39, which has structural and antigenic similarity to the NPNA-repeat

  17. Synthetic study on prion protein fragments using a SPPS and native chemical ligation

    Czech Academy of Sciences Publication Activity Database

    Zawada, Z.; Šebestík, Jaroslav; Bednárová, Lucie; Bouř, Petr; Hlaváček, Jan; Stibor, Ivan

    2009-01-01

    Roč. 37, Suppl. 1 (2009), s. 44-44 ISSN 0939-4451. [International Congress on Amino Acids, Peptides and Proteins /11./. 03.08.2009-07.08.2009, Vienna] Institutional research plan: CEZ:AV0Z40550506 Keywords : prion protein * SPPS * native chemical ligation * fragments Subject RIV: CC - Organic Chemistry

  18. Use of synthetic biology techniques to site-selective introduce posttranslational modifactions in proteins

    NARCIS (Netherlands)

    Bosmans, R.P.G.; Brunsveld, L.; Ryadnov, M.; Brunsveld, L.; Suga, H.

    2014-01-01

    Unravelling the influence of posttranslational modifications (PTMs) on protein functioning is of key interest to get understanding how complex cellular networks are regulated. The current biological toolbox to synthesize these modified proteins in a single form in decent quantities is insufficient,

  19. Antibacterial activity of synthetic peptides derived from lactoferricin against Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212.

    Science.gov (United States)

    León-Calvijo, María A; Leal-Castro, Aura L; Almanzar-Reina, Giovanni A; Rosas-Pérez, Jaiver E; García-Castañeda, Javier E; Rivera-Monroy, Zuly J

    2015-01-01

    Peptides derived from human and bovine lactoferricin were designed, synthesized, purified, and characterized using RP-HPLC and MALDI-TOF-MS. Specific changes in the sequences were designed as (i) the incorporation of unnatural amino acids in the sequence, the (ii) reduction or (iii) elongation of the peptide chain length, and (iv) synthesis of molecules with different number of branches containing the same sequence. For each peptide, the antibacterial activity against Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212 was evaluated. Our results showed that Peptides I.2 (RWQWRWQWR) and I.4 ((RRWQWR)4K2Ahx2C2) exhibit bigger or similar activity against E. coli (MIC 4-33 μM) and E. faecalis (MIC 10-33 μM) when they were compared with lactoferricin protein (LF) and some of its derivate peptides as II.1 (FKCRRWQWRMKKLGA) and IV.1 (FKCRRWQWRMKKLGAPSITCVRRAE). It should be pointed out that Peptides I.2 and I.4, containing the RWQWR motif, are short and easy to synthesize; our results demonstrate that it is possible to design and obtain synthetic peptides that exhibit enhanced antibacterial activity using a methodology that is fast and low-cost and that allows obtaining products with a high degree of purity and high yield.

  20. Antibacterial Activity of Synthetic Peptides Derived from Lactoferricin against Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212

    Directory of Open Access Journals (Sweden)

    María A. León-Calvijo

    2015-01-01

    Full Text Available Peptides derived from human and bovine lactoferricin were designed, synthesized, purified, and characterized using RP-HPLC and MALDI-TOF-MS. Specific changes in the sequences were designed as (i the incorporation of unnatural amino acids in the sequence, the (ii reduction or (iii elongation of the peptide chain length, and (iv synthesis of molecules with different number of branches containing the same sequence. For each peptide, the antibacterial activity against Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212 was evaluated. Our results showed that Peptides I.2 (RWQWRWQWR and I.4 ((RRWQWR4K2Ahx2C2 exhibit bigger or similar activity against E. coli (MIC 4–33 μM and E. faecalis (MIC 10–33 μM when they were compared with lactoferricin protein (LF and some of its derivate peptides as II.1 (FKCRRWQWRMKKLGA and IV.1 (FKCRRWQWRMKKLGAPSITCVRRAE. It should be pointed out that Peptides I.2 and I.4, containing the RWQWR motif, are short and easy to synthesize; our results demonstrate that it is possible to design and obtain synthetic peptides that exhibit enhanced antibacterial activity using a methodology that is fast and low-cost and that allows obtaining products with a high degree of purity and high yield.

  1. Reporter-Based Synthetic Genetic Array Analysis: A Functional Genomics Approach for Investigating Transcript or Protein Abundance Using Fluorescent Proteins in Saccharomyces cerevisiae.

    Science.gov (United States)

    Göttert, Hendrikje; Mattiazzi Usaj, Mojca; Rosebrock, Adam P; Andrews, Brenda J

    2018-01-01

    Fluorescent reporter genes have long been used to quantify various cell features such as transcript and protein abundance. Here, we describe a method, reporter synthetic genetic array (R-SGA) analysis, which allows for the simultaneous quantification of any fluorescent protein readout in thousands of yeast strains using an automated pipeline. R-SGA combines a fluorescent reporter system with standard SGA analysis and can be used to examine any array-based strain collection available to the yeast community. This protocol describes the R-SGA methodology for screening different arrays of yeast mutants including the deletion collection, a collection of temperature-sensitive strains for the assessment of essential yeast genes and a collection of inducible overexpression strains. We also present an alternative pipeline for the analysis of R-SGA output strains using flow cytometry of cells in liquid culture. Data normalization for both pipelines is discussed.

  2. Measurement of Hepatic Protein Fractional Synthetic Rate with Stable Isotope Labeling Technique in Thapsigargin Stressed HepG2 Cells

    Science.gov (United States)

    Song, Juquan; Zhang, Xiao-jun; Boehning, Darren; Brooks, Natasha C.; Herndon, David N.; Jeschke, Marc G.

    2012-01-01

    Severe burn-induced liver damage and dysfunction is associated with endoplasmic reticulum (ER) stress. ER stress has been shown to regulate global protein synthesis. In the current study, we induced ER stress in vitro and estimated the effect of ER stress on hepatic protein synthesis. The aim was two-fold: (1) to establish an in vitro model to isotopically measure hepatic protein synthesis and (2) to evaluate protein fractional synthetic rate (FSR) in response to ER stress. Human hepatocellular carcinoma cells (HepG2) were cultured in medium supplemented with stable isotopes 1,2-13C2-glycine and L-[ring-13C6]phenylalanine. ER stress was induced by exposing the cells to 100 nM of thapsigargin (TG). Cell content was collected from day 0 to 14. Alterations in cytosolic calcium were measured by calcium imaging and ER stress markers were confirmed by Western blotting. The precursor and product enrichments were detected by GC-MS analysis for FSR calculation. We found that the hepatic protein FSR were 0.97±0.02 and 0.99±0.05%/hr calculated from 1,2-13C2-glycine and L-[ring-13C6]phenylalanine, respectively. TG depleted ER calcium stores and induced ER stress by upregulating p-IRE-1 and Bip. FSR dramatically decreased to 0.68±0.03 and 0.60±0.06%/hr in the TG treatment group (pisotope tracer incorporation technique is a useful method for studying the effects of ER stress on hepatic protein synthesis. PMID:22298954

  3. Protein synthetic requirements for caffeine amelioration of radiation-induced G/sub 2/-arrest

    International Nuclear Information System (INIS)

    Rowley, R.; Colkitt, D.

    1984-01-01

    Irradiated cells are arrested in G/sub 2/ (transition point [TP] = 32 min before cell selection in mitosis). Irradiated cells do not recover from G/sub 2/ arrest in the presence of cycloheximide (CHM) indicating dependence of recovery on protein synthesis. Irradiated cells in the presence of caffeine progress to mitosis without arrest. The authors investigate whether irradiated cells in the presence of caffeine require protein synthesis to progress to mitosis. Mitotic cell selection was used to monitor the progression of irradiated CHO cells (150 rad) during exposure to 5 mM caffeine and/or 50 μg/ml CHM. Protein synthesis inhibition was confirmed using /sup 3/H-leucine incorporation. Cells exposed to CHM alone are arrested in G/sub 2/ (TP=49 min), thus cells beyond this point have synthesized all proteins necessary for entry into mitosis. In the presence of caffeine, unirradiated cells exposed to CHM are not arrested at all in G/sub 2/, instead arrest occurs near the S/G/sub 2/ boundary (TP=95 min) indicating that caffeine alleviates the dependence of G/sub 2/ cell progression on protein synthesis. However, irradiated cells exposed to both caffeine and CHM are only able to progress to mitosis if beyond the CHM-TP. Irradiated cells in the presence of caffeine thus behave as untreated cells and require protein synthesis for progression to mitosis when prior to the CHM-TP

  4. Engineering of kinase-based protein interacting devices: active expression of tyrosine kinase domains

    KAUST Repository

    Diaz Galicia, Miriam Escarlet

    2018-05-01

    Protein-protein interactions modulate cellular processes in health and disease. However, tracing weak or rare associations or dissociations of proteins is not a trivial task. Kinases are often regulated through interaction partners and, at the same time, themselves regulate cellular interaction networks. The use of kinase domains for creating a synthetic sensor device that reads low concentration protein-protein interactions and amplifies them to a higher concentration interaction which is then translated into a FRET (Fluorescence Resonance Energy Transfer) signal is here proposed. To this end, DNA constructs for interaction amplification (split kinases), positive controls (intact kinase domains), scaffolding proteins and phosphopeptide - SH2-domain modules for the reading of kinase activity were assembled and expression protocols for fusion proteins containing Lyn, Src, and Fak kinase domains in bacterial and in cell-free systems were optimized. Also, two non-overlapping methods for measuring the kinase activity of these proteins were stablished and, finally, a protein-fragment complementation assay with the split-kinase constructs was tested. In conclusion, it has been demonstrated that features such as codon optimization, vector design and expression conditions have an impact on the expression yield and activity of kinase-based proteins. Furthermore, it has been found that the defined PURE cell-free system is insufficient for the active expression of catalytic kinase domains. In contrast, the bacterial co-expression with phosphatases produced active kinase fusion proteins for two out of the three tested Tyrosine kinase domains.

  5. Multidrug resistance-selective antiproliferative activity of Piper amide alkaloids and synthetic analogues.

    Science.gov (United States)

    Wang, Yue-Hu; Goto, Masuo; Wang, Li-Ting; Hsieh, Kan-Yen; Morris-Natschke, Susan L; Tang, Gui-Hua; Long, Chun-Lin; Lee, Kuo-Hsiung

    2014-10-15

    Twenty-five amide alkaloids (1-25) from Piper boehmeriifolium and 10 synthetic amide alkaloid derivatives (39-48) were evaluated for antiproliferative activity against eight human tumor cell lines, including chemosensitive and multidrug-resistant (MDR) cell lines. The results suggested tumor type-selectivity. 1-[7-(3,4,5-Trimethoxyphenyl)heptanoyl]piperidine (46) exhibited the best inhibitory activity (IC50=4.94 μM) against the P-glycoprotein (P-gp)-overexpressing KBvin MDR sub-line, while it and all other tested compounds, except 9, were inactive (IC50 >40 μM) against MDA-MB-231 and SK-BR-3. Structure-activity relationships (SARs) indicated that (i) 3,4,5-trimethoxy phenyl substitution is critical for selectivity against KBvin, (ii) the 4-methoxy group in this pattern is crucial for antiproliferative activity, (iii) double bonds in the side chain are not needed for activity, and (iv), in arylalkenylacyl amide alkaloids, replacement of an isobutylamino group with pyrrolidin-1-yl or piperidin-1-yl significantly improved activity. Further study on Piper amides is warranted, particularly whether side chain length affects the ability to overcome the MDR cancer phenotype. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Discovery and Development of Synthetic and Natural Biomaterials for Protein Therapeutics and Medical Device Applications

    Science.gov (United States)

    Keefe, Andrew J.

    Controlling nonspecific protein interactions is important for applications from medical devices to protein therapeutics. The presented work is a compilation of efforts aimed at using zwitterionic (ionic yet charge neutral) polymers to modify and stabilize the surface of sensitive biomedical and biological materials. Traditionally, when modifying the surface of a material, the stability of the underlying substrate. The materials modified in this dissertation are unique due to their unconventional amorphous characteristics which provide additional challenges. These are poly(dimethyl siloxane) (PDMS) rubber, and proteins. These materials may seem dissimilar, but both have amorphous surfaces, that do not respond well to chemical modification. PDMS is a biomaterial extensively used in medical device manufacturing, but experiences unacceptably high levels of non-specific protein fouling when used with biological samples. To reduce protein fouling, surface modification is often needed. Unfortunately conventional surface modification methods, such as Poly(ethylene glycol) (PEG) coatings, do not work for PDMS due to its amorphous state. Herein, we demonstrate how a superhydrophilic zwitterionic material, poly(carboxybetaine methacrylate) (pCBMA), can provide a highly stable nonfouling coating with long term stability due to the sharp the contrast in hydrophobicity between pCBMA and PDMS. Biological materials, such as proteins, also require stabilization to improve shelf life, circulation time, and bioactivity. Conjugation of proteins with PEG is often used to increase protein stability, but has a detrimental effect on bioactivity. Here we have shown that pCBMA conjugation improves stability in a similar fashion to PEG, but also retains, or even improves, binding affinity due to enhanced protein-substrate hydrophobic interactions. Recognizing that pCBMA chemically resembles the combination of lysine (K) and glutamic acid (E) amino acids, we have shown how zwitterionic

  7. Modular design of synthetic protein mimics. Characterization of the helical conformation of a 13-residue peptide in crystals

    International Nuclear Information System (INIS)

    Karle, I.L.; Flippen-Anderson, J.L.; Uma, K.; Balaram, P.

    1989-01-01

    The incorporation of α-aminoisobutyryl (Aib) residues into peptide sequences facilitates helical folding. Aib-containing sequences have been chosen for the design of rigid helical segments in a modular approach to the construction of a synthetic protein mimic. The helical conformation of the synthetic peptide Boc-Aib-(Val-Ala-Leu-Aib) 3 -OMe in crystals is established by X-ray diffraction. The 13-residue apolar peptide adopts a helical form in the crystal with seven α-type hydrogen bonds in the middle and 3 10 -type hydrogen bonds at either end. The helices stack in columns, zigzag rather than linear, by means of direct NH hor-ellipsis OC head to tail hydrogen bonds. Leucyl side chains are extended on one side of the helix and valyl side chains on the other side. Water molecules form hydrogen bonds with several backbone carbonyl oxygens that also participate in α-helix hydrogen bonds. There is no apparent distortion of the helix caused by hydration

  8. "A Separation Theorem of Active Management and Synthetic Enhanced Active Strategies"(in Japanese)

    OpenAIRE

    Takao Kobayashi; Seiji Minami

    2008-01-01

    We propose a Separation Theorem of Active Management. It asserts that in the so-called Enhanced Active Portfolio framework the efficient frontier is linear in the active return/active risk space, and one can separate the determination of optimal active portfolio weights from the determination of optimal leverage ratio. The risk preference of investors does not play any role in the former decision. The theorem holds under a fairly general set of conditions on portfolio restrictions. As such it...

  9. Ellipsometric studies of synthetic albumin-binding chitosan-derivatives and selected blood plasma proteins

    Science.gov (United States)

    Sarkar, Sabyasachi

    This dissertation summarizes work on the synthesis of chitosan-derivatives and the development of ellipsometric methods to characterize materials of biological origin. Albumin-binding chitosan-derivatives were synthesized via addition reactions that involve amine groups naturally present in chitosan. These surfaces were shown to have an affinity towards human serum albumin via ELISA, UV spectroscopy and SDS PAGE. Modified surfaces were characterized with IR ellipsometry at various stages of their synthesis using appropriate optical models. It was found that spin cast chitosan films were anisotropic in nature. All optical models used for characterizing chitosan-derivatives were thus anisotropic. Chemical signal dependence on molecular structure and composition was illustrated via IR spectroscopic ellipsometry (IRSE). An anisotropic optical model of an ensemble of Lorentz oscillators were used to approximate material behavior. The presence of acetic acid in spin-cast non-neutralized chitosan samples was thus shown. IRSE application to biomaterials was also demonstrated by performing a step-wise chemical characterizations during synthesis stages. Protein adsorbed from single protein solutions on these modified surfaces was monitored by visible in-situ variable wavelength ellipsometry. Based on adsorption profiles obtained from single protein adsorption onto silicon surfaces, lumped parameter kinetic models were developed. These models were used to fit experimental data of immunoglobulin-G of different concentrations and approximate conformational changes in fibrinogen adsorption. Biomaterial characterization by ellipsometry was further extended to include characterization of individual protein solutions in the IR range. Proteins in an aqueous environment were characterized by attenuated total internal reflection (ATR) IR ellipsometry using a ZnSe prism. Parameterized dielectric functions were created for individual proteins using Lorentz oscillators. These

  10. Factors Influencing the Antifolate Activity of Synthetic Tea-Derived Catechins

    Directory of Open Access Journals (Sweden)

    José Neptuno Rodríguez-López

    2013-07-01

    Full Text Available Novel tea catechin derivatives have been synthesized, and a structure-activity study, related to the capacity of these and other polyphenols to bind dihydrofolate reductase (DHFR, has been performed. The data showed an effective binding between all molecules and the free enzyme, and the dissociation constants of the synthetic compounds and of the natural analogues were on the same order. Polyphenols with a catechin configuration were better DHFR inhibitors than those with an epicatechin configuration. Antiproliferative activity was also studied in cultured tumour cells, and the data showed that the activity of the novel derivatives was higher in catechin isomers. Derivatives with a hydroxyl group para on the ester-bonded gallate moiety presented a high in vitro binding to DHFR, but exhibited transport problems in cell culture due to ionization at physiologic pHs. The impact of the binding of catechins to serum albumin on their biological activity was also evaluated. The information provided in this study could be important for the design of novel medicinal active compounds derived from tea catechins. The data suggest that changes in their structure to avoid serum albumin interactions and to facilitate plasmatic membrane transport are essential for the intracellular functions of catechins.

  11. Parasite-Mediated Degradation of Synthetic Ozonide Antimalarials Impacts In Vitro Antimalarial Activity.

    Science.gov (United States)

    Giannangelo, Carlo; Stingelin, Lukas; Yang, Tuo; Tilley, Leann; Charman, Susan A; Creek, Darren J

    2018-03-01

    The peroxide bond of the artemisinins inspired the development of a class of fully synthetic 1,2,4-trioxolane-based antimalarials, collectively known as the ozonides. Similar to the artemisinins, heme-mediated degradation of the ozonides generates highly reactive radical species that are thought to mediate parasite killing by damaging critical parasite biomolecules. We examined the relationship between parasite dependent degradation and antimalarial activity for two ozonides, OZ277 (arterolane) and OZ439 (artefenomel), using a combination of in vitro drug stability and pulsed-exposure activity assays. Our results showed that drug degradation is parasite stage dependent and positively correlates with parasite load. Increasing trophozoite-stage parasitemia leads to substantially higher rates of degradation for both OZ277 and OZ439, and this is associated with a reduction in in vitro antimalarial activity. Under conditions of very high parasitemia (∼90%), OZ277 and OZ439 were rapidly degraded and completely devoid of activity in trophozoite-stage parasite cultures exposed to a 3-h drug pulse. This study highlights the impact of increasing parasite load on ozonide stability and in vitro antimalarial activity and should be considered when investigating the antimalarial mode of action of the ozonide antimalarials under conditions of high parasitemia. Copyright © 2018 American Society for Microbiology.

  12. Factors influencing the antifolate activity of synthetic tea-derived catechins.

    Science.gov (United States)

    Sáez-Ayala, Magalí; Fernández-Pérez, María Piedad; Chazarra, Soledad; Mchedlishvili, Nani; Tárraga-Tomás, Alberto; Rodríguez-López, José Neptuno

    2013-07-16

    Novel tea catechin derivatives have been synthesized, and a structure-activity study, related to the capacity of these and other polyphenols to bind dihydrofolate reductase (DHFR), has been performed. The data showed an effective binding between all molecules and the free enzyme, and the dissociation constants of the synthetic compounds and of the natural analogues were on the same order. Polyphenols with a catechin configuration were better DHFR inhibitors than those with an epicatechin configuration. Antiproliferative activity was also studied in cultured tumour cells, and the data showed that the activity of the novel derivatives was higher in catechin isomers. Derivatives with a hydroxyl group para on the ester-bonded gallate moiety presented a high in vitro binding to DHFR, but exhibited transport problems in cell culture due to ionization at physiologic pHs. The impact of the binding of catechins to serum albumin on their biological activity was also evaluated. The information provided in this study could be important for the design of novel medicinal active compounds derived from tea catechins. The data suggest that changes in their structure to avoid serum albumin interactions and to facilitate plasmatic membrane transport are essential for the intracellular functions of catechins.

  13. Measurement of Muscle Protein Fractional Synthetic Rate by Capillary Gas Chromatography/Combustion Isotope Ratio Mass Spectrometry

    Science.gov (United States)

    Yarasheski, Kevin E.; Smith, Kenneth; Rennie, Michael J.; Bier, Dennis M.

    2014-01-01

    The measurement of skeletal muscle protein fractional synthetic rate using an infusion of (1-13C)leucine and measuring the isotopic abundance of the tracer in skeletal muscle protein by preparative gas chromatography (GC)/ninhydrin isotope ratio mass spectrometry (IRMS) is laborious and subject to errors owing to contamination by 12C. The purpose of this study was to compare muscle (13C)leucine enrichment measured with the conventional preparative GC/ninhydrin IRMS approach to a new, continuous-flow technique using capillary GC/combustion IRMS. Quadriceps muscles were removed from four Sprague–Dawley rats after each was infused at a different rate with (1-13C)leucine for 6–8 h. Muscle leucine enrichment (at.% excess) measured by both methods differed by less than 4%, except at low (13C)leucine enrichments (IRMS was used to assess muscle (13C)leucine enrichment and fractional muscle protein synthesis rate in ten normal young men and women infused with (1,2-13C2)leucine for 12–14 h. This approach reduced the variability of the isotope abundance measure and gave estimates of muscle protein synthesis rate (0.050 ± 0.011% h−1 (mean ± SEM); range = 0.023–0.147% h−1) that agree with published values determined using the standard analytical approach. The measurement of (13C)leucine enrichment from skeletal muscle protein by capillary GC/combustion IRMS provides a simple, acceptable and practical alternative to preparative GC/ninhydrin IRMS. PMID:1420371

  14. Stability and Antioxidant Activity of Semi-synthetic Derivatives of 4-Nerolidylcatechol

    Directory of Open Access Journals (Sweden)

    Emerson Silva Lima

    2012-12-01

    Full Text Available 4-nerolidylcatechol (4-NC is an unstable natural product that exhibits important antioxidant, anti-inflammatory and other properties. It is readily obtainable on a multi-gram scale through straightforward solvent extraction of the roots of cultivated Piper peltatum or P. umbellatum, followed by column chromatography on the resulting extract. Semi-synthetic derivatives of 4-NC with one or two substituent groups (methyl, acetyl, benzyl, benzoyl on the O atoms have been introduced that have increased stability compared to 4-NC and significant in vitro inhibitory activity against the human malaria parasite Plasmodium falciparum. Antioxidant and anti-inflammatory properties may be important for the antiplasmodial mode of action of 4-NC derivatives. Thus, we decided to investigate the antioxidant properties, cytotoxicity and stability of 4-NC derivatives as a means to explore the potential utility of these compounds. 4-NC showed high antioxidant activity in the DPPH and ABTS assays and in 3T3-L1 cells (mouse embryonic fibroblast, however 4-NC was more cytotoxic (IC50 = 31.4 µM and more unstable than its derivatives and lost more than 80% of its antioxidant activity upon storage in solution at −20 °C for 30 days. DMSO solutions of mono-O-substituted derivatives of 4-NC exhibited antioxidant activity and radical scavenging activity in the DPPH and ABTS assays that was comparable to that of BHA and BHT. In the cell-based antioxidant model, most DMSO solutions of derivatives of 4-NC were less active on day 1 than 4-NC, quercetin and BHA and more active antioxidants than BHT. After storage for 30 days at −20 °C, DMSO solutions of most of the derivatives of 4-NC were more stable and exhibited more antioxidant activity than 4-NC, quercetin and BHA and exhibited comparable antioxidant activity to BHT. These findings point to the potential of derivatives of 4-NC as antioxidant compounds.

  15. Evaluation of dum palm kernel activated carbon in chromium and lead adsorption from synthetic waste water

    Directory of Open Access Journals (Sweden)

    HI Mohammed

    2016-09-01

    Full Text Available This study aimed at the co-current removal of chromium and lead ions from synthetic waste water using dum palm kernel activated carbon. The adsorption experiment was conducted by varying time, pH and concentrations of the simulated solution. The data obtained were analyzed, and the best conditions for the uptake were at pH of 6, equilibrium time of 40 minutes. The two best isotherms models for the adsorption system were Sip, and Dubinin–Radushkevich, models respectively. Based on the Temkin adsorption energy calculated as 9.5793 and 0.4997 J/mol, the uptake of lead and chromium were chemisorption and physico-sorption, respectively. The maximum uptake calculated from Dubinin–Radushkevich plots were 14.1696 and 7.7191 mg/g, for lead and chromium, respectively.International Journal of Environment Vol.5(3 2016, pp.104-118

  16. Identification and Biological Activity of Synthetic Macrophage Inducible C-Type Lectin Ligands

    Directory of Open Access Journals (Sweden)

    Chriselle D. Braganza

    2018-01-01

    Full Text Available The macrophage inducible C-type lectin (Mincle is a pattern recognition receptor able to recognize both damage-associated and pathogen-associated molecular patterns, and in this respect, there has been much interest in determining the scope of ligands that bind Mincle and how structural modifications to these ligands influence ensuing immune responses. In this review, we will present Mincle ligands of known chemical structure, with a focus on ligands that have been synthetically prepared, such as trehalose glycolipids, glycerol-based ligands, and 6-acylated glucose and mannose derivatives. The ability of the different classes of ligands to influence the innate, and consequently, the adaptive, immune response will be described, and where appropriate, structure–activity relationships within each class of Mincle ligands will be presented.

  17. Humoral and cellular immune responses to synthetic peptides of the Leishmania donovani kinetoplastid membrane protein-11

    DEFF Research Database (Denmark)

    Jensen, A T; Gasim, S; Ismail, A

    1998-01-01

    as solid-phase ligands in enzyme-linked immunosorbent assays (ELISAs) and as stimulating antigens in lymphoproliferative assays in order to evaluate humoral and cellular immune responses to well-defined sequences of the protein. Antibody reactivity against the three peptides was measured in plasma from 63...

  18. The use of synthetic peptides for detection of anti-citrullinated protein antibodies in rheumatoid arthritis

    DEFF Research Database (Denmark)

    Trier, Nicole Hartwig; Holm, Bettina Eide; Heiden, Julie

    2018-01-01

    Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology. A characteristic feature of RA is the presence of anti-citrullinated protein antibodies (ACPA). Since ACPAs are highly specific for RA and are often present before the onset of RA symptoms, they have become valuable diagnostic...

  19. Novel synthetic approach to the prion protein: Kinetic study optimization of a native chemical ligation

    Czech Academy of Sciences Publication Activity Database

    Zawada, Zbigniew; Šebestík, Jaroslav; Bouř, Petr; Hlaváček, Jan; Stibor, Ivan

    2008-01-01

    Roč. 14, č. 8 (2008), s. 76-77 ISSN 1075-2617. [European Peptide Symposium /30./. 31.08.2008-05.09.2008, Helsinki] R&D Projects: GA ČR GA203/07/1517 Institutional research plan: CEZ:AV0Z40550506 Keywords : prion protein * neurodegenerative diseases * chemical synthesis * ligation conditions Subject RIV: CC - Organic Chemistry

  20. Protein and synthetic polymer injection for induction of obstructive hydrocephalus in rats

    Directory of Open Access Journals (Sweden)

    Del Bigio Marc R

    2007-09-01

    Full Text Available Abstract Background The objective of this study was to develop a simple and inexpensive animal model of induced obstructive hydrocephalus with minimal tissue inflammation, as an alternative to kaolin injection. Materials Two-hundred and two male Sprague-Dawley rats aged 3 weeks received intracisternal injections of kaolin (25% suspension, Matrigel, type 1 collagen from rat tail, fibrin glue (Tisseel, n-butyl-cyanoacrylate (NBCA, or ethylene vinyl alcohol copolymer (Onyx-18 and Onyx-34. Magnetic resonance imaging was used to assess ventricle size. Animals were euthanized at 2, 5, 10 and 14 days post-injection for histological analysis. Results Kaolin was associated with 10% mortality and successful induction of hydrocephalus in 97% of survivors (ventricle area proportion 0.168 ± 0.018. Rapidly hardening agents (fibrin glue, NBCA, vinyl polymer had high mortality rates and low success rates in survivors. Only Matrigel had relatively low mortality (17% and moderate success rate (20%. An inflammatory response with macrophages and some lymphocytes was associated with kaolin. There was negligible inflammation associated with Matrigel. A severe inflammatory response with giant cell formation was associated with ethylene vinyl alcohol copolymer. Conclusion Kaolin predictably produces moderate to severe hydrocephalus with a mild chronic inflammatory reaction and fibrosis of the leptomeninges. Other synthetic polymers and biopolymers tested are unreliable and cause different types of inflammation.

  1. Photocatalytic Role of Zinc Oxide Nanoparticles on Synthetic Activated Carbon to Remove Antibiotic from Aquatic Environment

    Directory of Open Access Journals (Sweden)

    Mohammad Reza Samarghandi

    2017-10-01

    Full Text Available Background & Aims of the Study: The presence of antibiotics in the environment, especially in aquatic environments is a major concern for health and the environment. The advanced oxidation process due to the ease of use, economical advantages and high performance have attracted a lot of attention. The purpose of this study was Evaluating of the photocatalytic role of zinc oxide on synthetic activated carbon to remove antibiotic from aquatic environment. Materials & Methods: This experimental study was done in batch reactor that has a 1 L volume. In this study effect of parameters such as initial pH (3-9, initial concentration of cefazolin (20-200 mg/L, modified photocatalyst concentration (20-100 mg/L and reaction time (10-60 min was investigated. In this study a low-pressure mercury lamp with the power of 55 watts in stainless case has been used. The cefazolin concentrations in different steps were measured using UV-Vis spectrophotometer in Wavelength of 262 nm. Results: The results showed that the highest removal efficiency (96% of cefazolin was at the pH=3, 0.1 mg/L of modified photocatalyst, retention time of 60 min and cefazolin concentrations of 100 mg/L. In the case of changing any of the above mentioned values, process efficiency was decreased. Conclusion: The results showed that the photocatalytic process of zinc oxide nanoparticles on synthetic activated carbon can be used as an advanced oxidation process to effectively remove pollutants like cefazolin and other similar pollutants.

  2. Breast cancer stem cell selectivity of synthetic nanomolar-active salinomycin analogs

    International Nuclear Information System (INIS)

    Huang, Xiaoli; Borgström, Björn; Kempengren, Sebastian; Persson, Lo; Hegardt, Cecilia; Strand, Daniel; Oredsson, Stina

    2016-01-01

    Cancer stem cells (CSCs) have been invoked in resistance, recurrence and metastasis of cancer. Consequently, curative cancer treatments may be contingent on CSC selective approaches. Of particular interest in this respect is the ionophore salinomycin, a natural product shown to be 100-fold more active against CSCs than clinically used paclitaxel. We have previously reported that synthetic salinomycin derivatives display increased activity against breast cancer cell lines. Herein we specifically investigate the CSC selectivity of the most active member in each class of C20-O-acylated analogs as well as a C1-methyl ester analog incapable of charge-neutral metal ion transport. JIMT-1 breast cancer cells were treated with three C20-O-acylated analogs, the C1-methyl ester of salinomycin, and salinomycin. The effects of treatment on the CSC-related CD44 + /CD24 − and the aldehyde dehydrogenase positive (ALDH + ) populations were determined using flow cytometry. The survival ability of CSCs after treatment was investigated with a colony formation assay under serum free conditions. The effect of the compounds on cell migration was evaluated using wound-healing and Boyden chamber assays. The expression of vimentin, related to mesenchymal traits and expression of E-cadherin and β-catenin, related to the epithelial traits, were investigated using immunofluorescence microscopy. Treatment with each of the three C20-acylated analogs efficiently decreased the putative CSC population as reflected by reduction of the CD44 + /CD24 − and ALDH + populations already at a 50 nM concentration. In addition, colony forming efficiency and cell migration were reduced, and the expression of the epithelial markers E-cadherin and β-catenin at the cell surface were increased. In contrast, salinomycin used at the same concentration did not significantly influence the CSC population and the C1-methyl ester was inactive even at a 20 μM concentration. Synthetic structural analogs of

  3. Breast cancer stem cell selectivity of synthetic nanomolar-active salinomycin analogs.

    Science.gov (United States)

    Huang, Xiaoli; Borgström, Björn; Kempengren, Sebastian; Persson, Lo; Hegardt, Cecilia; Strand, Daniel; Oredsson, Stina

    2016-02-23

    Cancer stem cells (CSCs) have been invoked in resistance, recurrence and metastasis of cancer. Consequently, curative cancer treatments may be contingent on CSC selective approaches. Of particular interest in this respect is the ionophore salinomycin, a natural product shown to be 100-fold more active against CSCs than clinically used paclitaxel. We have previously reported that synthetic salinomycin derivatives display increased activity against breast cancer cell lines. Herein we specifically investigate the CSC selectivity of the most active member in each class of C20-O-acylated analogs as well as a C1-methyl ester analog incapable of charge-neutral metal ion transport. JIMT-1 breast cancer cells were treated with three C20-O-acylated analogs, the C1-methyl ester of salinomycin, and salinomycin. The effects of treatment on the CSC-related CD44(+)/CD24(-) and the aldehyde dehydrogenase positive (ALDH(+)) populations were determined using flow cytometry. The survival ability of CSCs after treatment was investigated with a colony formation assay under serum free conditions. The effect of the compounds on cell migration was evaluated using wound-healing and Boyden chamber assays. The expression of vimentin, related to mesenchymal traits and expression of E-cadherin and β-catenin, related to the epithelial traits, were investigated using immunofluorescence microscopy. Treatment with each of the three C20-acylated analogs efficiently decreased the putative CSC population as reflected by reduction of the CD44(+)/CD24(-) and ALDH(+) populations already at a 50 nM concentration. In addition, colony forming efficiency and cell migration were reduced, and the expression of the epithelial markers E-cadherin and β-catenin at the cell surface were increased. In contrast, salinomycin used at the same concentration did not significantly influence the CSC population and the C1-methyl ester was inactive even at a 20 μM concentration. Synthetic structural analogs of

  4. Petunia nectar proteins have ribonuclease activity.

    Science.gov (United States)

    Hillwig, Melissa S; Liu, Xiaoteng; Liu, Guangyu; Thornburg, Robert W; Macintosh, Gustavo C

    2010-06-01

    Plants requiring an insect pollinator often produce nectar as a reward for the pollinator's visitations. This rich secretion needs mechanisms to inhibit microbial growth. In Nicotiana spp. nectar, anti-microbial activity is due to the production of hydrogen peroxide. In a close relative, Petunia hybrida, limited production of hydrogen peroxide was found; yet petunia nectar still has anti-bacterial properties, suggesting that a different mechanism may exist for this inhibition. The nectar proteins of petunia plants were compared with those of ornamental tobacco and significant differences were found in protein profiles and function between these two closely related species. Among those proteins, RNase activities unique to petunia nectar were identified. The genes corresponding to four RNase T2 proteins from Petunia hybrida that show unique expression patterns in different plant tissues were cloned. Two of these enzymes, RNase Phy3 and RNase Phy4 are unique among the T2 family and contain characteristics similar to both S- and S-like RNases. Analysis of amino acid patterns suggest that these proteins are an intermediate between S- and S-like RNases, and support the hypothesis that S-RNases evolved from defence RNases expressed in floral parts. This is the first report of RNase activities in nectar.

  5. Resolving Electronic Transitions in Synthetic Fluorescent Protein Chromophores by Magnetic Circular Dichroism

    Czech Academy of Sciences Publication Activity Database

    Štěpánek, P.; Cowie, T. Y.; Šafařík, Martin; Šebestík, Jaroslav; Pohl, Radek; Bouř, Petr

    2016-01-01

    Roč. 17, č. 15 (2016), s. 2348-2354 ISSN 1439-4235 R&D Projects: GA ČR GA13-03978S; GA ČR(CZ) GA16-05935S Institutional support: RVO:61388963 Keywords : density functional calculations * fluorescence protein chromophores * magnetic circular dichroism * organic synthesis * spectral simulations Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.075, year: 2016

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

    Indian Academy of Sciences (India)

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

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

    International Nuclear Information System (INIS)

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

    2008-01-01

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

  8. Effects of Dietary Crude Protein Levels and Cysteamine Supplementation on Protein Synthetic and Degradative Signaling in Skeletal Muscle of Finishing Pigs.

    Directory of Open Access Journals (Sweden)

    Ping Zhou

    Full Text Available Dietary protein levels and cysteamine (CS supplementation can affect growth performance and protein metabolism of pigs. However, the influence of dietary protein intake on the growth response of CS-treated pigs is unclear, and the mechanisms involved in protein metabolism remain unknown. Hence, we investigated the interactions between dietary protein levels and CS supplementation and the effects of dietary crude protein levels and CS supplementation on protein synthetic and degradative signaling in skeletal muscle of finishing pigs. One hundred twenty barrows (65.84 ± 0.61 kg were allocated to a 2 × 2 factorial arrangement with five replicates of six pigs each. The primary variations were dietary crude protein (CP levels (14% or 10% and CS supplemental levels (0 or 700 mg/kg. The low-protein (LP diets (10% CP were supplemented with enough essential amino acids (EAA to meet the NRC AA requirements of pigs and maintain the balanced supply of eight EAA including lysine, methionine, threonine, tryptophan, valine, phenylalanine, isoleucine, and leucine. After 41 days, 10 pigs per treatment were slaughtered. We found that LP diets supplemented with EAA resulted in decreased concentrations of plasma somatostatin (SS (P<0.01 and plasma urea nitrogen (PUN (P<0.001, while dietary protein levels did not affect other traits. However, CS supplementation increased the average daily gain (P<0.001 and lean percentage (P<0.05, and decreased the feed conversion ratio (P<0.05 and back fat (P<0.05. CS supplementation also increased the concentrations of plasma insulin-like growth factor 1 (IGF-1 (P<0.001, and reduced the concentrations of leptin, SS, and PUN (P<0.001. Increased mRNA abundance of Akt1 and IGF-1 signaling (P<0.001 and decreased mRNA abundance of Forkhead Box O (FOXO 4 (P<0.01 and muscle atrophy F-box (P<0.001 were observed in pigs receiving CS. Additionally, CS supplementation increased the protein levels for the phosphorylated mammalian target of

  9. Bioorthogonal chemistry: applications in activity-based protein profiling.

    Science.gov (United States)

    Willems, Lianne I; van der Linden, Wouter A; Li, Nan; Li, Kah-Yee; Liu, Nora; Hoogendoorn, Sascha; van der Marel, Gijs A; Florea, Bogdan I; Overkleeft, Herman S

    2011-09-20

    of chemical biology research include contributions from many areas of the multifaceted discipline of chemistry, and particularly from organic chemistry. Researchers apply knowledge inherent to organic chemistry, such as reactivity and selectivity, to the manipulation of specific biomolecules in biological samples (cell extracts, living cells, and sometimes even animal models) to gain insight into the biological phenomena in which these molecules participate. In this Account, we highlight some of the recent developments in chemical biology research driven by organic chemistry, with a focus on bioorthogonal chemistry in relation to activity-based protein profiling. The rigorous demands of bioorthogonality have not yet been realized in a truly bioorthogonal reagent pair, but remarkable progress has afforded a range of tangible contributions to chemical biology research. Activity-based protein profiling, which aims to obtain information on the workings of a protein (or protein family) within the larger context of the full biological system, has in particular benefited from these advances. Both activity-based protein profiling and bioorthogonal chemistry have been around for approximately 15 years, and about 8 years ago the two fields very profitably intersected. We expect that each discipline, both separately and in concert, will continue to make important contributions to chemical biology research. © 2011 American Chemical Society

  10. Free-Radical-Scavenging, Antityrosinase, and Cellular Melanogenesis Inhibitory Activities of Synthetic Isoflavones.

    Science.gov (United States)

    Lu, Tzy-Ming; Ko, Horng-Huey; Ng, Lean-Teik; Hsieh, Yen-Pin

    2015-06-01

    In this study, we examined the potential of synthetic isoflavones for application in cosmeceuticals. Twenty-five isoflavones were synthesized and their capacities of free-radical-scavenging and mushroom tyrosinase inhibition, as well as their impact on cell viability of B16F10 murine melanoma cells and HaCaT human keratinocytes were evaluated. Isoflavones that showed significant mushroom tyrosinase inhibitory activities were further studied on reduction of cellular melanin formation and antityrosinase activities in B16F10 melanocytes in vitro. Among the isoflavones tested, 6-hydroxydaidzein (2) was the strongest scavenger of both ABTS(.+) and DPPH(.) radicals with SC50 values of 11.3 ± 0.3 and 9.4 ± 0.1 μM, respectively. Texasin (20) exhibited the most potent inhibition of mushroom tyrosinase (IC50 14.9 ± 4.5 μM), whereas retusin (17) showed the most efficient inhibition both of cellular melanin formation and antityrosinase activity in B16F10 melanocytes, respectively. In summary, both retusin (17) and texasin (20) exhibited potent free-radical-scavenging capacities as well as efficient inhibition of cellular melanogenesis, suggesting that they are valuable hit compounds with potential for advanced cosmeceutical development. Copyright © 2015 Verlag Helvetica Chimica Acta AG, Zürich.

  11. Semi-synthetic salinomycin analogs exert cytotoxic activity against human colorectal cancer stem cells.

    Science.gov (United States)

    Klose, Johannes; Kattner, Sarah; Borgström, Björn; Volz, Claudia; Schmidt, Thomas; Schneider, Martin; Oredsson, Stina; Strand, Daniel; Ulrich, Alexis

    2018-01-01

    Salinomycin, a polyether antibiotic, is a well-known inhibitor of human cancer stem cells. Chemical modification of the allylic C20 hydroxyl of salinomycin has enabled access to synthetic analogs that display increased cytotoxic activity compared to the native structure. The aim of this study was to investigate the activity of a cohort of C20-O-acyl analogs of salinomycin on human colorectal cancer cell lines in vitro. Two human colorectal cancer cell lines (SW480 and SW620) were exposed to three C20-O-acylated analogs and salinomycin. The impact of salinomycin and its analogs on tumor cell number, migration, cell death, and cancer stem cell specifity was analyzed. Exposure of human colorectal cancer cells to the C20-O-acylated analogs of salinomycin resulted in reduced tumor cell number and impaired tumor cell migration at lower concentrations than salinomycin. When used at higher (micromolar) concentrations, these effects were accompanied by induction of apoptotic cell death. Salinomycin analogs further expose improved activity against cancer stem cells compared to salinomycin. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. The Synthetic Lignan Secoisolariciresinol Diglucoside Prevents Asbestos-Induced NLRP3 Inflammasome Activation in Murine Macrophages

    Directory of Open Access Journals (Sweden)

    Ralph A. Pietrofesa

    2017-01-01

    Full Text Available Background. The interaction of asbestos with macrophages drives two key processes that are linked to malignancy: (1 the generation of reactive oxygen species (ROS/reactive nitrogen species (RNS and (2 the activation of an inflammation cascade that drives acute and chronic inflammation, with the NLRP3 inflammasome playing a key role. Synthetic secoisolariciresinol diglucoside (SDG, LGM2605, is a nontoxic lignan with anti-inflammatory and antioxidant properties and was evaluated for protection from asbestos in murine peritoneal macrophages (MF. Methods. MFs were exposed to crocidolite asbestos ± LGM2605 given 4 hours prior to exposure and evaluated at various times for NLRP3 expression, secretion of inflammasome-activated cytokines (IL-1β and IL-18, proinflammatory cytokines (IL-6, TNFα, and HMGB1, NF-κB activation, and levels of total nitrates/nitrites. Results. Asbestos induces a significant (p<0.0001 increase in the NLRP3 subunit, release of proinflammatory cytokines, NLRP3-activated cytokines, NF-κB, and levels of nitrates/nitrites. LGM2605 significantly reduced NLRP3 ranging from 40 to 81%, IL-1β by 89–96%, and TNFα by 67–78%, as well as activated NF-κB by 48-49% while decreasing levels of nitrates/nitrites by 85–93%. Conclusions. LGM2605 reduced asbestos-induced NLRP3 expression, proinflammatory cytokine release, NF-κB activation, and nitrosative stress in MFs supporting its possible use in preventing the asbestos-induced inflammatory cascade leading to malignancy.

  13. Effects of Synthetic Neural Adhesion Molecule Mimetic Peptides and Related Proteins on the Cardiomyogenic Differentiation of Mouse Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Ruodan Xu

    2015-04-01

    Full Text Available Background/Aims: Pluripotent stem cells differentiating into cardiomyocyte-like cells in an appropriate cellular environment have attracted significant attention, given the potential use of such cells for regenerative medicine. However, the precise mechanisms of lineage specification of pluripotent stem cells are still largely to be explored. Identifying the role of various small synthetic peptides involved in cardiomyogenesis may provide new insights into pathways promoting cardiomyogenesis. Methods: In the present study, using a transgenic murine embryonic stem (ES cell lineage expressing enhanced green fluorescent protein (EGFP under the control of α-myosin heavy chain (α-MHC promoter (pαMHC-EGFP, we investigated the cardiomyogenic effects of 7 synthetic peptides (Betrofin3, FGLs, FGLL, hNgf_C2, EnkaminE, Plannexin and C3 on cardiac differentiation. The expression of several cardiac-specific markers was determined by RT-PCR whereas the structural and functional properties of derived cardiomyocytes were examined by immunofluorescence and electrophysiology, respectively. Results: The results revealed that Betrofin3, an agonist of brain derived neurotrophic factor (BDNF peptide exerted the most striking pro-cardiomyogenic effect on ES cells. We found that BDNF receptor, TrkB expression was up-regulated during differentiation. Treatment of differentiating cells with Betrofin3 between days 3 and 5 enhanced the expression of cardiac-specific markers and improved cardiomyocyte differentiation and functionality as revealed by genes regulation, flow cytometry and patch clamp analysis. Thus Betrofin3 may exert its cardiomyogenic effects on ES cells via TrkB receptor. Conclusion: Taken together, the results suggest that Betrofin3 modulates BDNF signaling with positive cardiomyogenic effect in stage and dose-dependent manner providing an effective strategy to increase ES cell-based generation of cardiomyocytes and offer a novel therapeutic approach to

  14. A Synthetic Ecology Perspective: How Well Does Behavior of Model Organisms in the Laboratory Predict Microbial Activities in Natural Habitats?

    Science.gov (United States)

    Yu, Zheng; Krause, Sascha M B; Beck, David A C; Chistoserdova, Ludmila

    2016-01-01

    In this perspective article, we question how well model organisms, the ones that are easy to cultivate in the laboratory and that show robust growth and biomass accumulation, reflect the dynamics and interactions of microbial communities observed in nature. Today's -omics toolbox allows assessing the genomic potential of microbes in natural environments in a high-throughput fashion and at a strain-level resolution. However, understanding of the details of microbial activities and of the mechanistic bases of community function still requires experimental validation in simplified and fully controlled systems such as synthetic communities. We have studied methane utilization in Lake Washington sediment for a few decades and have identified a number of species genetically equipped for this activity. We have also identified co-occurring satellite species that appear to form functional communities together with the methanotrophs. Here, we compare experimental findings from manipulation of natural communities involved in metabolism of methane in this niche with findings from manipulation of synthetic communities assembled in the laboratory of species originating from the same study site, from very simple (two-species) to rather complex (50-species) synthetic communities. We observe some common trends in community dynamics between the two types of communities, toward representation of specific functional guilds. However, we also identify strong discrepancies between the dominant methane oxidizers in synthetic communities compared to natural communities, under similar incubation conditions. These findings highlight the challenges that exist in using the synthetic community approach to modeling dynamics and species interactions in natural communities.

  15. Synthetic secoisolariciresinol diglucoside (LGM2605) inhibits myeloperoxidase activity in inflammatory cells.

    Science.gov (United States)

    Mishra, Om P; Popov, Anatoliy V; Pietrofesa, Ralph A; Nakamaru-Ogiso, Eiko; Andrake, Mark; Christofidou-Solomidou, Melpo

    2018-06-01

    Myeloperoxidase (MPO) generates hypochlorous acid (HOCl) during inflammation and infection. We showed that secoisolariciresinol diglucoside (SDG) scavenges radiation-induced HOCl in physiological solutions. However, the action of SDG and its synthetic version, LGM2605, on MPO-catalyzed generation of HOCl is unknown. The present study evaluated the effect of LGM2605 on human MPO, and murine MPO from macrophages and neutrophils. MPO activity was determined fluorometrically using hypochlorite-specific 3'-(p-aminophenyl) fluorescein (APF). The effect of LGM2605 on (a) the peroxidase cycle of MPO was determined using Amplex Red while the effect on (b) the chlorination cycle was determined using a taurine chloramine assay. Using electron paramagnetic resonance (EPR) spectroscopy we determined the effect of LGM2605 on the EPR signals of MPO. Finally, computational docking of SDG was used to identify energetically favorable docking poses to enzyme's active site. LGM2605 inhibited human and murine MPO activity. MPO inhibition was observed in the absence and presence of Cl - . EPR confirmed that LGM2605 suppressed the formation of Compound I, an oxoiron (IV) intermediate [Fe(IV)O] containing a porphyrin π-radical of MPO's catalytic cycle. Computational docking revealed that SDG can act as an inhibitor by binding to the enzyme's active site. We conclude that LGM2605 inhibits MPO activity by suppressing both the peroxidase and chlorination cycles. EPR analysis demonstrated that LGM2605 inhibits MPO by decreasing the formation of the highly oxidative Compound I. This study identifies a novel mechanism of LGM2605 action as an inhibitor of MPO and indicates that LGM2605 may be a promising attenuator of oxidant-dependent inflammatory tissue damage. Copyright © 2018 Elsevier B.V. All rights reserved.

  16. Chloroquine inhibits accessory cell presentation of soluble natural and synthetic protein antigens

    DEFF Research Database (Denmark)

    Buus, S; Werdelin, O

    1984-01-01

    We have studied the in vitro effect of the lysosomotrophic agent, chloroquine, on the presentation of soluble protein antigens by guinea pig accessory cells. Chloroquine inhibited the capacity of antigen-pulsed accessory cells to stimulate proliferation in appropriately primed T cells. The effect...... was time- and dose-dependent. A brief treatment solely of the accessory cells with the drug compromised their ability to stimulate primed T cells in a subsequent culture provided the accessory cells were treated with chloroquine before their exposure to the antigen. These results suggest that chloroquine...... acts on an early event in the antigen handling by accessory cells. Chloroquine is a well known inhibitor of lysosomal proteolysis, and it is likely that its effect on antigen presentation is caused by an inhibition of antigen degradation....

  17. Topical application of the synthetic triterpenoid RTA 408 activates Nrf2 and induces cytoprotective genes in rat skin.

    Science.gov (United States)

    Reisman, Scott A; Lee, Chun-Yue I; Meyer, Colin J; Proksch, Joel W; Ward, Keith W

    2014-07-01

    RTA 408 is a member of the synthetic oleanane triterpenoid class of compounds known to potently activate the cytoprotective transcription factor Nrf2. Because skin is constantly exposed to external oxidative stress, such as that from ultraviolet radiation, from chemical exposure, during improper wound healing, and throughout the course of cancer radiation therapy, it may benefit from activation of Nrf2. This study was conducted to evaluate the transdermal penetration properties and Nrf2 activation potential of RTA 408 in normal rat skin. RTA 408 (0.1, 1.0, or 3.0%) was applied topically to the shaved skin of male Sprague-Dawley rats twice daily for 4 days and once on Day 5. Topical application of RTA 408 resulted in transdermal penetration, with low but dose-dependent plasma exposure with AUC(0-24 h) values of 3.6, 26.0, and 41.1 h ng/mL for the 0.1, 1.0, and 3.0% doses, respectively. Further, topical application of RTA 408 resulted in increased translocation of Nrf2 to the nucleus, dose-dependent mRNA induction of Nrf2 target genes (e.g. Nqo1, Srxn1, Gclc, and Gclm), and induction of the protein expression of the prototypical Nrf2 target gene Nqo1 and increased total glutathione (GSH) in normal rat skin. Immunohistochemistry demonstrated that increased staining for Nqo1 and total GSH of structures in both the epidermis and dermis was consistent with the full transdermal penetration of RTA 408. Finally, topically administered RTA 408 was well tolerated with no adverse in-life observations and normal skin histology. Thus, the data support the further development of RTA 408 for the potential treatment of skin diseases.

  18. Remotely Sensed Active Layer Thickness (ReSALT at Barrow, Alaska Using Interferometric Synthetic Aperture Radar

    Directory of Open Access Journals (Sweden)

    Kevin Schaefer

    2015-03-01

    Full Text Available Active layer thickness (ALT is a critical parameter for monitoring the status of permafrost that is typically measured at specific locations using probing, in situ temperature sensors, or other ground-based observations. Here we evaluated the Remotely Sensed Active Layer Thickness (ReSALT product that uses the Interferometric Synthetic Aperture Radar technique to measure seasonal surface subsidence and infer ALT around Barrow, Alaska. We compared ReSALT with ground-based ALT obtained using probing and calibrated, 500 MHz Ground Penetrating Radar at multiple sites around Barrow. ReSALT accurately reproduced observed ALT within uncertainty of the GPR and probing data in ~76% of the study area. However, ReSALT was less than observed ALT in ~22% of the study area with well-drained soils and in ~1% of the area where soils contained gravel. ReSALT was greater than observed ALT in some drained thermokarst lake basins representing ~1% of the area. These results indicate remote sensing techniques based on InSAR could be an effective way to measure and monitor ALT over large areas on the Arctic coastal plain.

  19. Activity of Genital Tract Secretions and Synthetic Antimicrobial Peptides against Group B Streptococcus.

    Science.gov (United States)

    Agarwal, Nidhi; Buckley, Niall; Nakra, Natasha; Gialanella, Philip; Yuan, Weirong; Ghartey, Jeny P

    2015-12-01

    Genital tract secretions inhibit Escherichia coli (E. coli) through antimicrobial peptides (AMP) secreted by the host and vaginal microbiota. However, there are limited data against group B Streptococcus (GBS). Group B Streptococcus were incubated with cervico-vaginal lavage (CVL) samples from healthy non-pregnant women (n = 12) or synthetic AMP and monitored for bacterial growth using a turbidimetric approach. E. coli inhibitory activity was determined by a colony-forming unit assay. None of the CVL samples inhibited GBS. The human neutrophil peptide-1 and human defensin 5 inhibited GBS growth by ≥80% at concentrations ≥20 μg/mL and ≥50 μg/mL, respectively, while human beta-defensin 2 and LL-37 did not inhibit at highest concentration tested (100 μg/mL). In contrast, all AMP inhibited E. coli. Antimicrobial peptides may protect against E. coli colonization but have more limited activity against GBS. Future studies will focus on augmenting host defense with specific AMP to prevent genitourinary infection with these pathogenic organisms. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  20. Raman optical activity of proteins and glycoproteins

    International Nuclear Information System (INIS)

    Smyth, E.

    2000-03-01

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

  1. Improving serodiagnosis of human and canine leishmaniasis with recombinant Leishmania braziliensis cathepsin l-like protein and a synthetic peptide containing its linear B-cell epitope.

    Directory of Open Access Journals (Sweden)

    Daniel Menezes-Souza

    2015-01-01

    Full Text Available The early and correct diagnosis of human leishmaniasis is essential for disease treatment. Another important step in the control of visceral leishmaniasis is the identification of infected dogs, which are the main domestic reservoir of L. infantum. Recombinant proteins and synthetic peptides based on Leishmania genes have emerged as valuable targets for serodiagnosis due to their increased sensitivity, specificity and potential for standardization. Cathepsin L-like genes are surface antigens that are secreted by amastigotes and have little similarity to host proteins, factors that enable this protein as a good target for serodiagnosis of the leishmaniasis.We mapped a linear B-cell epitope within the Cathepsin L-like protein from L. braziliensis. A synthetic peptide containing the epitope and the recombinant protein was evaluated for serodiagnosis of human tegumentary and visceral leishmaniasis, as well as canine visceral leishmaniasis.The recombinant protein performed best for human tegumentary and canine visceral leishmaniasis, with 96.30% and 89.33% accuracy, respectively. The synthetic peptide was the best to discriminate human visceral leishmaniasis, with 97.14% specificity, 94.55% sensitivity and 96.00% accuracy. Comparison with T. cruzi-infected humans and dogs suggests that the identified epitope is specific to Leishmania parasites, which minimizes the likelihood of cross-reactions.

  2. NanoShuttles: Harnessing Motor Proteins to Transport Cargo in Synthetic Environments

    Science.gov (United States)

    Vogel, V.; Hess, H.

    Motors have become a crucial commodity in our daily lives, from transportation to driving conveyor belts that enable the sequential assembly of cars and other industrial machines. For the sequential assembly of building blocks at the nanoscale that would not assemble spontaneously into larger functional systems, however, active transport systems are not yet available. In contrast, cells have evolved sophisticated molecular machinery that drives movement and active transport. Driven by the conversion of chemical into mechanical energy, namely through hydrolysis of the biological fuel ATP, molecular motors enable cells to operate far away from equilibrium by transporting organelles and molecules to designated locations within the cell, often against concentration gradients. Inspired by the biological concept of active transport, major efforts are underway to learn how to build nanoscale transport systems that are driven by molecular motors. Emerging engineering principles are discussed of how to build tracks and junctions to guide such nanoshuttles, how to load them with cargo and control their speed, how to use active transport to assemble mesoscopic structures that would otherwise not assemble spontaneously and what polymeric materials to choose to integrate motors into MEMS and other biohybrid devices. Finally, two applications that exploit the physical properties of microtubules are discussed, surface imaging by a swarm of microtubules and a self-assembled picoNewton force meter to probe receptor-ligand interactions.

  3. Insulinotropic and Muscle Protein Synthetic Effects of Branched-Chain Amino Acids: Potential Therapy for Type 2 Diabetes and Sarcopenia

    Directory of Open Access Journals (Sweden)

    Darren G. Candow

    2012-11-01

    Full Text Available The loss of muscle mass and strength with aging (i.e., sarcopenia has a negative effect on functional independence and overall quality of life. One main contributing factor to sarcopenia is the reduced ability to increase skeletal muscle protein synthesis in response to habitual feeding, possibly due to a reduction in postprandial insulin release and an increase in insulin resistance. Branched-chain amino acids (BCAA, primarily leucine, increases the activation of pathways involved in muscle protein synthesis through insulin-dependent and independent mechanisms, which may help counteract the “anabolic resistance” to feeding in older adults. Leucine exhibits strong insulinotropic characteristics, which may increase amino acid availability for muscle protein synthesis, reduce muscle protein breakdown, and enhance glucose disposal to help maintain blood glucose homeostasis.

  4. Response of Anastrepha suspensa to liquid protein baits and synthetic lure formulations

    International Nuclear Information System (INIS)

    Epsky, Nancy D.; Kendra, Paul E.; Heath, Robert R.

    2006-01-01

    Traps baited with AAPt captured more A. suspensa than traps baited with ABPt even when the ammonia release rates were similar. Reducing dosage of ammonia by 50% of the commercially available AA lure slightly increased female capture, but reducing dosage to 25% tended to decrease female capture. The 5% CPH/3% borax bait captured the same number of flies as TYB, and was more effective than 10% CPH/3% borax. Further decreasing the amount of borax added to CPH may improve its effectiveness. As has been observed in field tests, fresh TYB captures more A. suspensa than fresh Nulure/borax but this difference decreases as the bait solutions age. EAG analysis indicates that volatiles from fresh Nulure/ borax elicit a higher antennal response than TYB, but this difference decreases as the TYB solution ages. Chemical analysis will be needed to determine the nature of reduced capture by fresh Nulure/borax and to identify additional attractive chemicals emitted by these protein baits. (author)

  5. Response of Anastrepha suspensa to liquid protein baits and synthetic lure formulations

    Energy Technology Data Exchange (ETDEWEB)

    Epsky, Nancy D.; Kendra, Paul E.; Heath, Robert R., E-mail: Nancy.Epsky@ars.usda.go, E-mail: Paul.Kendra@ars.usda.go, E-mail: Bob.Heath@ars.usda.go [U.S. Department of Agriculture (USDA/ARS/SHRS), Miami, FL (United States). Agricultural Research Service. Subtropical Horticulture Research Station

    2006-07-01

    Traps baited with AAPt captured more A. suspensa than traps baited with ABPt even when the ammonia release rates were similar. Reducing dosage of ammonia by 50% of the commercially available AA lure slightly increased female capture, but reducing dosage to 25% tended to decrease female capture. The 5% CPH/3% borax bait captured the same number of flies as TYB, and was more effective than 10% CPH/3% borax. Further decreasing the amount of borax added to CPH may improve its effectiveness. As has been observed in field tests, fresh TYB captures more A. suspensa than fresh Nulure/borax but this difference decreases as the bait solutions age. EAG analysis indicates that volatiles from fresh Nulure/ borax elicit a higher antennal response than TYB, but this difference decreases as the TYB solution ages. Chemical analysis will be needed to determine the nature of reduced capture by fresh Nulure/borax and to identify additional attractive chemicals emitted by these protein baits. (author)

  6. Power efficiency of the active boundary layer control around the hump by a slotted synthetic jet generator

    Directory of Open Access Journals (Sweden)

    Pick Petr

    2015-01-01

    Full Text Available The present contribution summarizes the power efficiency of the active flow control of the boundary layer of air around a hump. The synthetic jet generator with a rectangular output part, i.e. a slot, is actuated using a modulated signal. The actuation of the synthetic jet is carried out by modulating the input voltage of acoustic transducers of the generator. This causes the decrease of the loss coefficient and the change of the mixing size area (e.g. wake. A comparison of three types of modulating signals and their influence on the loss coefficient is performed. The main advantages of modulated signal are then described.

  7. Comparative antimicrobial activity and mechanism of action of bovine lactoferricin-derived synthetic peptides.

    Science.gov (United States)

    Liu, Yifan; Han, Feifei; Xie, Yonggang; Wang, Yizhen

    2011-12-01

    Lactoferricin B (LfcinB), a 25 residue peptide derived from the N-terminal of bovine lactoferrin (bLF), causes depolarization of the cytoplasmic membrane in susceptible bacteria. Its mechanism of action, however, still needs to be elucidated. In the present study, synthetic LfcinB (without a disulfide bridge) and LfcinB (C-C; with a disulfide bridge) as well as three derivatives with 15-, 11- and 9-residue peptides were prepared to investigate their antimicrobial nature and mechanisms. The antimicrobial properties were measured via minimum inhibitory concentration (MIC) determinations, killing kinetics assays and synergy testing, and hemolytic activities were assessed by hemoglobin release. Finally, the morphology of peptide-treated bacteria was determined by atomic force microscopy (AFM). We found that there was no difference in MICs between LfcinB and LfcinB (C-C). Among the derivatives, only LfcinB15 maintained nearly the same level as LfcinB, in the MIC range of 16-128 μg/ml, and the MICs of LfcinB11 (64-256 μg/ml) were 4 times more than LfcinB, while LfcinB9 exhibited the lowest antimicrobial activity. When treated at MIC for 1 h, many blebs were formed and holes of various sizes appeared on the cell surface, but the cell still maintained its integrity. This suggested that LfcinB had a major permeability effect on the cytoplasmic membrane of both Gram-positive and Gram-negative bacteria, which also indicated it may be a possible intracellular target. Among the tested antibiotics, aureomycin increased the bactericidal activity of LfcinB against E. coli, S. aureus and P. aeruginosa, but neomycin did not have such an effect. We also found that the combination of cecropin A and LfcinB had synergistic effects against E. coli.

  8. A synthetic peptide blocking TRPV1 activation inhibits UV-induced skin responses.

    Science.gov (United States)

    Kang, So Min; Han, Sangbum; Oh, Jang-Hee; Lee, Young Mee; Park, Chi-Hyun; Shin, Chang-Yup; Lee, Dong Hun; Chung, Jin Ho

    2017-10-01

    Transient receptor potential type 1 (TRPV1) can be activated by ultraviolet (UV) irradiation, and mediates UV-induced matrix metalloproteinase (MMP)-1 and proinflammatory cytokines in keratinocytes. Various chemicals and compounds targeting TRPV1 activation have been developed, but are not in clinical use mostly due to their safety issues. We aimed to develop a novel TRPV1-targeting peptide to inhibit UV-induced responses in human skin. We designed and generated a novel TRPV1 inhibitory peptide (TIP) which mimics the specific site in TRPV1 (aa 701-709: Gln-Arg-Ala-Ile-Thr-Ile-Leu-Asp-Thr, QRAITILDT), Thr 705 , and tested its efficacy of blocking UV-induced responses in HaCaT, mouse, and human skin. TIP effectively inhibited capsaicin-induced calcium influx and TRPV1 activation. Treatment of HaCaT with TIP prevented UV-induced increases of MMP-1 and pro-inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor-α. In mouse skin in vivo, TIP inhibited UV-induced skin thickening and prevented UV-induced expression of MMP-13 and MMP-9. Moreover, TIP attenuated UV-induced erythema and the expression of MMP-1, MMP-2, IL-6, and IL-8 in human skin in vivo. The novel synthetic peptide targeting TRPV1 can ameliorate UV-induced skin responses in vitro and in vivo, providing a promising therapeutic approach against UV-induced inflammation and photoaging. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

  9. Volumetric localization of epileptic activities in tuberous sclerosis using synthetic aperture magnetometry

    Energy Technology Data Exchange (ETDEWEB)

    Xiao, Zheng [Hospital for Sick Children, Research Institute, Toronto (Canada); Hospital for Sick Children, Department of Diagnostic Imaging, Toronto (Canada); Xiang, Jing [Hospital for Sick Children, Research Institute, Toronto (Canada); Hospital for Sick Children, Department of Diagnostic Imaging, Toronto (Canada); Holowka, Stephanie; Chuang, Sylvester [Hospital for Sick Children, Department of Diagnostic Imaging, Toronto (Canada); Hunjan, Amrita; Sharma, Rohit; Otsubo, Hiroshi [Hospital for Sick Children, Division of Neurology, Toronto (Canada)

    2006-01-01

    Magnetoencephalography (MEG) is a novel noninvasive technique for localizing epileptic zones. Tuberous sclerosis complex (TSC) is often associated with medically refractory epilepsy with multiple epileptic zones. Surgical treatment of TSC requires accurate localization of epileptogenic tubers. The objective of this study was to introduce a new MEG technique, synthetic aperture magnetometry (SAM), to volumetrically localize irritable zones and clarify the correlations between SAM, dipole modeling and anatomical tubers. Eight pediatric patients with TSC confirmed by clinical and neuroimaging findings were retrospectively studied. MEG data were recorded using a whole-cortex CTF OMEGA system. Sleep deprivation was employed to provoke epileptiform activity. Irritable zones were localized using both dipole modeling and SAM. MRI detected 42 tubers in the eight patients. Dipole modeling localized 28 irritable zones, and 19 out of the 28 zones were near tubers (19/42, 45%). SAM found 51 irritable zones, and 31 out of the 51 zones were near tubers (31/42, 74%). Among the 51 irritable zones determined by SAM, thirty-five zones were in 1-35 Hz, nine zones were in 35-60 Hz, and seven zones were in 60-120 Hz. The new method, SAM, yielded very plausible equivalent sources for patients who showed anatomical tubers on MRI. Compared to conventional dipole modeling, SAM appeared to offer increased detection of irritable zones and beneficial volumetric and frequency descriptions. (orig.)

  10. Antimicrobial activity of the indolicidin-derived novel synthetic peptide In-58.

    Science.gov (United States)

    Vasilchenko, A S; Vasilchenko, A V; Pashkova, T M; Smirnova, M P; Kolodkin, N I; Manukhov, I V; Zavilgelsky, G B; Sizova, E A; Kartashova, O L; Simbirtsev, A S; Rogozhin, E A; Duskaev, G K; Sycheva, M V

    2017-12-01

    Natural peptides with antimicrobial activity are extremely diverse, and peptide synthesis technologies make it possible to significantly improve their properties for specific tasks. Here, we investigate the biological properties of the natural peptide indolicidin and the indolicidin-derived novel synthetic peptide In-58. In-58 was generated by replacing all tryptophan residues on phenylalanine in D-configuration; the α-amino group in the main chain also was modified by unsaturated fatty acid. Compared with indolicidin, In-58 is more bactericidal, more resistant to proteinase K, and less toxic to mammalian cells. Using molecular physics approaches, we characterized the action of In-58 on bacterial cells at the cellular level. Also, we have found that studied peptides damage bacterial membranes. Using the Escherichia coli luminescent biosensor strain MG1655 (pcolD'::lux), we investigated the action of indolicidin and In-58 at the subcellular level. At subinhibitory concentrations, indolicidin and In-58 induced an SOS response. Our data suggest that indolicidin damages the DNA, but bacterial membrane perturbation is its principal mode of action. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

  11. Antiparasitic activity of natural and semi-synthetic tirucallane triterpenoids from Schinus terebinthifolius (Anacardiaceae): structure/activity relationships.

    Science.gov (United States)

    Morais, Thiago R; da Costa-Silva, Thais A; Tempone, Andre G; Borborema, Samanta Etel T; Scotti, Marcus T; de Sousa, Raquel Maria F; Araujo, Ana Carolina C; de Oliveira, Alberto; de Morais, Sérgio Antônio L; Sartorelli, Patricia; Lago, João Henrique G

    2014-05-05

    Leishmaniasis and Chagas are diseases caused by parasitic protozoans that affect the poorest population in the World, causing a high mortality and morbidity. As a result of highly toxic and long-term treatments, the discovery of novel, safe and more efficacious drugs is essential. In this work, the in vitro antiparasitic activity and mammalian cytotoxicity of three natural tirucallane triterpenoids, isolated from leaves of Schinus terebinthifolius (Anacardiaceae), and nine semi-synthetic derivatives were investigated against Leishmania (L.) infantum and Trypanosoma cruzi. Trypomastigotes of T. cruzi were the most susceptible parasites and seven compounds demonstrated a trypanocidal activity with IC50 values in the range between 15 and 58 µg/mL. Four compounds demonstrated selectivity towards the intracellular amastigotes of Leishmania, with IC50 values in the range between 28 and 97 µg/mL. The complete characterization of triterpenoids was afforded after thorough analysis of nuclear magnetic resonance (NMR) data as well as electrospray ionization mass spectrometry (ESI-MS). Additionally, structure-activity relationships were performed using Decision Trees.

  12. Antiparasitic Activity of Natural and Semi-Synthetic Tirucallane Triterpenoids from Schinus terebinthifolius (Anacardiaceae: Structure/Activity Relationships

    Directory of Open Access Journals (Sweden)

    Thiago R. Morais

    2014-05-01

    Full Text Available Leishmaniasis and Chagas are diseases caused by parasitic protozoans that affect the poorest population in the World, causing a high mortality and morbidity. As a result of highly toxic and long-term treatments, the discovery of novel, safe and more efficacious drugs is essential. In this work, the in vitro antiparasitic activity and mammalian cytotoxicity of three natural tirucallane triterpenoids, isolated from leaves of Schinus terebinthifolius (Anacardiaceae, and nine semi-synthetic derivatives were investigated against Leishmania (L. infantum and Trypanosoma cruzi. Trypomastigotes of T. cruzi were the most susceptible parasites and seven compounds demonstrated a trypanocidal activity with IC50 values in the range between 15 and 58 µg/mL. Four compounds demonstrated selectivity towards the intracellular amastigotes of Leishmania, with IC50 values in the range between 28 and 97 µg/mL. The complete characterization of triterpenoids was afforded after thorough analysis of nuclear magnetic resonance (NMR data as well as electrospray ionization mass spectrometry (ESI-MS. Additionally, structure-activity relationships were performed using Decision Trees.

  13. A novel synthetic quinolinone inhibitor presents proteolytic and hemorrhagic inhibitory activities against snake venom metalloproteases.

    Science.gov (United States)

    Baraldi, Patrícia T; Magro, Angelo J; Matioli, Fábio F; Marcussi, Silvana; Lemke, Ney; Calderon, Leonardo A; Stábeli, Rodrigo G; Soares, Andreimar M; Correa, Arlene G; Fontes, Marcos R M

    2016-02-01

    Metalloproteases play a fundamental role in snake venom envenomation inducing hemorrhagic, fibrigen(ogen)olytic and myotoxic effects in their victims. Several snake venoms, such as those from the Bothrops genus, present important local effects which are not efficiently neutralized by conventional serum therapy. Consequently, these accidents may result in permanent sequelae and disability, creating economic and social problems, especially in developing countries, leading the attention of the World Health Organization that considered ophidic envenomations a neglected tropical disease. Aiming to produce an efficient inhibitor against bothropic venoms, we synthesized different molecules classified as quinolinones - a group of low-toxic chemical compounds widely used as antibacterial and antimycobacterial drugs - and tested their inhibitory properties against hemorrhage caused by bothropic venoms. The results from this initial screening indicated the molecule 2-hydroxymethyl-6-methoxy-1,4-dihydro-4-quinolinone (Q8) was the most effective antihemorrhagic compound among all of the assayed synthetic quinolinones. Other in vitro and in vivo experiments showed this novel compound was able to inhibit significantly the hemorrhagic and/or proteolytic activities of bothropic crude venoms and isolated snake venom metalloproteases (SVMPs) even at lower concentrations. Docking and molecular dynamic simulations were also performed to get insights into the structural basis of Q8 inhibitory mechanism against proteolytic and hemorrhagic SVMPs. These structural studies demonstrated that Q8 may form a stable complex with SVMPs, impairing the access of substrates to the active sites of these toxins. Therefore, both experimental and structural data indicate that Q8 compound is an interesting candidate for antiophidic therapy, particularly for the treatment of the hemorrhagic and necrotic effects induced by bothropic venoms. Copyright © 2015 Elsevier B.V. and Société Française de

  14. 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...... 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...... increased HCV IRES-mediated translation and MAPKAPK3-dependent HCV IRES activity was further increased by core protein. These data suggest that HCV core may modulate MAPKAPK3 to facilitate its own propagation....

  15. Differential activation of G-proteins by μ-opioid receptor agonists

    Science.gov (United States)

    Saidak, Zuzana; Blake-Palmer, Katherine; Hay, Debbie L; Northup, John K; Glass, Michelle

    2006-01-01

    We investigated the ability of the activated μ-opioid receptor (MOR) to differentiate between myristoylated Gαi1 and GαoA type Gα proteins, and the maximal activity of a range of synthetic and endogenous agonists to activate each Gα protein. Membranes from HEK293 cells stably expressing transfected MOR were chaotrope extracted to denature endogenous G-proteins and reconstituted with specific purified G-proteins. The Gα subunits were generated in bacteria and were demonstrated to be recognised equivalently to bovine brain purified Gα protein by CB1 cannabinoid receptors. The ability of agonists to catalyse the MOR-dependent GDP/[35S]GTPγS exchange was then compared for Gαi1 and GαoA. Activation of MOR by DAMGO produced a high-affinity saturable interaction for GαoA (Km=20±1 nM) but a low-affinity interaction with Gαi1 (Km=116±12 nM). DAMGO, met-enkephalin and leucine-enkephalin displayed maximal Gα activation among the agonists evaluated. Endomorphins 1 and 2, methadone and β-endorphin activated both Gα to more than 75% of the maximal response, whereas fentanyl partially activated both G-proteins. Buprenorphine and morphine demonstrated a statistically significant difference between the maximal activities between Gαi1 and GαoA. Interestingly, DAMGO, morphine, endomorphins 1 and 2, displayed significant differences in the potencies for the activation of the two Gα. Differences in maximal activity and potency, for Gαi1 versus GαoA, are both indicative of agonist selective activation of G-proteins in response to MOR activation. These findings may provide a starting point for the design of drugs that demonstrate greater selectivity between these two G-proteins and therefore produce a more limited range of effects. PMID:16415903

  16. Complement activation by ceramide transporter proteins.

    Science.gov (United States)

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

    2014-02-01

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

  17. Racemic crystallography of synthetic protein enantiomers used to determine the X-ray structure of plectasin by direct methods

    Science.gov (United States)

    Mandal, Kalyaneswar; Pentelute, Brad L; Tereshko, Valentina; Thammavongsa, Vilasak; Schneewind, Olaf; Kossiakoff, Anthony A; Kent, Stephen B H

    2009-01-01

    We describe the use of racemic crystallography to determine the X-ray structure of the natural product plectasin, a potent antimicrobial protein recently isolated from fungus. The protein enantiomers l-plectasin and d-plectasin were prepared by total chemical synthesis; interestingly, l-plectasin showed the expected antimicrobial activity, while d-plectasin was devoid of such activity. The mirror image proteins were then used for racemic crystallization. Synchrotron X-ray diffraction data were collected to atomic resolution from a racemic plectasin crystal; the racemate crystallized in the achiral centrosymmetric space group with one l-plectasin molecule and one d-plectasin molecule forming the unit cell. Dimer-like intermolecular interactions between the protein enantiomers were observed, which may account for the observed extremely low solvent content (13%–15%) and more highly ordered nature of the racemic crystals. The structure of the plectasin molecule was well defined for all 40 amino acids and was generally similar to the previously determined NMR structure, suggesting minimal impact of the crystal packing on the plectasin conformation. PMID:19472324

  18. Insulinotropic and Muscle Protein Synthetic Effects of Branched-Chain Amino Acids: Potential Therapy for Type 2 Diabetes and Sarcopenia

    OpenAIRE

    Darren G. Candow; Scott C. Forbes; Jonathan P. Little; Ralph J. Manders

    2012-01-01

    The loss of muscle mass and strength with aging (i.e., sarcopenia) has a negative effect on functional independence and overall quality of life. One main contributing factor to sarcopenia is the reduced ability to increase skeletal muscle protein synthesis in response to habitual feeding, possibly due to a reduction in postprandial insulin release and an increase in insulin resistance. Branched-chain amino acids (BCAA), primarily leucine, increases the activation of pathways involved in muscl...

  19. Multifunctional activities of KSLW synthetic antimicrobial decapeptide: Implications for wound healing

    Science.gov (United States)

    Williams, Richard Leroy

    Wound healing is a complex process leading to the maintenance of skin integrity. Stress is known to increase susceptibility to bacterial infection, alter proinflammatory cytokine expression, and delay wound closure. Recently, antimicrobial peptides have generated interest due to their prokaryotic selectivity, decreased microbial resistance and multifunctional roles in wound healing, including fibroblast stimulation, keratinocyte migration and leukocyte migration. The objective of this dissertation project was to evaluate the effect of a synthetic antimicrobial decapeptide (KSLW) on bacterial clearance inflammation, and wound closure during stress-impaired healing. SKH-1 mice were randomly assigned to either control or restraint-stressed (RST) groups. Punch biopsy wounds (3.5 mm in diameter) were created bilaterally on the dorsal skin. Wounds were injected with 50 microL of empty carriers or KSLW prepared in Pluronic-F68, phospholipid micelles, or saline. Bacterial assays of harvested wounds were conducted on BHI agar. Wound closure was determined by photoplanimetry. Cytokine and growth factor mRNA expression was assessed with real-time RT-PCR. Human neutrophil migration assays and checkerboard analyses were performed using Transweli plates, and counting on hemacytometer. Oxidative burst activity was measured by spectrophotometric analysis of 2,7-dichlorofluorescein oxidation. KSLW-treatment resulted in significant reductions in bacterial load among RST mice, with no difference from control after 24h. The effect was sustained 5 days post-wounding, in RST mice treated with KSLW-F68. Temporal analysis of gene induction revealed reversals of stress-induced altered expression of growth factors, proinflammatory cytokines, and chemokines essential for favorable wound healing, at various time points. KSLW-treatment in RST mice demonstrated faster wound closure throughout the stress period. KSLW, at micromolar concentrations, demonstrated a significant effect on neutrophil

  20. Antioxidative Activity of Tobacco Leaf Protein Hydrolysates

    Directory of Open Access Journals (Sweden)

    Guohua Rao

    2007-01-01

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

  1. Anticancer Activity of Bacterial Proteins and Peptides.

    Science.gov (United States)

    Karpiński, Tomasz M; Adamczak, Artur

    2018-04-30

    Despite much progress in the diagnosis and treatment of cancer, tumour diseases constitute one of the main reasons of deaths worldwide. The side effects of chemotherapy and drug resistance of some cancer types belong to the significant current therapeutic problems. Hence, searching for new anticancer substances and medicines are very important. Among them, bacterial proteins and peptides are a promising group of bioactive compounds and potential anticancer drugs. Some of them, including anticancer antibiotics (actinomycin D, bleomycin, doxorubicin, mitomycin C) and diphtheria toxin, are already used in the cancer treatment, while other substances are in clinical trials (e.g., p28, arginine deiminase ADI) or tested in in vitro research. This review shows the current literature data regarding the anticancer activity of proteins and peptides originated from bacteria: antibiotics, bacteriocins, enzymes, nonribosomal peptides (NRPs), toxins and others such as azurin, p28, Entap and Pep27anal2. The special attention was paid to the still poorly understood active substances obtained from the marine sediment bacteria. In total, 37 chemical compounds or groups of compounds with antitumor properties have been described in the present article.

  2. Comparative Molecular Dynamics Simulations of Mitogen-Activated Protein Kinase-Activated Protein Kinase 5

    Directory of Open Access Journals (Sweden)

    Inger Lindin

    2014-03-01

    Full Text Available The mitogen-activated protein kinase-activated protein kinase MK5 is a substrate of the mitogen-activated protein kinases p38, ERK3 and ERK4. Cell culture and animal studies have demonstrated that MK5 is involved in tumour suppression and promotion, embryogenesis, anxiety, cell motility and cell cycle regulation. In the present study, homology models of MK5 were used for molecular dynamics (MD simulations of: (1 MK5 alone; (2 MK5 in complex with an inhibitor; and (3 MK5 in complex with the interaction partner p38α. The calculations showed that the inhibitor occupied the active site and disrupted the intramolecular network of amino acids. However, intramolecular interactions consistent with an inactive protein kinase fold were not formed. MD with p38α showed that not only the p38 docking region, but also amino acids in the activation segment, αH helix, P-loop, regulatory phosphorylation region and the C-terminal of MK5 may be involved in forming a very stable MK5-p38α complex, and that p38α binding decreases the residual fluctuation of the MK5 model. Electrostatic Potential Surface (EPS calculations of MK5 and p38α showed that electrostatic interactions are important for recognition and binding.

  3. Synergistic Synthetic Biology: Units in Concert

    International Nuclear Information System (INIS)

    Trosset, Jean-Yves; Carbonell, Pablo

    2013-01-01

    Synthetic biology aims at translating the methods and strategies from engineering into biology in order to streamline the design and construction of biological devices through standardized parts. Modular synthetic biology devices are designed by means of an adequate elimination of cross-talk that makes circuits orthogonal and specific. To that end, synthetic constructs need to be adequately optimized through in silico modeling by choosing the right complement of genetic parts and by experimental tuning through directed evolution and craftsmanship. In this review, we consider an additional and complementary tool available to the synthetic biologist for innovative design and successful construction of desired circuit functionalities: biological synergies. Synergy is a prevalent emergent property in biological systems that arises from the concerted action of multiple factors producing an amplification or cancelation effect compared with individual actions alone. Synergies appear in domains as diverse as those involved in chemical and protein activity, polypharmacology, and metabolic pathway complementarity. In conventional synthetic biology designs, synergistic cross-talk between parts and modules is generally attenuated in order to verify their orthogonality. Synergistic interactions, however, can induce emergent behavior that might prove useful for synthetic biology applications, like in functional circuit design, multi-drug treatment, or in sensing and delivery devices. Synergistic design principles are therefore complementary to those coming from orthogonal design and may provide added value to synthetic biology applications. The appropriate modeling, characterization, and design of synergies between biological parts and units will allow the discovery of yet unforeseeable, novel synthetic biology applications.

  4. Synergistic Synthetic Biology: Units in Concert

    Science.gov (United States)

    Trosset, Jean-Yves; Carbonell, Pablo

    2013-01-01

    Synthetic biology aims at translating the methods and strategies from engineering into biology in order to streamline the design and construction of biological devices through standardized parts. Modular synthetic biology devices are designed by means of an adequate elimination of cross-talk that makes circuits orthogonal and specific. To that end, synthetic constructs need to be adequately optimized through in silico modeling by choosing the right complement of genetic parts and by experimental tuning through directed evolution and craftsmanship. In this review, we consider an additional and complementary tool available to the synthetic biologist for innovative design and successful construction of desired circuit functionalities: biological synergies. Synergy is a prevalent emergent property in biological systems that arises from the concerted action of multiple factors producing an amplification or cancelation effect compared with individual actions alone. Synergies appear in domains as diverse as those involved in chemical and protein activity, polypharmacology, and metabolic pathway complementarity. In conventional synthetic biology designs, synergistic cross-talk between parts and modules is generally attenuated in order to verify their orthogonality. Synergistic interactions, however, can induce emergent behavior that might prove useful for synthetic biology applications, like in functional circuit design, multi-drug treatment, or in sensing and delivery devices. Synergistic design principles are therefore complementary to those coming from orthogonal design and may provide added value to synthetic biology applications. The appropriate modeling, characterization, and design of synergies between biological parts and units will allow the discovery of yet unforeseeable, novel synthetic biology applications. PMID:25022769

  5. Antiulcerative Activity of Milk Proteins Hydrolysates.

    Science.gov (United States)

    Carrillo, Wilman; Monteiro, Karin Maia; Martínez-Maqueda, Daniel; Ramos, Mercedes; Recio, Isidra; Carvalho, João Ernesto de

    2018-04-01

    Several studies have shown the protective effect of dairy products, especially α-lactalbumin and derived hydrolysates, against induced gastric ulcerative lesions. The mucus strengthening represents an important mechanism in the defense of gastrointestinal mucosa. Previously, a hydrolysate from casein (CNH) and a hydrolysate from whey protein concentrate rich in β-lactoglobulin (WPH) demonstrated a stimulatory activity on mucus production in intestinal goblet cells. The aim of this work was to evaluate the possible antiulcerative activity of these two hydrolysates in an ethanol-induced ulcer model in rats. All tested samples significantly reduced the ulcerative lesions index (ULI), compared with the saline solution, using doses of 300 and 1000 mg kg -1 body weight with decreases up to 66.3% ULI. A dose-response relationship was found for both hydrolysates. The involvement of endogenous sulfhydryl (SH) groups and prostaglandins (PGs) in the antiulcerative activity was evaluated using their blockage. The antiulcerative activity of WPH showed a drastic decrease in presence of N-ethylmaleimide (from 41.4% to 9.2% ULI). However, the CNH antiulcerative properties were not significantly affected. The cytoprotective effect of WPH appears to depend on a PG-mediated mechanism. In conclusion, CNH and WPH demonstrated in vivo antiulcerative properties and represent a promising alternative as protectors of the gastric mucosa.

  6. Differential recruitment efficacy of patient-derived amyloidogenic and myeloma light chain proteins by synthetic fibrils-A metric for predicting amyloid propensity.

    Directory of Open Access Journals (Sweden)

    Emily B Martin

    Full Text Available Monoclonal free light chain (LC proteins are present in the circulation of patients with immunoproliferative disorders such as light chain (AL amyloidosis and multiple myeloma (MM. Light chain-associated amyloid is a complex pathology composed of proteinaceous fibrils and extracellular matrix proteins found in all patients with AL and in ~10-30% of patients who presented with MM. Amyloid deposits systemically in multiple organs and tissues leading to dysfunction and ultimately death. The overall survival of patients with amyloidosis is worse than for those with early stage MM.We have developed a sensitive binding assay quantifying the recruitment of full length, patient-derived LC proteins by synthetic amyloid fibrils, as a method for studying their amyloidogenic potential. In a survey of eight urinary LC, both AL and MM-associated proteins were recruited by synthetic amyloid fibrils; however, AL-associated LC bound significantly more efficiently (p < 0.05 than did MM LCs. The LC proteins used in this study were isolated from urine and presumed to represent a surrogate of serum free light chains.The binding of LC to synthetic fibrils in this assay accurately differentiated LC with amyloidogenic propensity from MM LC that were not associated with clinical amyloid disease. Notably, the LC from a MM patient who subsequently developed amyloid behaved as an AL-associated protein in the assay, indicating the possibility for identifying MM patients at risk for developing amyloidosis based on the light chain recruitment efficacy. With this information, at risk patients can be monitored more closely for the development of amyloidosis, allowing timely administration of novel, amyloid-directed immunotherapies-this approach may improve the prognosis for these patients.

  7. Inhibition of food stimulated acid secretion by misoprostol, an orally active synthetic E1 analogue prostaglandin.

    OpenAIRE

    Ramage, J K; Denton, A; Williams, J G

    1985-01-01

    The effect of 200 micrograms misoprostol (a synthetic prostaglandin E1 analogue) on food stimulated intragastric acidity has been monitored over a 9 h period in 16 normal volunteers. Misoprostol caused a significant inhibition of intragastric acidity for 2 h post-dosing, but no significant effect was seen thereafter on either basal or food stimulated acidity.

  8. Characterization of Exposure Potential during Activities on Synthetic Turf Fields with Recycled Tire Crumb Rubber Infill

    Science.gov (United States)

    The Federal Research Action Plan on Recycled Tire Crumb Used on Playing Fields and Playgrounds (FRAP), released in February 2016, is a multi-agency research plan in response to concerns over the use of tire crumb rubber as infill on synthetic turf fields. The FRAP outlines specif...

  9. Synthetic feeding stimulants enhance insecticide activity against western corn rootworm larvae, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae)

    Science.gov (United States)

    In behavioral bioassays, the addition of a synthetic feeding stimulant blend improved the efficacy of the insecticide thiamethoxam against neonate western corn rootworm, Diabrotica virgifera virgifera LeConte, larvae. In 4-h bioassays, the concentration of thiamethoxam required for 50% mortality (LC...

  10. An Experimental Study on Active Flow Control Using Synthetic Jet Actuators over S809 Airfoil

    International Nuclear Information System (INIS)

    Gul, M; Uzol, O; Akmandor, I S

    2014-01-01

    This study investigates the effect of periodic excitation from individually controlled synthetic jet actuators on the dynamics of the flow within the separation and re-attachment regions of the boundary layer over the suction surface of a 2D model wing that has S809 airfoil profile. Experiments are performed in METUWIND's C3 open-loop suction type wind tunnel that has a 1 m × 1 m cross-section test section. The synthetic jet array on the wing consists of three individually controlled actuators driven by piezoelectric diaphragms located at 28% chord location near the mid-span of the wing. In the first part of the study, surface pressure, Constant Temperature Anemometry (CTA) and Particle Image Velocimetry (PIV) measurements are performed over the suction surface of the airfoil to determine the size and characteristics of the separated shear layer and the re-attachment region, i.e. the laminar separation bubble, at 2.3x10 5 Reynolds number at zero angle of attack and with no flow control as a baseline case. For the controlled case, CTA measurements are carried out under the same inlet conditions at various streamwise locations along the suction surface of the airfoil to investigate the effect of the synthetic jet on the boundary layer properties. During the controlled case experiments, the synthetic jet actuators are driven with a sinusoidal frequency of 1.45 kHz and 300Vp-p. Results of this study show that periodic excitation from the synthetic jet actuators eliminates the laminar separation bubble formed over the suction surface of the airfoil at 2.3x10 5 Reynolds number at zero angle of attack

  11. Open questions in origin of life: experimental studies on the origin of nucleic acids and proteins with specific and functional sequences by a chemical synthetic biology approach

    DEFF Research Database (Denmark)

    Adamala, K.; Anella, F.; Wieczorek, R.

    2014-01-01

    sequences among a vast array of possible ones, the huge "sequence space", leading to the question "why these macromolecules, and not the others?" We have recently addressed these questions by using a chemical synthetic biology approach. In particular, we have tested the catalytic activity of small peptides...

  12. 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.

  13. Effect of gamma irradiation on nutritional components and Cry1Ab protein in the transgenic rice with a synthetic cry1Ab gene from Bacillus thuringiensis

    International Nuclear Information System (INIS)

    Wu Dianxing; Ye Qingfu; Wang Zhonghua; Xia Yingwu

    2004-01-01

    The effects of gamma irradiation on the transgenic rice containing a synthetic cry1Ab gene from Bacillus thuringiensis were investigated. There was almost no difference in the content of the major nutritional components, i.e. crude protein, crude lipid, eight essential amino acids and total ash between the irradiated grains and the non-irradiated transgenic rice. However, the amounts of Cry1Ab protein and apparent amylose in the irradiated transgenic rice were reduced significantly by the doses higher than 200 Gy. In vivo observation showed that Cry1Ab protein contents also decreased in the fresh leaf tissues of survival seedlings after irradiation with 200 Gy or higher doses and showed inhibition of seedling growth. The results indicate that gamma irradiation might improve the quality of transgenic rice due to removal of the toxic Cry1Ab protein

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

    Science.gov (United States)

    Gerasymenko, I M; Sheludko, Y V

    2017-07-01

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

  15. Antimalarial activity of synthetic 1,2,4-trioxanes and cyclic peroxy ketals, a quantum similarity study

    Science.gov (United States)

    Gironés, X.; Gallegos, A.; Carbó-Dorca, R.

    2001-12-01

    In this work, the antimalarial activity of two series of 20 and 7 synthetic 1,2,4-trioxanes and a set of 20 cyclic peroxy ketals are tested for correlation search by means of Molecular Quantum Similarity Measures (MQSM). QSAR models, dealing with different biological responses (IC90, IC50 and ED90) of the parasite Plasmodium Falciparum, are constructed using MQSM as molecular descriptors and are satisfactorily correlated. The statistical results of the 20 1,2,4-trioxanes are deeply analyzed to elucidate the relevant structural features in the biological activity, revealing the importance of phenyl substitutions.

  16. Scientific iconoclasm and active imagination: synthetic cells as techno-scientific mandalas.

    Science.gov (United States)

    Zwart, Hub

    2018-05-14

    Metaphors allow us to come to terms with abstract and complex information, by comparing it to something which is structured, familiar and concrete. Although modern science is "iconoclastic", as Gaston Bachelard phrases it (i.e. bent on replacing living entities by symbolic data: e.g. biochemical and mathematical symbols and codes), scientists are at the same time prolific producers of metaphoric images themselves. Synthetic biology is an outstanding example of a technoscientific discourse replete with metaphors, including textual metaphors such as the "Morse code" of life, the "barcode" of life and the "book" of life. This paper focuses on a different type of metaphor, however, namely on the archetypal metaphor of the mandala as a symbol of restored unity and wholeness. Notably, mandala images emerge in textual materials (papers, posters, PowerPoints, etc.) related to one of the new "frontiers" of contemporary technoscience, namely the building of a synthetic cell: a laboratory artefact that functions like a cell and is even able to replicate itself. The mandala symbol suggests that, after living systems have been successfully reduced to the elementary building blocks and barcodes of life, the time has now come to put these fragments together again. We can only claim to understand life, synthetic cell experts argue, if we are able to technically reproduce a fully functioning cell. This holistic turn towards the cell as a meaningful whole (a total work of techno-art) also requires convergence at the "subject pole": the building of a synthetic cell as a practice of the self, representing a turn towards integration, of multiple perspectives and various forms of expertise.

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

    Science.gov (United States)

    Kopyl'chuk, G P; Buchkovskaia, I M

    2014-01-01

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

  18. Synthetic Molecular Machines for Active Self-Assembly: Prototype Algorithms, Designs, and Experimental Study

    Science.gov (United States)

    Dabby, Nadine L.

    behaviors. This class of behaviors includes any behavior where a passive physical system simply does not have enough physical energy to perform the specified tasks in the requisite amount of time. As we will demonstrate and prove, a sufficiently expressive implementation of an "active" molecular self-assembly approach can achieve these behaviors. Using an external source of fuel solves part of the problem, so the system is not "energetically incomplete." But the programmable system also needs to have sufficient expressive power to achieve the specified behaviors. Perhaps surprisingly, some of these systems do not even require Turing completeness to be sufficiently expressive. Building on a large variety of work by other scientists in the fields of DNA nanotechnology, chemistry and reconfigurable robotics, this thesis introduces several research contributions in the context of active self-assembly. We show that simple primitives such as insertion and deletion are able to generate complex and interesting results such as the growth of a linear polymer in logarithmic time and the ability of a linear polymer to treadmill. To this end we developed a formal model for active-self assembly that is directly implementable with DNA molecules. We show that this model is computationally equivalent to a machine capable of producing strings that are stronger than regular languages and, at most, as strong as context-free grammars. This is a great advance in the theory of active self-assembly as prior models were either entirely theoretical or only implementable in the context of macro-scale robotics. We developed a chain reaction method for the autonomous exponential growth of a linear DNA polymer. Our method is based on the insertion of molecules into the assembly, which generates two new insertion sites for every initial one employed. The building of a line in logarithmic time is a first step toward building a shape in logarithmic time. We demonstrate the first construction of a synthetic

  19. Sulfated glycopeptide nanostructures for multipotent protein activation

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-06-19

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

  20. Novel activation domain derived from Che-1 cofactor coupled with the artificial protein Jazz drives utrophin upregulation.

    Science.gov (United States)

    Desantis, Agata; Onori, Annalisa; Di Certo, Maria Grazia; Mattei, Elisabetta; Fanciulli, Maurizio; Passananti, Claudio; Corbi, Nicoletta

    2009-02-01

    Our aim is to upregulate the expression level of the dystrophin related gene utrophin in Duchenne muscular dystrophy, thus complementing the lack of dystrophin functions. To this end, we have engineered synthetic zinc finger based transcription factors. We have previously shown that the artificial three-zinc finger protein named Jazz fused with the Vp16 activation domain, is able to bind utrophin promoter A and to increase the endogenous level of utrophin in transgenic mice. Here, we report on an innovative artificial protein, named CJ7, that consists of Jazz DNA binding domain fused to a novel activation domain derived from the regulatory multivalent adaptor protein Che-1/AATF. This transcriptional activation domain is 100 amino acids in size and it is very powerful as compared to the Vp16 activation domain. We show that CJ7 protein efficiently promotes transcription and accumulation of the acetylated form of histone H3 on the genomic utrophin promoter locus.

  1. Structure-Activity Relationship Study of Sesquiterpene Lactones and Their Semi-Synthetic Amino Derivatives as Potential Antitrypanosomal Products

    Directory of Open Access Journals (Sweden)

    Stefanie Zimmermann

    2014-03-01

    Full Text Available Sesquiterpene lactones (STLs are natural products that have potent antitrypanosomal activity in vitro and, in the case of cynaropicrin, also reduce parasitemia in the murine model of trypanosomiasis. To explore their structure-antitrypanosomal activity relationships, a set of 34 natural and semi-synthetic STLs and amino-STLs was tested in vitro against T. b. rhodesiense (which causes East African sleeping sickness and mammalian cancer cells (rat bone myoblast L6 cells. It was found that the α-methylene-γ-lactone moiety is necessary for both antitrypanosomal effects and cytotoxicity. Antitrypanosomal selectivity is facilitated by 2-(hydroxymethylacrylate or 3,4-dihydroxy-2-methylenebutylate side chains, and by the presence of cyclopentenone rings. Semi-synthetic STL amines with morpholino and dimethylamino groups showed improved in vitro activity over the native STLs. The dimethylamino derivative of cynaropicrin was prepared and tested orally in the T. b. rhodesiense acute mouse model, where it showed reduced toxicity over cynaropicrin, but also lost antitrypanosomal activity.

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

    NARCIS (Netherlands)

    Koppelman, S.J.

    1995-01-01

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

  3. Generation of dTALEs and Libraries of Synthetic TALE-Activated Promoters for Engineering of Gene Regulatory Networks in Plants.

    Science.gov (United States)

    Schreiber, Tom; Tissier, Alain

    2017-01-01

    Transcription factors with programmable DNA-binding specificity constitute valuable tools for the design of orthogonal gene regulatory networks for synthetic biology. Transcription activator-like effectors (TALEs), as natural transcription regulators, were used to design, build, and test libraries of synthetic TALE-activated promoters (STAPs) that show a broad range of expression levels in plants. In this chapter, we present protocols for the construction of artificial TALEs and corresponding STAPs.

  4. Synthetic Peptides Analogue to Enamel Proteins Promote Osteogenic Differentiation of MC3T3-E1 and Mesenchymal Stem Cells

    Czech Academy of Sciences Publication Activity Database

    Rubert, M.; Ramis, J. M.; Vondrášek, Jiří; Gaya, A.; Lyngstadaas, S. P.; Monjo, M.

    2011-01-01

    Roč. 1, č. 2 (2011), s. 198-209 ISSN 2157-9083 Grant - others:GA ČR(CZ) GAP302/10/0427 Institutional research plan: CEZ:AV0Z40550506 Keywords : proline-rich regions * synthetic peptides * bone formation * mineralization * In Vitro Subject RIV: EI - Biotechnology ; Bionics

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

    NARCIS (Netherlands)

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

    1996-01-01

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

  6. Enhancement of the Enterocin CRL35 Activity by a Synthetic Peptide Derived from the NH2-Terminal Sequence

    Science.gov (United States)

    Saavedra, Lucila; Minahk, Carlos; de Ruiz Holgado, Aída P.; Sesma, Fernando

    2004-01-01

    The enterocin CRL35 biosynthetic gene cluster was cloned and sequenced. The sequence was revealed to be highly identical to that of the mundticin KS gene cluster (S. Kawamoto, J. Shima, R. Sato, T. Eguchi, S. Ohmomo, J. Shibato, N. Horikoshi, K. Takeshita, and T. Sameshima, Appl. Environ. Microbiol. 68:3830-3840, 2002). Short synthetic peptides were designed based on the bacteriocin sequence and were evaluated in antimicrobial competitive assays. The peptide KYYGNGVSCNKKGCS produced an enhancement of enterocin CRL35 antimicrobial activity in a buffer system. PMID:15215149

  7. Comparison of antimicrobial activities of natural essential oils and synthetic fragrances against selected environmental pathogens.

    Science.gov (United States)

    Vieira-Brock, Paula L; Vaughan, Brent M; Vollmer, David L

    2017-12-01

    Plant essential oils (EOs) are known to inhibit the growth of bacteria and fungi. Whether these antimicrobial effects are comparable to synthetic household products is less clear. Furthermore, limited research is available on the potential additive effect of blending EOs. In this investigation, a new EO blend containing orange, patchouli, peppermint, and clary sage was compared to its individual single oils and to three household products-air freshener, liquid soap, and body spray-for their ability to inhibit the growth of Staphylococcus aureus, Streptococcus pneumoniae, Pseudonomas aeruginosa, and Aspergillus brasiliensis in the disc-diffusion assay. The new EO blend significantly inhibited the growth of the four microorganisms. The zones of inhibition of new EO blend were greater than the air freshener and similar to the liquid soap and body spray, with the exception of Str. pneumoniae in which the body spray provided greater inhibitory zone. The new EO blend and the single oils, with the exception of peppermint, equally inhibited the growth of S. aureus and Str. pneumoniae suggesting no additive effect. P. aeruginosa and A. brasiliensis showed variable susceptibility to all EOs except for no susceptibility to orange and limonene. No difference was found between (-) and (+)-limonene; whereas, (+)-menthol showed greater effect than (-)-menthol. In conclusion, blending the EO of orange, patchouli, peppermint, and clary sage was beneficial in inhibiting the growth of S. aureus, Str. pneumoniae, P. aeruginosa, and A. brasiliensis providing a natural antimicrobial fragrance option over synthetics fragrances used in soaps, body sprays, and air fresheners.

  8. Catalytically-active inclusion bodies-Carrier-free protein immobilizates for application in biotechnology and biomedicine.

    Science.gov (United States)

    Krauss, Ulrich; Jäger, Vera D; Diener, Martin; Pohl, Martina; Jaeger, Karl-Erich

    2017-09-20

    Bacterial inclusion bodies (IBs) consist of unfolded protein aggregates and represent inactive waste products often accumulating during heterologous overexpression of recombinant genes in Escherichia coli. This general misconception has been challenged in recent years by the discovery that IBs, apart from misfolded polypeptides, can also contain substantial amounts of active and thus correctly or native-like folded protein. The corresponding catalytically-active inclusion bodies (CatIBs) can be regarded as a biologically-active sub-micrometer sized biomaterial or naturally-produced carrier-free protein immobilizate. Fusion of polypeptide (protein) tags can induce CatIB formation paving the way towards the wider application of CatIBs in synthetic chemistry, biocatalysis and biomedicine. In the present review we summarize the history of CatIBs, present the molecular-biological tools that are available to induce CatIB formation, and highlight potential lines of application. In the second part findings regarding the formation, architecture, and structure of (Cat)IBs are summarized. Finally, an overview is presented about the available bioinformatic tools that potentially allow for the prediction of aggregation and thus (Cat)IB formation. This review aims at demonstrating the potential of CatIBs for biotechnology and hopefully contributes to a wider acceptance of this promising, yet not widely utilized, protein preparation. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Human T-cell recognition of synthetic peptides representing conserved and variant sequences from the merozoite surface protein 2 of Plasmodium falciparum

    DEFF Research Database (Denmark)

    Theander, T G; Hviid, L; Dodoo, D

    1997-01-01

    Merozoite surface protein 2 (MSP2) is a malaria vaccine candidate currently undergoing clinical trials. We analyzed the peripheral blood mononuclear cell (PBMC) response to synthetic peptides corresponding to conserved and variant regions of the FCQ-27 allelic form of MSP2 in Ghanaian individuals....... The findings are encouraging for the development of a vaccine based on these T-epitope containing regions of MSP2, as the peptides were broadly recognized suggesting that they can bind to diverse HLA alleles and also because they include conserved MSP2 sequences. Immunisation with a vaccine construct...

  10. Pharmacological Characterization of 30 Human Melanocortin-4 Receptor Polymorphisms with the Endogenous Proopiomelanocortin Derived Agonists, Synthetic Agonists, and the Endogenous Agouti-Related Protein (AGRP) Antagonist

    Science.gov (United States)

    Xiang, Zhimin; Proneth, Bettina; Dirain, Marvin L.; Litherland, Sally A.; Haskell-Luevano, Carrie

    2010-01-01

    The melanocortin-4 receptor (MC4R) is a G-protein coupled receptor (GPCR) that is expressed in the central nervous system and has a role in regulating feeding behavior, obesity, energy homeostasis, male erectile response, and blood pressure. Since the report of the MC4R knockout mouse in 1997, the field has been searching for links between this genetic bio marker and human obesity and type 2 diabetes. More then 80 single nucleotide polymorphisms (SNPs) have been identified from human patients, both obese and non-obese controls. Many significant studies have been performed examining the pharmacological characteristics of these hMC4R SNPs in attempts to identify a molecular defects/insights that might link a genetic factor to the obese phenotype observed in patients possessing these mutations. Our laboratory has previously reported the pharmacological characterization of 40 of these polymorphic hMC4 receptors with multiple endogenous and synthetic ligands. The goal of the current study is to perform a similar comprehensive side-by-side characterization of 30 additional human hMC4R with single nucleotide polymorphisms using multiple endogenous agonists [α-, β, γ2-melanocyte stimulating hormones (MSH) and adrenocorticotropin (ACTH)], the antagonist agouti-related protein hAGRP(87-132), and synthetic agonists [NDP-MSH, MTII, and the tetrapeptide Ac-His-DPhe-Arg-Trp-NH2 (JRH887-9)]. These in vitro data, in some cases, provide a putative molecular link between dysfunctional hMC4R's and human obesity. These 30 hMC4R SNPs include R7H, R18H, R18L, S36Y, P48S, V50M, F51L, E61K, I69T, D90N, S94R, G98R, I121T, A154D, Y157S, W174C, G181D, F202L, A219V, I226T, G231S, G238D, N240S, C271R, S295P, P299L, E308K, I317V, L325F and 750DelGA. All but the N240S hMC4R were identified in obese patients. Additionally, we have characterized a double I102T/V103I hMC4R. In addition to the pharmacological characterization, the hMC4R variants were evaluated for cell surface expression by flow

  11. Pharmacological characterization of 30 human melanocortin-4 receptor polymorphisms with the endogenous proopiomelanocortin-derived agonists, synthetic agonists, and the endogenous agouti-related protein antagonist.

    Science.gov (United States)

    Xiang, Zhimin; Proneth, Bettina; Dirain, Marvin L; Litherland, Sally A; Haskell-Luevano, Carrie

    2010-06-08

    The melanocortin-4 receptor (MC4R) is a G-protein-coupled receptor (GPCR) that is expressed in the central nervous system and has a role in regulating feeding behavior, obesity, energy homeostasis, male erectile response, and blood pressure. Since the report of the MC4R knockout mouse in 1997, the field has been searching for links between this genetic biomarker and human obesity and type 2 diabetes. More then 80 single nucleotide polymorphisms (SNPs) have been identified from human patients, both obese and nonobese controls. Many significant studies have been performed examining the pharmacological characteristics of these hMC4R SNPs in attempts to identify a molecular defects/insights that might link a genetic factor to the obese phenotype observed in patients possessing these mutations. Our laboratory has previously reported the pharmacological characterization of 40 of these polymorphic hMC4 receptors with multiple endogenous and synthetic ligands. The goal of the current study is to perform a similar comprehensive side-by-side characterization of 30 additional human hMC4R with single nucleotide polymorphisms using multiple endogenous agonists [alpha-, beta-, and gamma(2)-melanocyte stimulating hormones (MSH) and adrenocorticotropin (ACTH)], the antagonist agouti-related protein hAGRP(87-132), and synthetic agonists [NDP-MSH, MTII, and the tetrapeptide Ac-His-dPhe-Arg-Trp-NH(2) (JRH887-9)]. These in vitro data, in some cases, provide a putative molecular link between dysfunctional hMC4R's and human obesity. These 30 hMC4R SNPs include R7H, R18H, R18L, S36Y, P48S, V50M, F51L, E61K, I69T, D90N, S94R, G98R, I121T, A154D, Y157S, W174C, G181D, F202L, A219 V, I226T, G231S, G238D, N240S, C271R, S295P, P299L, E308K, I317V, L325F, and 750DelGA. All but the N240S hMC4R were identified in obese patients. Additionally, we have characterized a double I102T/V103I hMC4R. In addition to the pharmacological characterization, the hMC4R variants were evaluated for cell surface

  12. Protein C activity and antigen levels in childhood

    NARCIS (Netherlands)

    van Teunenbroek, A.; Peters, M.; Sturk, A.; Borm, J. J.; Breederveld, C.

    1990-01-01

    Hereditary protein C deficiency is an important risk factor for thrombosis. To enable its diagnosis shortly after birth, we determined reference values of protein C antigen and activity levels for the first 3 months of life. To establish an age-related range of protein C levels we also determined

  13. Efficient generation of dopamine neuron-like cells from skin-derived precursors with a synthetic peptide derived from von Hippel-Lindau protein.

    Science.gov (United States)

    Kubo, Atsuhiko; Yoshida, Tetsuhiko; Kobayashi, Nahoko; Yokoyama, Takaakira; Mimura, Toshiro; Nishiguchi, Takao; Higashida, Tetsuhiro; Yamamoto, Isao; Kanno, Hiroshi

    2009-12-01

    Skin-derived precursors (SKPs) from mammalian dermis represent neural crest-related stem cells capable of differentiating into both neural and mesodermal progency. SKPs are of clinical interest because they serve as accessible autologous donor cells for neuronal repair for neuronal intractable diseases. However, little is known about the efficient generation of neurons from SKPs, and phenotypes of neurons generated from SKPs have been restricted. In addition, the neuronal repair using their generated neurons as donor cells has not been achieved. The von Hippel-Lindau protein (pVHL) is one of the proteins that play an important role during neuronal differentiation, and recently neuronal differentiation of neural progenitor cells by intracellular delivery of a synthetic VHL peptide derived from elongin BC-binding site has been demonstrated. In the present study, a synthetic VHL peptide derived from elongin BC-binding site was conjugated to the protein transduction domain (PTD) of HIV-TAT protein (TATVHL peptide) to facilitate entry into cells, and we demonstrate the efficient generation of cells with dopaminergic phenotype from SKPs with the intracellular delivery of TATVHL peptide, and characterized the generated cells. The TATVHL peptide-treated SKPs expressed neuronal marker proteins, particularly dopamine neuron markers, and also up-regulated mRNA levels of proneural basic helix-loop-helix factors. After the TATVHL peptide treatment, transplanted SKPs into Parkinson's disease (PD) model rats sufficiently differentiated into dopamine neuron-like cells in PD model rats, and partially but significantly corrected behavior of PD model rats. The generated dopamine neuron-like cells are expected to serve as donor cells for neuronal repair for PD.

  14. Neuronal Functions of Activators of G Protein Signaling

    Directory of Open Access Journals (Sweden)

    Man K. Tse

    2012-05-01

    Full Text Available G protein-coupled receptors (GPCRs are one of the most important gateways for signal transduction across the plasma membrane. Over the past decade, several classes of alternative regulators of G protein signaling have been identified and reported to activate the G proteins independent of the GPCRs. One group of such regulators is the activator of G protein signaling (AGS family which comprises of AGS1-10. They have entirely different activation mechanisms for G proteins as compared to the classic model of GPCR-mediated signaling and confer upon cells new avenues of signal transduction. As GPCRs are widely expressed in our nervous system, it is believed that the AGS family plays a major role in modulating the G protein signaling in neurons. In this article, we will review the current knowledge on AGS proteins in relation to their potential roles in neuronal regulations.

  15. Synthetic Strigolactone Analogues Reveal Anti-Cancer Activities on Hepatocellular Carcinoma Cells

    KAUST Repository

    Hasan, Mohammed Nihal

    2018-02-09

    Hepatocellular carcinoma (HCC) remains one of the leading causes of death worldwide. The complex etiology is attributed to many factors like heredity, cirrhosis, hepatitis infections or the dysregulation of the different molecular pathways. Nevertheless, the current treatment regimens have either severe side effects or tumors gradually acquire resistance upon prolonged use. Thus, developing a new selective treatment for HCC is the need of the hour. Many anticancer agents derived from plants have been evaluated for their cytotoxicity towards many human cancer cell lines. Strigolactones (SLs)-a newly discovered class of phytohormones, play a crucial role in the development of plant-root and shoot. Recently, many synthetic analogues of SLs have demonstrated pro-apoptotic effects on different cancer cell lines like prostate, breast, colon and lung. In this study, we tested synthetic SLs analogues on HCC cell line-HepG2 and evaluated their capability to induce cell proliferation inhibition and apoptosis. Primary WST-1 assays, followed by annexin-V/7AAD staining, demonstrated the anti-proliferative effects. The SLs analogues TIT3 and TIT7 were found to significantly reduce HepG2 cell viability in a dose- and time-dependent manner and induce apoptosis. Interestingly, though TIT3 and TIT7 strongly affected cancer cell proliferation, both compounds showed moderate anti-proliferative effect on normal cells. Further, migration of cancer cells was suppressed upon treatment with TIT3 and TIT7 in a wound healing assay. In summary, these findings suggest that two SLs analogues TIT3 and TIT7 exert selective inhibitory effects on cancer cells most likely through targeting microtubules. SLs analogues could be used in future as potential anti-cancer candidates in chemotherapy.

  16. Comparison of antimicrobial activities of natural essential oils and synthetic fragrances against selected environmental pathogens

    Directory of Open Access Journals (Sweden)

    Paula L. Vieira-Brock

    2017-12-01

    Full Text Available Plant essential oils (EOs are known to inhibit the growth of bacteria and fungi. Whether these antimicrobial effects are comparable to synthetic household products is less clear. Furthermore, limited research is available on the potential additive effect of blending EOs. In this investigation, a new EO blend containing orange, patchouli, peppermint, and clary sage was compared to its individual single oils and to three household products–air freshener, liquid soap, and body spray–for their ability to inhibit the growth of Staphylococcus aureus, Streptococcus pneumoniae, Pseudonomas aeruginosa, and Aspergillus brasiliensis in the disc-diffusion assay. The new EO blend significantly inhibited the growth of the four microorganisms. The zones of inhibition of new EO blend were greater than the air freshener and similar to the liquid soap and body spray, with the exception of Str. pneumoniae in which the body spray provided greater inhibitory zone. The new EO blend and the single oils, with the exception of peppermint, equally inhibited the growth of S. aureus and Str. pneumoniae suggesting no additive effect. P. aeruginosa and A. brasiliensis showed variable susceptibility to all EOs except for no susceptibility to orange and limonene. No difference was found between (− and (+-limonene; whereas, (+-menthol showed greater effect than (−-menthol. In conclusion, blending the EO of orange, patchouli, peppermint, and clary sage was beneficial in inhibiting the growth of S. aureus, Str. pneumoniae, P. aeruginosa, and A. brasiliensis providing a natural antimicrobial fragrance option over synthetics fragrances used in soaps, body sprays, and air fresheners. Keywords: Essential oils, Soap, Body spray, Air freshener

  17. Synthetic Strigolactone Analogues Reveal Anti-Cancer Activities on Hepatocellular Carcinoma Cells

    KAUST Repository

    Hasan, Mohammed Nihal; Choudhry, Hani; Razvi, Syed Shoeb; Moselhy, Said Salama; Kumosani, Taha Abduallah; Zamzami, Mazin A.; Omran, Ziad; Halwani, Majed A.; Al-Babili, Salim; Abualnaja, Khalid Omer; Al-Malki, Abdulrahman Labeed; Alhosin, Mahmoud; Asami, Tadao

    2018-01-01

    Hepatocellular carcinoma (HCC) remains one of the leading causes of death worldwide. The complex etiology is attributed to many factors like heredity, cirrhosis, hepatitis infections or the dysregulation of the different molecular pathways. Nevertheless, the current treatment regimens have either severe side effects or tumors gradually acquire resistance upon prolonged use. Thus, developing a new selective treatment for HCC is the need of the hour. Many anticancer agents derived from plants have been evaluated for their cytotoxicity towards many human cancer cell lines. Strigolactones (SLs)-a newly discovered class of phytohormones, play a crucial role in the development of plant-root and shoot. Recently, many synthetic analogues of SLs have demonstrated pro-apoptotic effects on different cancer cell lines like prostate, breast, colon and lung. In this study, we tested synthetic SLs analogues on HCC cell line-HepG2 and evaluated their capability to induce cell proliferation inhibition and apoptosis. Primary WST-1 assays, followed by annexin-V/7AAD staining, demonstrated the anti-proliferative effects. The SLs analogues TIT3 and TIT7 were found to significantly reduce HepG2 cell viability in a dose- and time-dependent manner and induce apoptosis. Interestingly, though TIT3 and TIT7 strongly affected cancer cell proliferation, both compounds showed moderate anti-proliferative effect on normal cells. Further, migration of cancer cells was suppressed upon treatment with TIT3 and TIT7 in a wound healing assay. In summary, these findings suggest that two SLs analogues TIT3 and TIT7 exert selective inhibitory effects on cancer cells most likely through targeting microtubules. SLs analogues could be used in future as potential anti-cancer candidates in chemotherapy.

  18. Large-scale production and study of a synthetic G protein-coupled receptor: Human olfactory receptor 17-4

    Science.gov (United States)

    Cook, Brian L.; Steuerwald, Dirk; Kaiser, Liselotte; Graveland-Bikker, Johanna; Vanberghem, Melanie; Berke, Allison P.; Herlihy, Kara; Pick, Horst; Vogel, Horst; Zhang, Shuguang

    2009-01-01

    Although understanding of the olfactory system has progressed at the level of downstream receptor signaling and the wiring of olfactory neurons, the system remains poorly understood at the molecular level of the receptors and their interaction with and recognition of odorant ligands. The structure and functional mechanisms of these receptors still remain a tantalizing enigma, because numerous previous attempts at the large-scale production of functional olfactory receptors (ORs) have not been successful to date. To investigate the elusive biochemistry and molecular mechanisms of olfaction, we have developed a mammalian expression system for the large-scale production and purification of a functional OR protein in milligram quantities. Here, we report the study of human OR17-4 (hOR17-4) purified from a HEK293S tetracycline-inducible system. Scale-up of production yield was achieved through suspension culture in a bioreactor, which enabled the preparation of >10 mg of monomeric hOR17-4 receptor after immunoaffinity and size exclusion chromatography, with expression yields reaching 3 mg/L of culture medium. Several key post-translational modifications were identified using MS, and CD spectroscopy showed the receptor to be ≈50% α-helix, similar to other recently determined G protein-coupled receptor structures. Detergent-solubilized hOR17-4 specifically bound its known activating odorants lilial and floralozone in vitro, as measured by surface plasmon resonance. The hOR17-4 also recognized specific odorants in heterologous cells as determined by calcium ion mobilization. Our system is feasible for the production of large quantities of OR necessary for structural and functional analyses and research into OR biosensor devices. PMID:19581598

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

    NARCIS (Netherlands)

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

    2015-01-01

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

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

    NARCIS (Netherlands)

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

    1993-01-01

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

  1. Phase I hydroxylated metabolites of the K2 synthetic cannabinoid JWH-018 retain in vitro and in vivo cannabinoid 1 receptor affinity and activity.

    Directory of Open Access Journals (Sweden)

    Lisa K Brents

    Full Text Available K2 products are synthetic cannabinoid-laced, marijuana-like drugs of abuse, use of which is often associated with clinical symptoms atypical of marijuana use, including hypertension, agitation, hallucinations, psychosis, seizures and panic attacks. JWH-018, a prevalent K2 synthetic cannabinoid, is structurally distinct from Δ(9-THC, the main psychoactive ingredient in marijuana. Since even subtle structural differences can lead to differential metabolism, formation of novel, biologically active metabolites may be responsible for the distinct effects associated with K2 use. The present study proposes that K2's high adverse effect occurrence is due, at least in part, to distinct JWH-018 metabolite activity at the cannabinoid 1 receptor (CB1R.JWH-018, five potential monohydroxylated metabolites (M1-M5, and one carboxy metabolite (M6 were examined in mouse brain homogenates containing CB1Rs, first for CB1R affinity using a competition binding assay employing the cannabinoid receptor radioligand [(3H]CP-55,940, and then for CB1R intrinsic efficacy using an [(35S]GTPγS binding assay. JWH-018 and M1-M5 bound CB1Rs with high affinity, exhibiting K(i values that were lower than or equivalent to Δ(9-THC. These molecules also stimulated G-proteins with equal or greater efficacy relative to Δ(9-THC, a CB1R partial agonist. Most importantly, JWH-018, M2, M3, and M5 produced full CB1R agonist levels of activation. CB1R-mediated activation was demonstrated by blockade with O-2050, a CB1R-selective neutral antagonist. Similar to Δ(9-THC, JWH-018 and M1 produced a marked depression of locomotor activity and core body temperature in mice that were both blocked by the CB1R-preferring antagonist/inverse agonist AM251.Unlike metabolites of most drugs, the studied JWH-018 monohydroxylated compounds, but not the carboxy metabolite, retain in vitro and in vivo activity at CB1Rs. These observations, combined with higher CB1R affinity and activity relative to Δ(9

  2. Toxic influence of silver and uranium salts on activated sludge of wastewater treatment plants and synthetic activated sludge associates modeled on its pure cultures.

    Science.gov (United States)

    Tyupa, Dmitry V; Kalenov, Sergei V; Skladnev, Dmitry A; Khokhlachev, Nikolay S; Baurina, Marina M; Kuznetsov, Alexander Ye

    2015-01-01

    Toxic impact of silver and uranium salts on activated sludge of wastewater treatment facilities has been studied. Some dominating cultures (an active nitrogen fixer Agrobacterium tumifaciens (A.t) and micromyces such as Fusarium nivale, Fusarium oxysporum, and Penicillium glabrum) have been isolated and identified as a result of selection of the activated sludge microorganisms being steadiest under stressful conditions. For these cultures, the lethal doses of silver amounted 1, 600, 50, and 300 µg/l and the lethal doses of uranium were 120, 1,500, 1,000, and 1,000 mg/l, respectively. A.tumifaciens is shown to be more sensitive to heavy metals than micromyces. Synthetic granular activated sludge was formed on the basis of three cultures of the isolated micromyces steadiest against stress. Its granules were much more resistant to silver than the whole native activated sludge was. The concentration of silver causing 50 % inhibition of synthetic granular activated sludge growth reached 160-170 μg/l as far as for the native activated sludge it came only to 100-110 μg/l.

  3. Comparison of human myofibrillar protein catabolic rate derived from 3-methylhistidine excretion with synthetic rate from muscle biopsies during L-(. cap alpha. -/sup 15/N)lysine infusion

    Energy Technology Data Exchange (ETDEWEB)

    McKeran, R O; Halliday, D; Purkiss, P [Clinical Research Centre, Harrow (UK). Div. of Inherited Metabolic Diseases and Clinical Investigation

    1978-05-01

    Urine was collected in five healthy men over 10 to 14 days, with fasting blood samples on days 1, 5 and 10, whilst they consumed a standard creatine-free diet, which was quantitatively related to their body surface area. The urinary excretion of 3-methylhistidine fell to a plateau by day 5 in all subjects. Myofibrillar protein catabolic rate calculated from the mean value of 3-methylhistidine excretion from day 5 to day 10 averaged 1.21 g day/sup -1/ kg/sup -1/ body weight. The average turnover of muscle myofibrillar protein was calculated to be 2.16%/day. From a previous study using continuous intravenous infusion of L-(a-/sup 15/N)lysine with serial muscle biopsies on the same subjects, the mean myofibrillar protein synthetic rate was calculated to be 0.82 g day/sup -1/ kg/sup -1/ body weight, and the mean turnover rate was 1.47%/day of total muscle myofibrillar protein. The estimations of myofibrillar protein turnover rate derived from the two methods are compared and the differences discussed.

  4. Gc protein (vitamin D-binding protein): Gc genotyping and GcMAF precursor activity.

    Science.gov (United States)

    Nagasawa, Hideko; Uto, Yoshihiro; Sasaki, Hideyuki; Okamura, Natsuko; Murakami, Aya; Kubo, Shinichi; Kirk, Kenneth L; Hori, Hitoshi

    2005-01-01

    The Gc protein (human group-specific component (Gc), a vitamin D-binding protein or Gc globulin), has important physiological functions that include involvement in vitamin D transport and storage, scavenging of extracellular G-actin, enhancement of the chemotactic activity of C5a for neutrophils in inflammation and macrophage activation (mediated by a GalNAc-modified Gc protein (GcMAF)). In this review, the structure and function of the Gc protein is focused on especially with regard to Gc genotyping and GcMAF precursor activity. A discussion of the research strategy "GcMAF as a target for drug discovery" is included, based on our own research.

  5. Light-activated control of protein channel assembly mediated by membrane mechanics

    Science.gov (United States)

    Miller, David M.; Findlay, Heather E.; Ces, Oscar; Templer, Richard H.; Booth, Paula J.

    2016-12-01

    Photochemical processes provide versatile triggers of chemical reactions. Here, we use a photoactivated lipid switch to modulate the folding and assembly of a protein channel within a model biological membrane. In contrast to the information rich field of water-soluble protein folding, there is only a limited understanding of the assembly of proteins that are integral to biological membranes. It is however possible to exploit the foreboding hydrophobic lipid environment and control membrane protein folding via lipid bilayer mechanics. Mechanical properties such as lipid chain lateral pressure influence the insertion and folding of proteins in membranes, with different stages of folding having contrasting sensitivities to the bilayer properties. Studies to date have relied on altering bilayer properties through lipid compositional changes made at equilibrium, and thus can only be made before or after folding. We show that light-activation of photoisomerisable di-(5-[[4-(4-butylphenyl)azo]phenoxy]pentyl)phosphate (4-Azo-5P) lipids influences the folding and assembly of the pentameric bacterial mechanosensitive channel MscL. The use of a photochemical reaction enables the bilayer properties to be altered during folding, which is unprecedented. This mechanical manipulation during folding, allows for optimisation of different stages of the component insertion, folding and assembly steps within the same lipid system. The photochemical approach offers the potential to control channel assembly when generating synthetic devices that exploit the mechanosensitive protein as a nanovalve.

  6. A synthetic arabinose-inducible promoter confers high levels of recombinant protein expression in hyperthermophilic archaeon Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Peng, Nan; Deng, Ling; Mei, Yuxia

    2012-01-01

    Despite major progresses in genetic studies of hyperthermophilic archaea, recombinant protein production in these organisms always suffers from low yields and a robust expression system is still in great demand. Here we report a versatile vector that confers high levels of protein expression...... to remove the peptide tags from expressed recombinant proteins. While pEXA employed an araS promoter for protein expression, pSeSD utilized P(araS-SD), an araS derivative promoter carrying an engineered ribosome-binding site (RBS; a Shine-Dalgarno [SD] sequence). We found that P(araS-SD) directed high...... levels of target gene expression. More strikingly, N-terminal amino acid sequencing of recombinant proteins unraveled that the protein synthesized from pEXA-N-lacS lacked the designed 6×His tag and that translation initiation did not start at the ATG codon of the fusion gene. Instead, it started...

  7. Synthetic Cannabinoids

    Directory of Open Access Journals (Sweden)

    Aslihan Okan Ibiloglu

    2017-09-01

    Full Text Available Synthetic cannabinoids which is a subgroup of cannabinoids are commonly used for recreational drug use throughout the whole world. Although both marijuana and synthetic cannabinoids stimulate the same receptors, cannabinoid receptor 1 (CB1 and cannabinoid receptor 2 (CB2, studies have shown that synthetic cannabinoids are much more potent than marijuana. The longer use of synthetic cannabinoids can cause severe physical and psychological symptoms that might even result in death, similar to many known illicit drugs. Main treatment options mostly involve symptom management and supportive care. The aim of this article is to discuss clinical and pharmacological properties of the increasingly used synthetic cannabinoids. [Psikiyatride Guncel Yaklasimlar - Current Approaches in Psychiatry 2017; 9(3.000: 317-328

  8. Use of adsorption using granular activated carbon (GAC) for the enhancement of removal of chromium from synthetic wastewater by electrocoagulation.

    Science.gov (United States)

    Vivek Narayanan, N; Ganesan, Mahesh

    2009-01-15

    The present work deals with removal of hexavalent chromium from synthetic effluents in a batch stirred electrocoagulation cell with iron-aluminium electrode pair coupled with adsorption using granular activated carbon (GAC). Several working parameters such as pH, current density, adsorbent concentration and operating time were studied in an attempt to achieve higher removal capacity. Results obtained with synthetic wastewater revealed that most effective removal capacities of chromium (VI) could be achieved when the initial pH was near 8. The removal of chromium (VI) during electrocoagulation, is due to the combined effect of chemical precipitation, coprecipitation, sweep coagulation and adsorption. In addition, increasing current density in a range of 6.7-26.7mA/cm2 and operating time from 20 to 100min enhanced the treatment rate to reduce metal ion concentration below admissible legal levels. The addition of GAC as adsorbent resulted in remarkable increase in the removal rate of chromium at lower current densities and operating time, than the conventional electrocoagulation process. The method was found to be highly efficient and relatively fast compared to existing conventional techniques.

  9. Complement Activation by Ceramide Transporter Proteins

    NARCIS (Netherlands)

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

    2014-01-01

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

  10. Synthetic biology era: Improving antibiotic's world.

    Science.gov (United States)

    Guzmán-Trampe, Silvia; Ceapa, Corina D; Manzo-Ruiz, Monserrat; Sánchez, Sergio

    2017-06-15

    The emergence of antibiotic-resistant pathogen microorganisms is problematic in the context of the current spectrum of available medication. The poor specificity and the high toxicity of some available molecules have made imperative the search for new strategies to improve the specificity and to pursue the discovery of novel compounds with increased bioactivity. Using living cells as platforms, synthetic biology has counteracted this problem by offering novel pathways to create synthetic systems with improved and desired functions. Among many other biotechnological approaches, the advances in synthetic biology have made it possible to design and construct novel biological systems in order to look for new drugs with increased bioactivity. Advancements have also been made in the redesigning of RNA and DNA molecules in order to engineer antibiotic clusters for antibiotic overexpression. As for the production of these antibacterial compounds, yeasts and filamentous fungi as well as gene therapy are utilized to enhance protein solubility. Specific delivery is achieved by creating chimeras using plant genes into bacterial hosts. Some of these synthetic systems are currently in clinical trials, proving the proficiency of synthetic biology in terms of both pharmacological activities as well as an increase in the biosafety of treatments. It is possible that we may just be seeing the tip of the iceberg, and synthetic biology applications will overpass expectations beyond our present knowledge. Copyright © 2017. Published by Elsevier Inc.

  11. Locomotor activity and discriminative stimulus effects of a novel series of synthetic cathinone analogs in mice and rats.

    Science.gov (United States)

    Gatch, Michael B; Dolan, Sean B; Forster, Michael J

    2017-04-01

    Recent years have seen an increase in the recreational use of novel, synthetic psychoactive substances. There are little or no data on the abuse liability of many of the newer compounds. The current study investigated the discriminative stimulus and locomotor effects of a series of synthetic analogs of cathinone: α-pyrrolidinopropiophenone (α-PPP), α-pyrrolidinohexiophenone (α-PHP), α-pyrrolidinopentiothiophenone (α-PVT), 3,4-methylenedioxybutiophenone (MDPBP), and ethylone. Locomotor activity was assessed in an open-field assay using Swiss-Webster mice. Discriminative stimulus effects were assessed in Sprague-Dawley rats trained to discriminate either cocaine or methamphetamine from vehicle. Each of the compounds produced an inverted-U dose-effect on locomotor activity. Maximal effects were similar among the test compounds, but potencies varied with relative potencies of MDPBP > α-PPP = α-PHP > ethylone > α-PVT. Each of the test compounds substituted fully for the discriminative stimulus effects of methamphetamine. α-PPP, α-PHP, and ethylone fully substituted for cocaine. α-PVT produced a maximum of 50% cocaine-appropriate responding, and MDPBP produced an inverted-U-shaped dose-effect curve with maximum effects of 67%. These data provide initial evidence that these structurally similar, emerging novel psychoactive substances demonstrate potential for abuse and may be utilized for their stimulant-like effects, given their ability to stimulate locomotor activity and their substitution for the discriminative stimulus effects of the classical psychostimulants cocaine and/or methamphetamine.

  12. Anti-Bacterial and Anti-Fungal Activity of Xanthones Obtained via Semi-Synthetic Modification of α-Mangostin from Garcinia mangostana

    Directory of Open Access Journals (Sweden)

    Srinivasan Narasimhan

    2017-02-01

    Full Text Available The microbial contamination in food packaging has been a major concern that has paved the way to search for novel, natural anti-microbial agents, such as modified α-mangostin. In the present study, twelve synthetic analogs were obtained through semi-synthetic modification of α-mangostin by Ritter reaction, reduction by palladium-carbon (Pd-C, alkylation, and acetylation. The evaluation of the anti-microbial potential of the synthetic analogs showed higher bactericidal activity than the parent molecule. The anti-microbial studies proved that I E showed high anti-bacterial activity whereas I I showed the highest anti-fungal activity. Due to their microbicidal potential, modified α-mangostin derivatives could be utilized as active anti-microbial agents in materials for the biomedical and food industry.

  13. Sensitive detection of proteasomal activation using the Deg-On mammalian synthetic gene circuit.

    Science.gov (United States)

    Zhao, Wenting; Bonem, Matthew; McWhite, Claire; Silberg, Jonathan J; Segatori, Laura

    2014-04-08

    The ubiquitin proteasome system (UPS) has emerged as a drug target for diverse diseases characterized by altered proteostasis, but pharmacological agents that enhance UPS activity have been challenging to establish. Here we report the Deg-On system, a genetic inverter that translates proteasomal degradation of the transcriptional regulator TetR into a fluorescent signal, thereby linking UPS activity to an easily detectable output, which can be tuned using tetracycline. We demonstrate that this circuit responds to modulation of UPS activity in cell culture arising from the inhibitor MG-132 and activator PA28γ. Guided by predictive modelling, we enhanced the circuit's signal sensitivity and dynamic range by introducing a feedback loop that enables self-amplification of TetR. By linking UPS activity to a simple and tunable fluorescence output, these genetic inverters will enable a variety of applications, including screening for UPS activating molecules and selecting for mammalian cells with different levels of proteasome activity.

  14. Antifungal activity of synthetic di(hetero)arylamines based on the benzo[b]thiophene moiety.

    Science.gov (United States)

    Pinto, Eugénia; Queiroz, Maria-João R P; Vale-Silva, Luís A; Oliveira, João F; Begouin, Agathe; Begouin, Jeanne-Marie; Kirsch, Gilbert

    2008-09-01

    The antifungal activity of several di(hetero)arylamine derivatives of the benzo[b]thiophene system was evaluated against clinically relevant Candida, Aspergillus, and dermatophyte species by a broth macrodilution test based on CLSI (formerly NCCLS) guidelines. The most active compound showed a broad spectrum of activity (against all tested fungal strains, including fluconazole-resistant fungi), with particularly low MICs for dermatophytes. Results from the inhibition of the dimorphic transition in Candida albicans and flow cytometry studies further confirmed their biological activity. With this study it was possible to establish some structure-activity relationships (SARs). The hydroxy groups proved to be essential for the activity in the aryl derivatives. Furthermore, the spectrum of activity in the pyridine derivatives was broadened by the absence of the ester group on position 2 of the benzo[b]thiophene system.

  15. Activity and Safety of Synthetic Lectins Based on Benzoboroxole-Functionalized Polymers for Inhibition of HIV Entry

    Science.gov (United States)

    Mahalingam, Alamelu; Geonnotti, Anthony R.; Balzarini, Jan; Kiser, Patrick F.

    2011-01-01

    Lectins derived from plant and microbial sources constitute a vital class of entry inhibitors that target the oligomannose residues on the HIV envelope gp120. Despite their potency and specificity, success of lectin-based entry inhibitors may be impeded by issues in regards to economical production, formulation and potential mitogenicity. Therefore, there exists a gap in the HIV therapeutics pipeline that underscores the need for mass producible, synthetic, broad-spectrum, and biocomptabile inhibitors of HIV entry. Here, we present the development of a polymeric synthetic lectin, based on benzoboroxole (BzB), which exhibits weak affinity (~25 M−1) for non-reducing sugars, similar to those found on the HIV envelope. High molecular weight BzB-functionalized polymers demonstrated antiviral activity that increased with an increase in ligand density and molecular weight of the polymer construct; revealing that polyvalency improves activity. Polymers showed significant increase in activity from 25 to 75 mol% BzB functionalization with EC50 of 15 μM and 15 nM, respectively. A further increase in mole functionalization to 90% resulted in an increase of the EC50 (59 ± 5 nM), likely due to the elongated rigid structure of the polymer chain compelled by electrostatic repulsion between the boronic acid groups. An increase in molecular weight of the polymer at 50 mol% BzB functionalization showed a gradual but significant increase in antiviral activity, with the highest activity seen with the 382 kDa polymer (EC50 of 1.1 ± 0.5 nM in CEM cells and 11 ± 3 nM in TZM-bl cells). Supplementing the polymer backbone with 10 mol% sulfonic acid not only increased the aqueous solubility of the polymers by at least 50-fold, but also demonstrated a synergistic increase in anti-HIV activity (4.0 ± 1.5 nM in TZM-bl cells), possibly due to electrostatic interactions between the negatively charged polymer backbone and the positively charged V3-loop in the gp120. The benzoboroxole

  16. Larvicidal activity and structure activity relationship of cinnamoyl amides from Zanthoxylum armatum and their synthetic analogues against diamondback moth, Plutella xylostella.

    Science.gov (United States)

    Kumar, Vishal; Reddy, S G Eswara; Bhardwaj, Anuja; Dolma, Shudh Kirti; Kumar, Neeraj

    2016-01-01

    Cinnamoyl amides isolated from Zanthoxylum armatum (Rutaceae) and their synthetic analogues were tested for their insecticidal activity against the second instar larvae of diamondback moth, Plutella xylostella (L.) (Lepidoptera: Yponomeutidae) to determine the promising structures with insecticidal activity. Most of the test compounds showed promising activity against larvae of P. xylostella. However, the activities of different compounds varied depending on the presence of different substituents at various positions of both the aromatic rings A and B. Among the tested compounds, 8, N-(3-bromo-4-methoxyphenethyl)cinnamamide showed best larvicidal activity with an LC50 = 62.13 mg/L followed by 6, N-(3׳-bromophenethyl)cinnamamide (LC50=128.49 mg/L) and 2 N-(4׳-methoxyphenylethyl)cinnamamide (LC50 = 225.65 mg/L).

  17. Enhancement of aerodynamic performance of a heaving airfoil using synthetic-jet based active flow control.

    Science.gov (United States)

    Wang, Chenglei; Tang, Hui

    2018-05-25

    In this study, we explore the use of synthetic jet (SJ) in manipulating the vortices around a rigid heaving airfoil, so as to enhance its aerodynamic performance. The airfoil heaves at two fixed pitching angles, with the Strouhal number, reduced frequency and Reynolds number chosen as St  =  0.3, k  =  0.25 and Re  =  100, respectively, all falling in the ranges for natural flyers. As such, the vortex force plays a dominant role in determining the airfoil's aerodynamic performance. A pair of in-phase SJs is implemented on the airfoil's upper and lower surfaces, operating with the same strength but in opposite directions. Such a fluid-structure interaction problem is numerically solved using a lattice Boltzmann method based numerical framework. It is found that, as the airfoil heaves with zero pitching angle, its lift and drag can be improved concurrently when the SJ phase angle [Formula: see text] relative to the heave motion varies between [Formula: see text] and [Formula: see text]. But this concurrent improvement does not occur as the airfoil heaves with [Formula: see text] pitching angle. Detailed inspection of the vortex evolution and fluid stress over the airfoil surface reveals that, if at good timing, the suction and blowing strokes of the SJ pair can effectively delay or promote the shedding of leading edge vortices, and mitigate or even eliminate the generation of trailing edge vortices, so as to enhance the airfoil's aerodynamic performance. Based on these understandings, an intermittent operation of the SJ pair is then proposed to realize concurrent lift and drag improvement for the heaving airfoil with [Formula: see text] pitching angle.

  18. Activity of Selected Formulated Biorational and Synthetic Insecticides Against Larvae of Helicoverpa armigera (Lepidoptera: Noctuidae).

    Science.gov (United States)

    Vivan, L M; Torres, J B; Fernandes, P L S

    2017-02-01

    This work studied 17 insecticides belonging to nucleopolyhedrovirus (NPV), Bacillus thuringiensis (Bt kurstaki and Bt aizawai), benzoylureas (insect growth regulators [IGRs]), carbamates, organophosphates, spinosyns, and diamides against larvae of Helicoverpa armigera (Hübner), invasive species in the South American continent. Larvae of different instars were fed for 7 d with untreated or insecticide-treated diets. Mortality was recorded daily for 7 d, and surviving larvae were individually weighed on the seventh day. The NPV and Bt insecticides caused 100% mortality of first-instar larvae and first-instar and second-instar larvae, respectively. However, both NPV and Bt-based products caused low mortality of third-instar larvae and did not kill older larvae. The IGR lufenuron was highly effective against all three ages of larvae tested, whereas teflubenzuron and triflumuron produced maximum 60% mortality of second-instar larvae and lower than 50% to older larvae. Thiodicarb, chlorantraniliprole, indoxacarb, chlorpyrifos, and chlorfenapyr, irrespective of tested age, caused 100% mortality of larvae, with the last two insecticides reaching 100% mortality within 2 d of feeding on the treated diet. Flubendiamide caused lower mortality but significantly affected the weight of surviving larvae, whereas neither spinosad nor methomyl produced significant mortality or affected the weight of larvae. Based on the results, the age of H. armigera larvae plays an important role in the recommendation of NPV and Bt insecticides. Furthermore, there are potential options between biological and synthetic insecticides tested against H. armigera, and recording larval size during monitoring, in addition to the infestation level, should be considered when recommending biological-based insecticides to control this pest. © The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  19. Antifouling Activity of Simple Synthetic Diterpenoids against Larvae of the Barnacle Balanus albicostatus Pilsbry

    Directory of Open Access Journals (Sweden)

    Dan-Qing Feng

    2010-11-01

    Full Text Available Five new pimarane diterpenoids 1-5 were synthesized using ent-8(14-pimarene-15R,16-diol as starting material. The structures were elucidated by means of extensive NMR and MS analysis. The antifouling activity against larval settlement of the barnacle Balanus albicostatus were evaluated using capsaicin as a positive control. Compounds 1-3 and 5 showed more potent antifouling activity than capsaicin. Compound 5, which exhibited almost the same antifouling activity as starting material, showed better stability than starting material. These compounds all showed antifouling activity in a non-toxic way against larval settlement of the barnacle B. albicostatus. Analysis of structure-activity relationships (SAR demonstrated that the substituents on the C-15 and C-16 position of pimarane diterpenoid were responsible for the antifouling activity.

  20. Venom Protein C activators as diagnostic agents for defects of protein C System.

    Science.gov (United States)

    Ramzan, Faiqah; Asmat, Andleeb

    2018-06-18

    Background Protein C is a vitamin K dependent plasma zymogen. It prevents clotting by inhibiting clotting by inactivating factor V and factor VIII. Protein C activation pathway involves three steps: (i) Activation of protein C; (ii) Inhibition of coagulation through inactivating factor V and VIII by activated protein C and (iii) Inhibition of activated protein C by plasma protease inhibitors specific for this enzyme. Proteinases converts the zymogen Protein C (PC) of vertebrates into activated PC, which has been detected in several snake venoms. Most PC activators have been purified from venom of snake species belonging to the genera of the Agkistrodon complex. Unlike the physiological thrombin-catalyzed PC activation reaction which requires thrombomodulin as a cofactor, most snake venom activators directly convert the zymogen PC into the catalytically active form which can easily be determined by means of coagulation or chromogenic substrate techniques. Conclusion The fast-acting PC activator Protac® from Agkistrodon contortrix (southern copperhead snake) venom has been found to have broad application in diagnostic practice for the determination of disorders in the PC pathway. Recently, screening assays for the PC pathway have been introduced, based on the observation that the PC pathway is probably the most important physiological barrier against thrombosis. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  1. In Vitro Screening of Synthetic Fluorogenic Substrates for Detection of Cancer Procoagulant Activity.

    Science.gov (United States)

    Krause, Jason; Frost, Carminita L

    2018-04-01

    Cancer procoagulant (CP), a direct activator of coagulation factor X, is among one of the tumour cell products or activities which may promote fibrin formation and has been suggested to be selectively associated with the malignant phenotype. At present, the most reliable assay for the quantification of CP activity is the three-stage chromogenic assay which utilises the ability of CP to activate factor X. In this assay, the activation of factor X leads to the formation of activated thrombin from prothrombin and the eventual hydrolyses of a thrombin chromogenic substrate which contains a p-nitroaniline leaving group. The complexity of the three-stage chromogenic assay suggests a need for a direct method of assaying CP activity. This study focuses on the design of a fluorogenic substrate that would enable the direct quantification of CP activity. The results of the study show two promising substrates for the determination of CP activity: Boc-PQVR-AMC and PQVR-AMC. Further analysis showed that Boc-PQVR-AMC could be excluded as a potential substrate for CP since it was also cleaved by collagenase.

  2. Protein kinase and phosphatase activities of thylakoid membranes

    International Nuclear Information System (INIS)

    Michel, H.; Shaw, E.K.; Bennett, J.

    1987-01-01

    Dephosphorylation of the 25 and 27 kDa light-harvesting Chl a/b proteins (LHCII) of the thylakoid membranes is catalyzed by a phosphatase which differs from previously reported thylakoid-bound phosphatases in having an alkaline pH optimum (9.0) and a requirement for Mg 2+ ions. Dephosphorylation of the 8.3 kDa psb H gene product requires a Mg 2+ ion concentration more than 200 fold higher than that for dephosphorylation of LHC II. The 8.3 kDa and 27 kDa proteins appear to be phosphorylated by two distinct kinases, which differ in substrate specificity and sensitivity to inhibitors. The plastoquinone antagonist 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone (DBMIB) inhibits phosphorylation of the 27 kDa LHC II much more readily than phosphorylation of the 8.3 kDa protein. A similar pattern of inhibition is seen for two synthetic oligopeptides (MRKSATTKKAVC and ATQTLESSSRC) which are analogs of the phosphorylation sites of the two proteins. Possible modes of action of DBMIB are discussed. 45 refs., 7 figs., 3 tabs

  3. Mitogen-activated protein kinase signaling in plants

    DEFF Research Database (Denmark)

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

    2010-01-01

    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...... 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...

  4. Protein stability and enzyme activity at extreme biological temperatures

    International Nuclear Information System (INIS)

    Feller, Georges

    2010-01-01

    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 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)

  5. Building synthetic cellular organization

    OpenAIRE

    Polka, Jessica K.; Silver, Pamela A.

    2013-01-01

    The elaborate spatial organization of cells enhances, restricts, and regulates protein–protein interactions. However, the biological significance of this organization has been difficult to study without ways of directly perturbing it. We highlight synthetic biology tools for engineering novel cellular organization, describing how they have been, and can be, used to advance cell biology.

  6. 2-propen-1-amine derivatives and their synthetic intermediates: activity against pathogenic trypanosomatids.

    Science.gov (United States)

    de Souza, A O; Hemerly, F P; Gomes-Cardoso, L; Santa-Rita, R M; Leon, L L; de Castro, S L; Durán, N

    2004-12-01

    The potential activity of three new derivatives of 3-(4'-Y-[1,1'-biphenyl]-4-yl)-3-(4-X-phenyl)-N,N-dimethyl-2-propen-1-amine (2-PAMs) was assayed against Trypanosoma cruzi and Leishmania amazonensis. They showed higher activity against trypomastigotes and epimastigotes of T. cruzi than the standard drugs, crystal violet and nifurtimox. Besides these derivatives, a series of eleven 2-PAMs derivatives and the corresponding intermediates, biphenyl methanones (BPMs) were assayed against promastigotes of L. amazonensis, showing that the 2-PAMs were remarkably more active than the BPMs. The PAMs 2c, 2e and 2j were about 2-fold more active that pentamidine isothionate and between 27.2- and 46.4-fold less toxic to V79 mammalian cells. The present results encourage further studies, especially against intracellular parasites and in experimental animals.

  7. 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......), 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...... 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...

  8. Metofluthrin: a potent new synthetic pyrethroid with high vapor activity against mosquitoes.

    Science.gov (United States)

    Ujihara, Kazuya; Mori, Tatsuya; Iwasaki, Tomonori; Sugano, Masayo; Shono, Yoshinori; Matsuo, Noritada

    2004-01-01

    (1R)-trans-Norchrysanthemic acid fluorobenzyl esters are synthesized and their structure-activity relationships are discussed. These esters show outstanding insecticidal activity against mosquitoes. In particular, the 2,3,5,6-tetrafluoro-4-methoxymethylbenzyl analog (metofluthrin) exhibits the highest potency, being approximately forty times as potent as d-allethrin in a mosquito coil formulation when tested against southern house mosquitoes (Culex quinquefasciatus). Metofluthrin also exhibits a significant vapor action at room temperature.

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

    NARCIS (Netherlands)

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

    1992-01-01

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

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

    International Nuclear Information System (INIS)

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

    2014-01-01

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

  11. Positioning cell wall synthetic complexes by the bacterial morphogenetic proteins MreB and MreD.

    Science.gov (United States)

    White, Courtney L; Kitich, Aleksandar; Gober, James W

    2010-05-01

    In Caulobacter crescentus, intact cables of the actin homologue, MreB, are required for the proper spatial positioning of MurG which catalyses the final step in peptidoglycan precursor synthesis. Similarly, in the periplasm, MreC controls the spatial orientation of the penicillin binding proteins and a lytic transglycosylase. We have now found that MreB cables are required for the organization of several other cytosolic murein biosynthetic enzymes such as MraY, MurB, MurC, MurE and MurF. We also show these proteins adopt a subcellular pattern of localization comparable to MurG, suggesting the existence of cytoskeletal-dependent interactions. Through extensive two-hybrid analyses, we have now generated a comprehensive interaction map of components of the bacterial morphogenetic complex. In the cytosol, this complex contains both murein biosynthetic enzymes and morphogenetic proteins, including RodA, RodZ and MreD. We show that the integral membrane protein, MreD, is essential for lateral peptidoglycan synthesis, interacts with the precursor synthesizing enzymes MurG and MraY, and additionally, determines MreB localization. Our results suggest that the interdependent localization of MreB and MreD functions to spatially organize a complex of peptidoglycan precursor synthesis proteins, which is required for propagation of a uniform cell shape and catalytically efficient peptidoglycan synthesis.

  12. Guanylation of thiosemicarbazones: a new synthetic route to polysubstituted guanylhydrazones with antimicrobial activity

    International Nuclear Information System (INIS)

    Cunha, Silvio; Macedo Junior, Fernando Cesar de; Costa, Giselle A.N.; Neves, Daniela C.; Souza Neta, Lourdes Cardoso de

    2009-01-01

    Thiosemicarbazones were employed for the first time as electrophiles in the guanylation reaction promoted by HgCl 2 , affording polysubstituted guanylhydrazones, with regioselective introduction of each nitrogen substituent. The antibacterial and antifungal activities of guanylhydrazones were evaluated by determination of minimal inhibitory concentrations. Some of them exhibited very low minimal inhibitory concentrations (MIC) and broad-spectrum activities. The configurations of two guanylhydrazones were assigned by X-ray analysis that also revealed intramolecular interactions of the type N-H...N and C-H...N. (author)

  13. [Cardiotropic activity of synthetic peptide CH3CO-Lys-Lys-Arg-Arg-NH2 (protectin)].

    Science.gov (United States)

    Sazhin, A I; Zaĭtseva, M A; Melikhova, M E; Ezhov, N F; Sadovnikov, V B; Navolotskaia, E V

    2011-01-01

    Peptide CH3CO-Lys-Lys-Arg-Arg-NH2 (protectin) was synthesized and its activity was studied on the model of experimental myocardial infarction in rats in comparison to the reference antihypoxant drug riboxin. Intranasal injections ofprotectin at doses within 2-20 microg/kg once a day by course of 7 days produced a pronounced anti-ischemic action, improved coronary circulation of the blood, increases contractile activity of myocardium, reduced intensity of lipid peroxidation, and improved antioxidant protection. In some respects (improved coronary circulation of the blood, increased antioxidant protection), protectin was more effective than riboxin.

  14. Adsorption uptake of synthetic organic chemicals by carbon nanotubes and activated carbons

    Science.gov (United States)

    Brooks, A. J.; Lim, Hyung-nam; Kilduff, James E.

    2012-07-01

    Carbon nanotubes (CNTs) have shown great promise as high performance materials for adsorbing priority pollutants from water and wastewater. This study compared uptake of two contaminants of interest in drinking water treatment (atrazine and trichloroethylene) by nine different types of carbonaceous adsorbents: three different types of single walled carbon nanotubes (SWNTs), three different sized multi-walled nanotubes (MWNTs), two granular activated carbons (GACs) and a powdered activated carbon (PAC). On a mass basis, the activated carbons exhibited the highest uptake, followed by SWNTs and MWNTs. However, metallic impurities in SWNTs and multiple walls in MWNTs contribute to adsorbent mass but do not contribute commensurate adsorption sites. Therefore, when uptake was normalized by purity (carbon content) and surface area (instead of mass), the isotherms collapsed and much of the CNT data was comparable to the activated carbons, indicating that these two characteristics drive much of the observed differences between activated carbons and CNT materials. For the limited data set here, the Raman D:G ratio as a measure of disordered non-nanotube graphitic components was not a good predictor of adsorption from solution. Uptake of atrazine by MWNTs having a range of lengths and diameters was comparable and their Freundlich isotherms were statistically similar, and we found no impact of solution pH on the adsorption of either atrazine or trichloroethylene in the range of naturally occurring surface water (pH = 5.7-8.3). Experiments were performed using a suite of model aromatic compounds having a range of π-electron energy to investigate the role of π-π electron donor-acceptor interactions on organic compound uptake by SWNTs. For the compounds studied, hydrophobic interactions were the dominant mechanism in the uptake by both SWNTs and activated carbon. However, comparing the uptake of naphthalene and phenanthrene by activated carbon and SWNTs, size exclusion effects

  15. Adsorption uptake of synthetic organic chemicals by carbon nanotubes and activated carbons

    International Nuclear Information System (INIS)

    Brooks, A J; Kilduff, James E; Lim, Hyung-nam

    2012-01-01

    Carbon nanotubes (CNTs) have shown great promise as high performance materials for adsorbing priority pollutants from water and wastewater. This study compared uptake of two contaminants of interest in drinking water treatment (atrazine and trichloroethylene) by nine different types of carbonaceous adsorbents: three different types of single walled carbon nanotubes (SWNTs), three different sized multi-walled nanotubes (MWNTs), two granular activated carbons (GACs) and a powdered activated carbon (PAC). On a mass basis, the activated carbons exhibited the highest uptake, followed by SWNTs and MWNTs. However, metallic impurities in SWNTs and multiple walls in MWNTs contribute to adsorbent mass but do not contribute commensurate adsorption sites. Therefore, when uptake was normalized by purity (carbon content) and surface area (instead of mass), the isotherms collapsed and much of the CNT data was comparable to the activated carbons, indicating that these two characteristics drive much of the observed differences between activated carbons and CNT materials. For the limited data set here, the Raman D:G ratio as a measure of disordered non-nanotube graphitic components was not a good predictor of adsorption from solution. Uptake of atrazine by MWNTs having a range of lengths and diameters was comparable and their Freundlich isotherms were statistically similar, and we found no impact of solution pH on the adsorption of either atrazine or trichloroethylene in the range of naturally occurring surface water (pH = 5.7–8.3). Experiments were performed using a suite of model aromatic compounds having a range of π-electron energy to investigate the role of π–π electron donor–acceptor interactions on organic compound uptake by SWNTs. For the compounds studied, hydrophobic interactions were the dominant mechanism in the uptake by both SWNTs and activated carbon. However, comparing the uptake of naphthalene and phenanthrene by activated carbon and SWNTs, size exclusion

  16. Activation of the polyomavirus enhancer by a murine activator protein 1 (AP1) homolog and two contiguous proteins.

    OpenAIRE

    Martin, M E; Piette, J; Yaniv, M; Tang, W J; Folk, W R

    1988-01-01

    The polyomavirus enhancer is composed of multiple DNA sequence elements serving as binding sites for proteins present in mouse nuclear extracts that activate transcription and DNA replication. We have identified three such proteins and their binding sites and correlate them with enhancer function. Mutation of nucleotide (nt) 5140 in the enhancer alters the binding site (TGACTAA, nt 5139-5145) for polyomavirus enhancer A binding protein 1 (PEA1), a murine homolog of the human transcription fac...

  17. Quantitative structure--property relationships for enhancing predictions of synthetic organic chemical removal from drinking water by granular activated carbon.

    Science.gov (United States)

    Magnuson, Matthew L; Speth, Thomas F

    2005-10-01

    Granular activated carbon is a frequently explored technology for removing synthetic organic contaminants from drinking water sources. The success of this technology relies on a number of factors based not only on the adsorptive properties of the contaminant but also on properties of the water itself, notably the presence of substances in the water which compete for adsorption sites. Because it is impractical to perform field-scale evaluations for all possible contaminants, the pore surface diffusion model (PSDM) has been developed and used to predict activated carbon column performance using single-solute isotherm data as inputs. Many assumptions are built into this model to account for kinetics of adsorption and competition for adsorption sites. This work further evaluates and expands this model, through the use of quantitative structure-property relationships (QSPRs) to predict the effect of natural organic matter fouling on activated carbon adsorption of specific contaminants. The QSPRs developed are based on a combination of calculated topographical indices and quantum chemical parameters. The QSPRs were evaluated in terms of their statistical predictive ability,the physical significance of the descriptors, and by comparison with field data. The QSPR-enhanced PSDM was judged to give results better than what could previously be obtained.

  18. Mineralisation of 14C-labelled synthetic lignin and ligninolytic enzyme activities of litter-decomposing basidiomycetous fungi.

    Science.gov (United States)

    Steffen, K T; Hofrichter, M; Hatakka, A

    2000-12-01

    Within a screening program, 27 soil litter-decomposing basidiomycetes were tested for ligninolytic enzyme activities using agar-media containing 2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonate), a humic acid or Mn2+ ions as indicator substrates. Most active species were found within the family Strophariaceae (Agrocybe praecox, Stropharia coronilla, S. rugosoannulata) and used for mineralisation experiments with a 14C-ring-labelled synthetic lignin (14C-DHP). The fungi mineralised around 25% of the lignin to 14CO2 within 12 weeks of incubation in a straw environment; about 20% of the lignin was converted to water-soluble fragments. Mn-peroxidase was found to be the predominant ligninolytic enzyme of all three fungi in liquid culture and its production was strongly enhanced in the presence of Mn2+ ions. The results of this study demonstrate that certain ubiquitous litter-decomposing basidiomycetes possess ligninolytic activities similar to the wood-decaying white-rot fungi, the most efficient lignin degraders in nature.

  19. Activity of the pterophyllins 2 and 4 against postharvest fruit pathogenic fungi. Comparison with a synthetic analog and related intermediates.

    Science.gov (United States)

    Pergomet, Jorgelina L; Di Liberto, Melina G; Derita, Marcos G; Bracca, Andrea B J; Kaufman, Teodoro S

    2018-03-01

    The antifungal activity of pterophyllin 2, pterophyllin 4, a 5-desmethyl analog of the latter and some of their synthetic intermediates, against three postharvest phytopathogenic fungi, was evaluated. The target fungi were Rhizopus stolonifer, Botrytis cinerea and Monilinia fructicola, which affect fruits worldwide, causing important economic losses. The tests were carried out with imazalil and carbendazim as positive controls. Minimum inhibitory concentrations and minimum fungicidal concentrations were determined, and the morphology of the colonies was examined microscopically. In liquid medium, it was found that pterophyllin 4 exhibited selective fungicidal activity toward M. fructicola, whereas its congener pterophyllin 2 proved to be less potent and not selective and the 5-desmethyl analog of pterophyllin 4 displayed a different activity profile. Morphological changes were observed in the colonies exposed to pterophyllin 4. The results highlighted the importance of small structural features for the antifungal behavior and also suggested that, in Nature, the pterophyllins may act as plant defenses against pathogens. Copyright © 2018 Elsevier B.V. All rights reserved.

  20. Development, evaluation, and laboratory validation of immunoassays for the diagnosis of equine infectious anemia (EIA) using recombinant protein produced from a synthetic p26 gene of EIA virus.

    Science.gov (United States)

    Singha, Harisankar; Goyal, Sachin K; Malik, Praveen; Khurana, Sandip K; Singh, Raj K

    2013-12-01

    Equine infectious anemia (EIA)-a retroviral disease caused by equine infectious anemia virus (EIAV)-is a chronic, debilitating disease of horses, mules, and donkeys. EIAV infection has been reported worldwide and is recognized as pathogen of significant economic importance to the horse industry. This disease falls under regulatory control program in many countries including India. Control of EIA is based on identification of inapparent carriers by detection of antibodies to EIAV in serologic tests and "Stamping Out" policy. The current internationally accepted test for diagnosis of EIA is the agar gel immune-diffusion test (AGID), which detects antibodies to the major gag gene (p26) product. The objective of this study was to develop recombinant p26 based in-house immunoassays [enzyme linked immunosorbent assays (ELISA), and AGID] for EIA diagnosis. The synthetic p26 gene of EIAV was expressed in Escherichia coli and diagnostic potential of recombinant p26 protein were evaluated in ELISA and AGID on 7,150 and 1,200 equine serum samples, respectively, and compared with commercial standard AGID kit. The relative sensitivity and specificity of the newly developed ELISA were 100 and 98.6 %, respectively. Whereas, relative sensitivity and specificity of the newly developed AGID were in complete agreement in respect to commercial AGID kit. Here, we have reported the validation of an ELISA and AGID on large number of equine serum samples using recombinant p26 protein produced from synthetic gene which does not require handling of pathogenic EIAV. Since the indigenously developed reagents would be economical than commercial diagnostic kit, the rp26 based-immunoassays could be adopted for the sero-diagnosis and control of EIA in India.

  1. In Vitro Activities against Cystic Fibrosis Pathogens of Synthetic Host Defence Propeptides Processed by Neutrophil Elastase.

    LENUS (Irish Health Repository)

    Desgranges, Stephane

    2011-02-22

    The antimicrobial and haemolytic activities of a host defence peptide can be controlled by modification as a propeptide of reduced net charge which can be processed by neutrophil elastase, a serine protease involved in chronic airway inflammation and infections associated with cystic fibrosis.

  2. 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.

  3. Chimeric microbial rhodopsins for optical activation of Gs-proteins

    Science.gov (United States)

    Yoshida, Kazuho; Yamashita, Takahiro; Sasaki, Kengo; Inoue, Keiichi; Shichida, Yoshinori; Kandori, Hideki

    2017-01-01

    We previously showed that the chimeric proteins of microbial rhodopsins, such as light-driven proton pump bacteriorhodopsin (BR) and Gloeobacter rhodopsin (GR) that contain cytoplasmic loops of bovine rhodopsin, are able to activate Gt protein upon light absorption. These facts suggest similar protein structural changes in both the light-driven proton pump and animal rhodopsin. Here we report two trials to engineer chimeric rhodopsins, one for the inserted loop, and another for the microbial rhodopsin template. For the former, we successfully activated Gs protein by light through the incorporation of the cytoplasmic loop of β2-adrenergic receptor (β2AR). For the latter, we did not observe any G-protein activation for the light-driven sodium pump from Indibacter alkaliphilus (IndiR2) or a light-driven chloride pump halorhodopsin from Natronomonas pharaonis (NpHR), whereas the light-driven proton pump GR showed light-dependent G-protein activation. This fact suggests that a helix opening motion is common to G protein coupled receptor (GPCR) and GR, but not to IndiR2 and NpHR. Light-induced difference FTIR spectroscopy revealed similar structural changes between WT and the third loop chimera for each light-driven pump. A helical structural perturbation, which was largest for GR, was further enhanced in the chimera. We conclude that similar structural dynamics that occur on the cytoplasmic side of GPCR are needed to design chimeric microbial rhodopsins. PMID:29362703

  4. Biological Activity of Peanut (Arachis hypogaea) Phytoalexins and Selected Natural and Synthetic Stilbenoids

    Science.gov (United States)

    2011-02-11

    prenylated flavonoids have been identified as constituents in plants, and display biological activities, such as anticancer, antiandrogen, anti-Leishmania, and...to opioid receptors was examined. ’MATERIALS AND METHODS General Experimental Procedures. HPLC -grade solvents used in the preparation of mobile phases...were obtained from Fisher (Suwanee, GA). HPLC -grade H2Owas prepared with a ZD20 four-bowl Milli-Q water system (Millipore). Deuterium oxide (99.9 atom

  5. On the problem of radiation purification of waste waters containing synthetic surface-active agents

    International Nuclear Information System (INIS)

    Buslaeva, S.P.; Kon'kov, N.G.; Makarochkina, L.M.; Panin, Yu.A.; Upadyshev, L.B.; Filippov, M.T.

    1975-01-01

    Radiation decomposition of artificial solutions containing surface-active substances and the real sewage of textile factories is studied. Experiments on treatment of sewage in a foamed state were conducted (since the irradiation of sewage in a liquid form did not result in the desired effect) with a laboratory installation that made it possible to irradiate sewage by an electron beam both under stationary and flowing conditions. The dose rate was about 140 w/kg. The results of determination of the dependence of the decomposition rate for solutions that contain surface-active substances on the absorbed dose and the magnitude of the beam current are presented. It is demonstrated that the decrease of dose rate is accompanied by the increase of the yield of the surface-active substance decomposition in the foam. During irradiation of real sewage the improvement of their coloring as well as the acceleration of coagulation and the sedimentation of dissolved and suspended substances were observed. Recommendations are suggested for design of an experimental-industrial installation for irradiation of sewage. It is established that with the efficiency of 1000 m 3 /day the cost of sewage treatment will be 35-40 kop/m 3

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

    Indian Academy of Sciences (India)

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

    two various rotor speeds: 16,000 and 22,000 r.p.m. at ambient temperature. Anal- yses of the particle ... tribomechanical activation can be also applied for the treatment of some organic materials. Physical and ... Lactose (%). Fat (%). Water (%).

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

    Indian Academy of Sciences (India)

    2012-01-19

    Jan 19, 2012 ... effector molecules and also in the control of intracellular bacterial replication ..... H37Ra in THP-1 cells. The fall and rise in the activation of .... use this distinct role of p38 MAPK to balance the expression of CD44 during ...

  8. Antimicrobial activity of synthetic cationic peptides and lipopeptides derived from human lactoferricin against Pseudomonas aeruginosa planktonic cultures and biofilms.

    Science.gov (United States)

    Sánchez-Gómez, Susana; Ferrer-Espada, Raquel; Stewart, Philip S; Pitts, Betsey; Lohner, Karl; Martínez de Tejada, Guillermo

    2015-07-07

    Infections by Pseudomonas aeruginosa constitute a serious health threat because this pathogen -particularly when it forms biofilms - can acquire resistance to the majority of conventional antibiotics. This study evaluated the antimicrobial activity of synthetic peptides based on LF11, an 11-mer peptide derived from human lactoferricin against P. aeruginosa planktonic and biofilm-forming cells. We included in this analysis selected N-acylated derivatives of the peptides to analyze the effect of acylation in antimicrobial activity. To assess the efficacy of compounds against planktonic bacteria, microdilution assays to determine the minimal inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time-kill studies were conducted. The anti-biofilm activity of the agents was assessed on biofilms grown under static (on microplates) and dynamic (in a CDC-reactor) flow regimes. The antimicrobial activity of lipopeptides differed from that of non-acylated peptides in their killing mechanisms on planktonic and biofilm-forming cells. Thus, acylation enhanced the bactericidal activity of the parental peptides and resulted in lipopeptides that were uniformly bactericidal at their MIC. In contrast, acylation of the most potent anti-biofilm peptides resulted in compounds with lower anti-biofilm activity. Both peptides and lipopeptides displayed very rapid killing kinetics and all of them required less than 21 min to reduce 1,000 times the viability of planktonic cells when tested at 2 times their MBC. The peptides, LF11-215 (FWRIRIRR) and LF11-227 (FWRRFWRR), displayed the most potent anti-biofilm activity causing a 10,000 fold reduction in cell viability after 1 h of treatment at 10 times their MIC. At that concentration, these two compounds exhibited low citotoxicity on human cells. In addition to its bactericidal activity, LF11-227 removed more that 50 % of the biofilm mass in independent assays. Peptide LF11-215 and two of the shortest and least

  9. 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.

  10. Synthetic environments

    Science.gov (United States)

    Lukes, George E.; Cain, Joel M.

    1996-02-01

    The Advanced Distributed Simulation (ADS) Synthetic Environments Program seeks to create robust virtual worlds from operational terrain and environmental data sources of sufficient fidelity and currency to interact with the real world. While some applications can be met by direct exploitation of standard digital terrain data, more demanding applications -- particularly those support operations 'close to the ground' -- are well-served by emerging capabilities for 'value-adding' by the user working with controlled imagery. For users to rigorously refine and exploit controlled imagery within functionally different workstations they must have a shared framework to allow interoperability within and between these environments in terms of passing image and object coordinates and other information using a variety of validated sensor models. The Synthetic Environments Program is now being expanded to address rapid construction of virtual worlds with research initiatives in digital mapping, softcopy workstations, and cartographic image understanding. The Synthetic Environments Program is also participating in a joint initiative for a sensor model applications programer's interface (API) to ensure that a common controlled imagery exploitation framework is available to all researchers, developers and users. This presentation provides an introduction to ADS and the associated requirements for synthetic environments to support synthetic theaters of war. It provides a technical rationale for exploring applications of image understanding technology to automated cartography in support of ADS and related programs benefitting from automated analysis of mapping, earth resources and reconnaissance imagery. And it provides an overview and status of the joint initiative for a sensor model API.

  11. Protein corona between nanoparticles and bacterial proteins in activated sludge: Characterization and effect on nanoparticle aggregation.

    Science.gov (United States)

    Zhang, Peng; Xu, Xiao-Yan; Chen, You-Peng; Xiao, Meng-Qian; Feng, Bo; Tian, Kai-Xun; Chen, Yue-Hui; Dai, You-Zhi

    2018-02-01

    In this work, the protein coronas of activated sludge proteins on TiO 2 nanoparticles (TNPs) and ZnO nanoparticles (ZNPs) were characterized. The proteins with high affinity to TNPs and ZNPs were identified by shotgun proteomics, and their effects of on the distributions of TNPs and ZNPs in activated sludge were concluded. In addition, the effects of protein coronas on the aggregations of TNPs and ZNPs were evaluated. Thirty and nine proteins with high affinities to TNPs and ZNPs were identified, respectively. The proteomics and adsorption isotherms demonstrated that activated sludge had a higher affinity to TNPs than to ZNPs. The aggregation percentages of ZNPs at 35, 53, and 106 mg/L of proteins were 13%, 14%, and 18%, respectively, whereas those of TNPs were 21%, 30%, 41%, respectively. The proteins contributed to ZNPs aggregation by dissolved Zn ion-bridging, whereas the increasing protein concentrations enhanced the TNPs aggregation through macromolecule bridging flocculation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Free-Standing Metal Oxide Nanoparticle Superlattices Constructed with Engineered Protein Containers Show in Crystallo Catalytic Activity.

    Science.gov (United States)

    Lach, Marcel; Künzle, Matthias; Beck, Tobias

    2017-12-11

    The construction of defined nanostructured catalysts is challenging. In previous work, we established a strategy to assemble binary nanoparticle superlattices with oppositely charged protein containers as building blocks. Here, we show that these free-standing nanoparticle superlattices are catalytically active. The metal oxide nanoparticles inside the protein scaffold are accessible for a range of substrates and show oxidase-like and peroxidase-like activity. The stable superlattices can be reused for several reaction cycles. In contrast to bulk nanoparticle-based catalysts, which are prone to aggregation and difficult to characterize, nanoparticle superlattices based on engineered protein containers provide an innovative synthetic route to structurally defined heterogeneous catalysts with control over nanoparticle size and composition. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Synthetic ciguatoxins selectively activate Nav1.8-derived chimeric sodium channels expressed in HEK293 cells.

    Science.gov (United States)

    Yamaoka, Kaoru; Inoue, Masayuki; Miyazaki, Keisuke; Hirama, Masahiro; Kondo, Chie; Kinoshita, Eiji; Miyoshi, Hiroshi; Seyama, Issei

    2009-03-20

    The synthetic ciguatoxin CTX3C has been shown to activate tetrodotoxin (TTX)-sensitive sodium channels (Na(v)1.2, Na(v)1.4, and Na(v)1.5) by accelerating activation kinetics and shifting the activation curve toward hyperpolarization (Yamaoka, K., Inoue, M., Miyahara, H., Miyazaki, K., and Hirama, M. (2004) Br. J. Pharmacol. 142, 879-889). In this study, we further explored the effects of CTX3C on the TTX-resistant sodium channel Na(v)1.8. TTX-resistant channels have been shown to be involved in transducing pain and related sensations (Akopian, A. N., Sivilotti, L., and Wood, J. N. (1996) Nature 379, 257-262). Thus, we hypothesized that ciguatoxin-induced activation of the Na(v)1.8 current would account for the neurological symptoms of ciguatera poisoning. We found that 0.1 mum CTX3C preferentially affected the activation process of the Na(v)1.8 channel compared with those of the Na(v)1.2 and Na(v)1.4 channels. Importantly, without stimulation, 0.1 mum CTX3C induced a large leakage current (I (L)). The conductance of the I (L) calculated relative to the maximum conductance (G (max)) was 10 times larger than that of Na(v)1.2 or Na(v)1.4. To determine the molecular domain of Na(v)1.8 responsible for conferring higher sensitivity to CTX3C, we made two chimeric constructs from Na(v)1.4 and Na(v)1.8. Chimeras containing the N-terminal half of Na(v)1.8 exhibited a large response similar to wild-type Na(v)1.8, indicating that the region conferring high sensitivity to ciguatoxin action is located in the D1 or D2 domains.

  14. Synthetic Ciguatoxins Selectively Activate Nav1.8-derived Chimeric Sodium Channels Expressed in HEK293 Cells*

    Science.gov (United States)

    Yamaoka, Kaoru; Inoue, Masayuki; Miyazaki, Keisuke; Hirama, Masahiro; Kondo, Chie; Kinoshita, Eiji; Miyoshi, Hiroshi; Seyama, Issei

    2009-01-01

    The synthetic ciguatoxin CTX3C has been shown to activate tetrodotoxin (TTX)-sensitive sodium channels (Nav1.2, Nav1.4, and Nav1.5) by accelerating activation kinetics and shifting the activation curve toward hyperpolarization (Yamaoka, K., Inoue, M., Miyahara, H., Miyazaki, K., and Hirama, M. (2004) Br. J. Pharmacol. 142, 879–889). In this study, we further explored the effects of CTX3C on the TTX-resistant sodium channel Nav1.8. TTX-resistant channels have been shown to be involved in transducing pain and related sensations (Akopian, A. N., Sivilotti, L., and Wood, J. N. (1996) Nature 379, 257–262). Thus, we hypothesized that ciguatoxin-induced activation of the Nav1.8 current would account for the neurological symptoms of ciguatera poisoning. We found that 0.1 μm CTX3C preferentially affected the activation process of the Nav1.8 channel compared with those of the Nav1.2 and Nav1.4 channels. Importantly, without stimulation, 0.1 μm CTX3C induced a large leakage current (IL). The conductance of the IL calculated relative to the maximum conductance (Gmax) was 10 times larger than that of Nav1.2 or Nav1.4. To determine the molecular domain of Nav1.8 responsible for conferring higher sensitivity to CTX3C, we made two chimeric constructs from Nav1.4 and Nav1.8. Chimeras containing the N-terminal half of Nav1.8 exhibited a large response similar to wild-type Nav1.8, indicating that the region conferring high sensitivity to ciguatoxin action is located in the D1 or D2 domains. PMID:19164297

  15. Therapeutic Activity of an Engineered Synthetic Killer Antiidiotypic Antibody Fragment against Experimental Mucosal and Systemic Candidiasis

    OpenAIRE

    Polonelli, Luciano; Magliani, Walter; Conti, Stefania; Bracci, Luisa; Lozzi, Luisa; Neri, Paolo; Adriani, Daniela; De Bernardis, Flavia; Cassone, Antonio

    2003-01-01

    Peptides derived from the sequence of a single-chain, recombinant, antiidiotypic antibody (IdAb; KT-scFv) acting as a functional internal image of a microbicidal, wide-spectrum yeast killer toxin (KT) were synthesized and studied for their antimicrobial activity by using the KT-susceptible Candida albicans as model organism. A decapeptide containing the first three amino acids (SAS) of the light chain CDR1 was selected and optimized by alanine replacement of a single residue. This peptide exe...

  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. Unnatural amino acids increase activity and specificity of synthetic substrates for human and malarial cathepsin C.

    Science.gov (United States)

    Poreba, Marcin; Mihelic, Marko; Krai, Priscilla; Rajkovic, Jelena; Krezel, Artur; Pawelczak, Malgorzata; Klemba, Michael; Turk, Dusan; Turk, Boris; Latajka, Rafal; Drag, Marcin

    2014-04-01

    Mammalian cathepsin C is primarily responsible for the removal of N-terminal dipeptides and activation of several serine proteases in inflammatory or immune cells, while its malarial parasite ortholog dipeptidyl aminopeptidase 1 plays a crucial role in catabolizing the hemoglobin of its host erythrocyte. In this report, we describe the systematic substrate specificity analysis of three cathepsin C orthologs from Homo sapiens (human), Bos taurus (bovine) and Plasmodium falciparum (malaria parasite). Here, we present a new approach with a tailored fluorogenic substrate library designed and synthesized to probe the S1 and S2 pocket preferences of these enzymes with both natural and a broad range of unnatural amino acids. Our approach identified very efficiently hydrolyzed substrates containing unnatural amino acids, which resulted in the design of significantly better substrates than those previously known. Additionally, in this study significant differences in terms of the structures of optimal substrates for human and malarial orthologs are important from the therapeutic point of view. These data can be also used for the design of specific inhibitors or activity-based probes.

  18. Anti-Leishmania and cytotoxic activities of perillaldehyde epoxide synthetic positional isomers.

    Science.gov (United States)

    Keesen, Tatjana Souza Lima; da Silva, Larisse Virgolino; da Câmara Rocha, Juliana; Andrade, Luciana Nalone; Lima, Tamires Cardoso; de Sousa, Damião Pergentino

    2018-03-13

    Leishmaniasis belongs to a complex of zoonotic disease caused by protozoa of the genus Leishmania and is considered a major public health problem. Several essential oil chemical components have inhibitory effect against protozoa, including Leishmania donovani. Thus, the aim of this study was to evaluate for the first time the anti-Leishmania activity of two p-menthane monoterpene isomers (EPER-1: perillaldehyde 1,2-epoxide and EPER-2: perillaldehyde 8,9-epoxide) against L. donovani promastigotes as well as evaluating cytotoxic effect on mononuclear peripheral blood cells. Results of anti-Leishmania assay revealed that EPER-2 (IC 50  = 3.8 μg.mL -1 ) was 16-fold more potent than its isomer EPER-1 (IC 50  = 64.6 μg.mL -1 ). In contrast to PBMC cells, EPER-2 was not cytotoxic (IC 50  > 400 μg.mL -1 ) when compared to positive control. These data suggest that the disposition of epoxide group into the p-menthane skeleton affects the anti-Leishmania activity, being that the presence of the exocyclic epoxide group considerably increased potency. Thus, it was possible to observe that the location of the epoxide group into the p-menthane skeleton resulted in different potencies.

  19. Inhibition of Poly(A)-binding protein with a synthetic RNA mimic reduces pain sensitization in mice.

    Science.gov (United States)

    Barragán-Iglesias, Paulino; Lou, Tzu-Fang; Bhat, Vandita D; Megat, Salim; Burton, Michael D; Price, Theodore J; Campbell, Zachary T

    2018-01-02

    Nociceptors rely on cap-dependent translation to rapidly induce protein synthesis in response to pro-inflammatory signals. Comparatively little is known regarding the role of the regulatory factors bound to the 3' end of mRNA in nociceptor sensitization. Poly(A)-binding protein (PABP) stimulates translation initiation by bridging the Poly(A) tail to the eukaryotic initiation factor 4F complex associated with the mRNA cap. Here, we use unbiased assessment of PABP binding specificity to generate a chemically modified RNA-based competitive inhibitor of PABP. The resulting RNA mimic, which we designated as the Poly(A) SPOT-ON, is more stable than unmodified RNA and binds PABP with high affinity and selectivity in vitro. We show that injection of the Poly(A) SPOT-ON at the site of an injury can attenuate behavioral response to pain. Collectively, these results suggest that PABP is integral for nociceptive plasticity. The general strategy described here provides a broad new source of mechanism-based inhibitors for RNA-binding proteins and is applicable for in vivo studies.

  20. Activated Protein C Drives the Hyperfibrinolysis of Acute Traumatic Coagulopathy.

    Science.gov (United States)

    Davenport, Ross A; Guerreiro, Maria; Frith, Daniel; Rourke, Claire; Platton, Sean; Cohen, Mitchell; Pearse, Rupert; Thiemermann, Chris; Brohi, Karim

    2017-01-01

    Major trauma is a leading cause of morbidity and mortality worldwide with hemorrhage accounting for 40% of deaths. Acute traumatic coagulopathy exacerbates bleeding, but controversy remains over the degree to which inhibition of procoagulant pathways (anticoagulation), fibrinogen loss, and fibrinolysis drive the pathologic process. Through a combination of experimental study in a murine model of trauma hemorrhage and human observation, the authors' objective was to determine the predominant pathophysiology of acute traumatic coagulopathy. First, a prospective cohort study of 300 trauma patients admitted to a single level 1 trauma center with blood samples collected on arrival was performed. Second, a murine model of acute traumatic coagulopathy with suppressed protein C activation via genetic mutation of thrombomodulin was used. In both studies, analysis for coagulation screen, activated protein C levels, and rotational thromboelastometry (ROTEM) was performed. In patients with acute traumatic coagulopathy, the authors have demonstrated elevated activated protein C levels with profound fibrinolytic activity and early depletion of fibrinogen. Procoagulant pathways were only minimally inhibited with preservation of capacity to generate thrombin. Compared to factors V and VIII, proteases that do not undergo activated protein C-mediated cleavage were reduced but maintained within normal levels. In transgenic mice with reduced capacity to activate protein C, both fibrinolysis and fibrinogen depletion were significantly attenuated. Other recognized drivers of coagulopathy were associated with less significant perturbations of coagulation. Activated protein C-associated fibrinolysis and fibrinogenolysis, rather than inhibition of procoagulant pathways, predominate in acute traumatic coagulopathy. In combination, these findings suggest a central role for the protein C pathway in acute traumatic coagulopathy and provide new translational opportunities for management of

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

    NARCIS (Netherlands)

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

    2015-01-01

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

  2. The cartilage protein melanoma inhibitory activity contributes to inflammatory arthritis

    NARCIS (Netherlands)

    Yeremenko, Nataliya; Härle, Peter; Cantaert, Tineke; van Tok, Melissa; van Duivenvoorde, Leonie M.; Bosserhoff, Anja; Baeten, Dominique

    2014-01-01

    Melanoma inhibitory activity (MIA) is a small chondrocyte-specific protein with unknown function. MIA knockout mice (MIA(-/-)) have a normal phenotype with minor microarchitectural alterations of cartilage. Our previous study demonstrated that immunodominant epitopes of MIA are actively presented in

  3. Energy transfer at the active sites of heme proteins

    International Nuclear Information System (INIS)

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

    1995-01-01

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

  4. Liposomal packaging generates Wnt protein with in vivo biological activity.

    Directory of Open Access Journals (Sweden)

    Nathan T Morrell

    2008-08-01

    Full Text Available Wnt signals exercise strong cell-biological and regenerative effects of considerable therapeutic value. There are, however, no specific Wnt agonists and no method for in vivo delivery of purified Wnt proteins. Wnts contain lipid adducts that are required for activity and we exploited this lipophilicity by packaging purified Wnt3a protein into lipid vesicles. Rather than being encapsulated, Wnts are tethered to the liposomal surface, where they enhance and sustain Wnt signaling in vitro. Molecules that effectively antagonize soluble Wnt3a protein but are ineffective against the Wnt3a signal presented by a cell in a paracrine or autocrine manner are also unable to block liposomal Wnt3a activity, suggesting that liposomal packaging mimics the biological state of active Wnts. When delivered subcutaneously, Wnt3a liposomes induce hair follicle neogenesis, demonstrating their robust biological activity in a regenerative context.

  5. Theoretical Study of Phosphoethanolamine: A Synthetic Anticancer Agent with Broad Antitumor Activity

    Directory of Open Access Journals (Sweden)

    Vitor Prates Lorenzo

    2016-01-01

    Full Text Available Cancer is a major public health problem with limited success of available treatments, pointing to the need for new strategies to be developed. Phosphoethanolamine exhibits broad antitumor activity in a variety of tumor cells and potent inhibitor effects on tumor progress in vivo. Once-used organophosphates inhibit acetylcholinesterase (AChE, resulting in toxic effects to the user. As this group is present in phosphoethanolamine, we perform prediction of the in silico metabolism of phosphoethanolamine and submit this series to a docking study on AChE. A total of 10 metabolites were indicated by the prediction, including ammonia and hydroxylamine, which were not included in the study. Using a group of 8 organophosphorus whose pIC50 values ranged from 5.92 to 9.47 as template, we observed that no compound present in the phosphoethanolamine series had a binding energy lower than that of organophosphorus, suggesting that the series has low inhibitory power on AChE. In light of this, we conclude that phosphoethanolamine and its predicted metabolites do not significantly inhibit AChE to cause a cholinergic crisis. This finding highlights the importance of investigating this compound as lead for potential anticancer agents.

  6. BDA-410: a novel synthetic calpain inhibitor active against blood stage malaria.

    Science.gov (United States)

    Li, Xuerong; Chen, Huiqing; Jeong, Jong-Jin; Chishti, Athar H

    2007-09-01

    Falcipains, the papain-family cysteine proteases of the Plasmodium falciparum, are potential drug targets for malaria parasite. Pharmacological inhibition of falcipains can block the hydrolysis of hemoglobin, parasite development, and egress, suggesting that falcipains play a key role at the blood stage of parasite life cycle. In the present study, we evaluated the anti-malarial effects of BDA-410, a novel cysteine protease inhibitor as a potential anti-malarial drug. Recombinant falcipain (MBP-FP-2B) and P. falciparum trophozoite extract containing native falcipains were used for enzyme inhibition studies in vitro. The effect of BDA-410 on the malaria parasite development in vitro as well as its anti-malarial activity in vivo was evaluated using the Plasmodium chabaudi infection rodent model. The 50% inhibitory concentrations of BDA-410 were determined to be 628 and 534nM for recombinant falcipain-2B and parasite extract, respectively. BDA-410 inhibited the malaria parasite growth in vitro with an IC(50) value of 173nM causing irreversible damage to the intracellular parasite. In vivo, the BDA-410 delayed the progression of malaria infection significantly using a mouse model of malaria pathogenesis. The characterization of BDA-410 as a potent inhibitor of P. falciparum cysteine proteases, and the demonstration of its efficacy in blocking parasite growth both in vitro and in vivo assays identifies BDA-410 is an important lead compound for the development of novel anti-malarial drugs.

  7. PtPb nanoparticle electrocatalysts: control of activity through synthetic methods

    International Nuclear Information System (INIS)

    Ghosh, Tanushree; Matsumoto, Futoshi; McInnis, Jennifer; Weiss, Marilyn; Abruna, Hector D.; DiSalvo, Francis J.

    2009-01-01

    Solution phase synthesis of intermetallic nanoparticles without using surfactants (for catalytic applications) and subsequent control of size distribution remains a challenge: of growing interest, but not widely explored yet. To understand the questions in the syntheses of Pt containing intermetallic nanoparticles (as electrocatalysts for direct fuel cells) by using sodium naphthalide as the reducing agent, the effects of the Pt precursors' organic ligands were investigated. PtPb syntheses were studied as the model case. In particular, methods that lead to nanoparticles that are independent single crystals are desirable. Platinum acetylacetonate, which is soluble in many organic solvents, has ligands that may interfere less with nanoparticle growth and ordering. Interesting trends, contrary to expectations, were observed when precursors were injected into a reducing agent solution at high temperatures. The presence of acetylacetonate, from the precursor, on the nanoparticles was confirmed by ATR, while SEM imaging showed evidence of morphological changes in the nanoparticles with increasing reaction temperature. A definite relationship between domain size and extent of observed residue (organic material and sodium) present on the particles could be established. By varying post-reaction solvent removal techniques, room temperature crystallization of PtPb nanoparticles was also achieved. Electrochemical activity of the nanoparticles was also much higher than that of nanoparticles synthesized by previous reaction schemes using sodium naphthalide as the reducing agent. Along with the above mentioned techniques, BET, TEM, CBED, SAED, and XRD were used as characterization tools for the prepared nanoparticles.

  8. Coordination chemistry of oxovanadium(V) complexes with active Schiff bases: synthetic, spectral, and antimicrobial approach

    International Nuclear Information System (INIS)

    Garg, R; Fahmi, N.; Singh, R.V.

    2007-01-01

    The Schiff bases, 3-(indolin-2-one)hydrazinecarbothioamide, 3-(indolin-2-one)hydrazinecarboxamide, 5,6-dimethyl-3-(indolin-2-one)hydrazinecarbothioamide, and 5,6-dimethyl-3-(indolin-2-one)hydrazinecarboxamide, have been synthesized by the condensation of 1H-indol-2,3-dione and 5,6-dimethyl-1H-indol-2,3-dione with the corresponding hydrazinecarbothioamide and hydrazinecarboxamide, respectively. The complexes of oxovanadium and ligands have been characterized by elemental analyses, melting points, conductance measurements, molecular weight determinations, and IR, 1 H NMR and UV spectral studies. These studies showed that the ligands coordinated to the oxovanadium in a monobasic bidentate fashion through oxygen or sulfur and the nitrogen donor system. Thus, penta- and hexa coordinated environment around the vanadium atom has been proposed. All the complexes and their parent organic moieties have been screened for their biological activity on several pathogenic fungi and bacteria and were found to possess appreciable fungicidal and bactericidal properties [ru

  9. Enhancing the antimicrobial activity of natural extraction using the synthetic ultrasmall metal nanoparticles

    Science.gov (United States)

    Li, Huanhuan; Chen, Quansheng; Zhao, Jiewen; Urmila, Khulal

    2015-01-01

    The use of Catechin as an antibacterial agent is becoming ever-more common, whereas unstable and easy oxidation, have limited its application. A simple and low-energy-consuming approach to synthesize highly stable and dispersive Catechin-Cu nanoparticles(NPs) has been developed, in which the stability and dispersivity of the NPs are varied greatly with the pH value and temperature of the reaction. The results demonstrate that the optimal reaction conditions are pH 11 at room temperature. As-synthesized NPs display excellent antimicrobial activity, the survival rates of bacterial cells exposed to the NPs were evaluated using live/dead Bacterial Viability Kit. The results showed that NPs at the concentration of 10 ppm and 20 ppm provided rapid and effective killing of up to 90% and 85% of S. aureus and E. coli within 3 h, respectively. After treatment with 20 ppm and 40 ppm NPs, the bacteria are killed completely. Furthermore, on the basis of assessing the antibacterial effects by SEM, TEM, and AFM, it was found the cell membrane damage of the bacteria caused by direct contact of the bacteria with the NPs was the effective mechanism in the bacterial inactivation. PMID:26046938

  10. Natural products induce a G protein-mediated calcium pathway activating p53 in cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Ginkel, Paul R. van; Yan, Michael B. [UW Carbone Cancer Center, University of Wisconsin, Madison, WI 53792 (United States); Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI 53792 (United States); Bhattacharya, Saswati [UW Carbone Cancer Center, University of Wisconsin, Madison, WI 53792 (United States); Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI 53792 (United States); Department of Pediatrics, University of Wisconsin, Madison, WI 53792 (United States); Polans, Arthur S., E-mail: aspolans@wisc.edu [UW Carbone Cancer Center, University of Wisconsin, Madison, WI 53792 (United States); Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI 53792 (United States); Kenealey, Jason D. [UW Carbone Cancer Center, University of Wisconsin, Madison, WI 53792 (United States); Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI 53792 (United States); Department of Nutrition, Dietetics and Food Science, Brigham Young University, Provo, UT 84602 (United States)

    2015-11-01

    Paclitaxel, etoposide, vincristine and doxorubicin are examples of natural products being used as chemotherapeutics but with adverse side effects that limit their therapeutic window. Natural products derived from plants and having low toxicity, such as quercetin, resveratrol, epigallocatechin gallate and piceatannol, have been shown to inhibit tumor cell growth both in vitro and in pre-clinical models of cancer, but their mechanisms of action have not been fully elucidated, thus restricting their use as prototypes for developing synthetic analogs with improved anti-cancer properties. We and others have demonstrated that one of the earliest and consistent events upon exposure of tumor cells to these less toxic natural products is a rise in cytoplasmic calcium, activating several pro-apoptotic pathways. We describe here a G protein/inositol 1,4,5-trisphosphate pathway (InsP3) in MDA-MB-231 human breast cancer cells that mediates between these less toxic natural products and the release of calcium from the endoplasmic reticulum. Further, we demonstrate that this elevation of intracellular calcium modulates p53 activity and the subsequent transcription of several pro-apoptotic genes encoding PIG8, CD95, PIDD, TP53INP, RRM2B, Noxa, p21 and PUMA. We conclude from our findings that less toxic natural products likely bind to a G protein coupled receptor that activates a G protein-mediated and calcium-dependent pathway resulting selectively in tumor cell death. - Highlights: • Natural products having low toxicity increase cytoplasmic calcium in cancer cells. • A G-protein/IP{sub 3} pathway mediates the release of calcium from the ER. • The elevation of intracellular calcium modulates p53 activity. • p53 and other Ca{sup 2+}-dependent pro-apoptotic pathways inhibit cancer cell growth.

  11. Natural products induce a G protein-mediated calcium pathway activating p53 in cancer cells

    International Nuclear Information System (INIS)

    Ginkel, Paul R. van; Yan, Michael B.; Bhattacharya, Saswati; Polans, Arthur S.; Kenealey, Jason D.

    2015-01-01

    Paclitaxel, etoposide, vincristine and doxorubicin are examples of natural products being used as chemotherapeutics but with adverse side effects that limit their therapeutic window. Natural products derived from plants and having low toxicity, such as quercetin, resveratrol, epigallocatechin gallate and piceatannol, have been shown to inhibit tumor cell growth both in vitro and in pre-clinical models of cancer, but their mechanisms of action have not been fully elucidated, thus restricting their use as prototypes for developing synthetic analogs with improved anti-cancer properties. We and others have demonstrated that one of the earliest and consistent events upon exposure of tumor cells to these less toxic natural products is a rise in cytoplasmic calcium, activating several pro-apoptotic pathways. We describe here a G protein/inositol 1,4,5-trisphosphate pathway (InsP3) in MDA-MB-231 human breast cancer cells that mediates between these less toxic natural products and the release of calcium from the endoplasmic reticulum. Further, we demonstrate that this elevation of intracellular calcium modulates p53 activity and the subsequent transcription of several pro-apoptotic genes encoding PIG8, CD95, PIDD, TP53INP, RRM2B, Noxa, p21 and PUMA. We conclude from our findings that less toxic natural products likely bind to a G protein coupled receptor that activates a G protein-mediated and calcium-dependent pathway resulting selectively in tumor cell death. - Highlights: • Natural products having low toxicity increase cytoplasmic calcium in cancer cells. • A G-protein/IP 3 pathway mediates the release of calcium from the ER. • The elevation of intracellular calcium modulates p53 activity. • p53 and other Ca 2+ -dependent pro-apoptotic pathways inhibit cancer cell growth.

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

    Science.gov (United States)

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

    2005-07-01

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

  13. Synthetic arylquinuclidine derivatives exhibit antifungal activity against Candida albicans, Candida tropicalis and Candida parapsilopsis

    Directory of Open Access Journals (Sweden)

    Gilbert Ian

    2011-01-01

    Full Text Available Abstract Background Sterol biosynthesis is an essential pathway for fungal survival, and is the biochemical target of many antifungal agents. The antifungal drugs most widely used to treated fungal infections are compounds that inhibit cytochrome P450-dependent C14α-demethylase (CYP51, but other enzymes of this pathway, such as squalene synthase (SQS which catalyses the first committed step in sterol biosynthesis, could be viable targets. The aim of this study was to evaluate the antifungal activity of SQS inhibitors on Candida albicans, Candida tropicalis and Candida parapsilopsis strains. Methods Ten arylquinuclidines that act as SQS inhibitors were tested as antiproliferative agents against three ATCC strains and 54 clinical isolates of Candida albicans, Candida tropicalis and Candida parapsilopsis. Also, the morphological alterations induced in the yeasts by the experimental compounds were evaluated by fluorescence and transmission electron microscopy. Results The most potent arylquinuclidine derivative (3-[1'-{4'-(benzyloxy-phenyl}]-quinuclidine-2-ene (WSP1267 had a MIC50 of 2 μg/ml for all species tested and MIC90 varying from 4 μg/ml to 8 μg/ml. Ultrathin sections of C. albicans treated with 1 μg/ml of WSP1267 showed several ultrastructural alterations, including (a loss of cell wall integrity, (b detachment of the plasma membrane from the fungal cell wall, (c accumulation of small vesicles in the periplasmic region, (d presence of large electron-dense vacuoles and (e significantly increased cell size and cell wall thickness. In addition, fluorescence microscopy of cells labelled with Nile Red showed an accumulation of lipid droplets in the cytoplasm of treated yeasts. Nuclear staining with DAPI revealed the appearance of uncommon yeast buds without a nucleus or with two nuclei. Conclusion Taken together, our data demonstrate that arylquinuclidine derivatives could be useful as lead compounds for the rational synthesis of new

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

    International Nuclear Information System (INIS)

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

    2010-01-01

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

  15. Synthetic Rutile

    International Nuclear Information System (INIS)

    Burastero, J.

    1975-01-01

    This work is about the laboratory scale investigation of the conditions in the rutile synthetic production from one me nita in Aguas Dulces reservoir. The iron mineral is chlorinated and volatilized selectively leaving a residue enriched in titanium dioxide which can be used as a substitute of rutile mineral

  16. The effects of dissolved natural organic matter on the adsorption of synthetic organic chemicals by activated carbons and carbon nanotubes.

    Science.gov (United States)

    Zhang, Shujuan; Shao, Ting; Karanfil, Tanju

    2011-01-01

    Understanding the influence of natural organic matter (NOM) on synthetic organic contaminant (SOC) adsorption by carbon nanotubes (CNTs) is important for assessing the environmental implications of accidental CNT release and spill to natural waters, and their potential use as adsorbents in engineered systems. In this study, adsorption of two SOCs by three single-walled carbon nanotubes (SWNTs), one multi-walled carbon nanotube (MWNT), a microporous activated carbon fiber (ACF) [i.e., ACF10] and a bimodal porous granular activated carbon (GAC) [i.e., HD4000] was compared in the presence and absence of NOM. The NOM effect was found to depend strongly on the pore size distribution of carbons. Minimal NOM effect occurred on the macroporous MWNT, whereas severe NOM effects were observed on the microporous HD4000 and ACF10. Although the single-solute adsorption capacities of the SWNTs were much lower than those of HD4000, in the presence of NOM the SWNTs exhibited adsorption capacities similar to those of HD4000. Therefore, if released into natural waters, SWNTs can behave like an activated carbon, and will be able to adsorb, carry, and transfer SOCs to other systems. However, from an engineering application perspective, CNTs did not exhibit a major advantage, in terms of adsorption capacities, over the GAC and ACF. The NOM effect was also found to depend on molecular properties of SOCs. NOM competition was more severe on the adsorption of 2-phenylphenol, a nonplanar and hydrophilic SOC, than phenanthrene, a planar and hydrophobic SOC, tested in this study. In terms of surface chemistry, both adsorption affinity to SOCs and NOM effect on SOC adsorption were enhanced with increasing hydrophobicity of the SWNTs. Copyright © 2010 Elsevier Ltd. All rights reserved.

  17. Protein determination in soya bean by fast neutron activation analysis

    International Nuclear Information System (INIS)

    Szegedi, S.; Mosbah, D.S.; Varadi, M.; Szaloki, I.

    1988-01-01

    For a non-destructive determination of the protein content in soya bean samples, 14-MeV neutron activation analysis was applied. To check the method, the results obtained by X-ray fluorescence analysis and the Kjeldahl procedure were compared. For pressed pellet samples of about 1 g with 15 min irradiation and 10 min measuring times the accuracy of the protein determination was found to be 15%. (author) 7 refs.; 4 figs.; 3 tabs

  18. Synthetic 6B di-, tri-, and tetrasaccharide-protein conjugates contain pneumococcal type 6A and 6B common and 6B-specific epitopes that elicit protective antibodies in mice

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Jansen, W.T.M.; Hogenboom, S.; Thijssen, M.J.L.; Kamerling, J.P.; Verhoef, J.; Snippe, H.; Verheul, A.F.M.

    2001-01-01

    The immunogenicity and protective capacity of Streptococcus pneumoniae 6B capsular polysaccharide (PS)-derived synthetic phosphate-containing disaccharide (Rha-ribitol-P-), trisaccharide (ribitol-P-Gal-Glc-), and tetrasaccharide (Rha-ribitol-P-Gal-Glc-)-protein conjugates in rabbits and mice were

  19. Monoaddition of dictamnine to synthetic double-stranded polydeoxyribonucleotides in UVA and the effect of photomodified DNA on template activity

    Energy Technology Data Exchange (ETDEWEB)

    Pfyffer, G.E.; Pfyffer, B.U.; Towers, G.H.N. (British Columbia Univ., Vancouver (Canada))

    1982-06-01

    The photoreactivity of dictamnine, a furoquinoline alkaloid, towards different synthetic DNAs has been studied. The ratio of the photobinding of (/sup 3/H)-dictamnine to poly(dA-dT).poly(dA-dT):poly(dG-dC).poly(dG-dC):poly(dA-dU).poly(dA-dU):poly(dA).poly(dT), in relation to that of calf thymus DNA, is 18:1:0.5:0.3. Prior treatment of calf thymus DNA with dictamnine in light inhibits the subsequent incorporation of 8-methoxypsoralen (8-MOP). These results suggest that the sites in DNA for the photobinding of dictamnine are probably identical with those for monoadducts of 8-MOP. Furthermore, the template activity of photomodified DNA in the RNA polymerase reaction is considerably inhibited for poly(dA-dT).poly(dA-dT), to a lesser extent for calf thymus DNA, but almost not affected for the linear copolymer, poly(dA).poly(dT).

  20. Rapid isolation of antibody from a synthetic human antibody library by repeated fluorescence-activated cell sorting (FACS.

    Directory of Open Access Journals (Sweden)

    Sung Sun Yim

    Full Text Available Antibodies and their derivatives are the most important agents in therapeutics and diagnostics. Even after the significant progress in the technology for antibody screening from huge libraries, it takes a long time to isolate an antibody, which prevents a prompt action against the spread of a disease. Here, we report a new strategy for isolating desired antibodies from a combinatorial library in one day by repeated fluorescence-activated cell sorting (FACS. First, we constructed a library of synthetic human antibody in which single-chain variable fragment (scFv was expressed in the periplasm of Escherichia coli. After labeling the cells with fluorescent antigen probes, the highly fluorescent cells were sorted by using a high-speed cell sorter, and these cells were reused without regeneration in the next round of sorting. After repeating this sorting, the positive clones were completely enriched in several hours. Thus, we screened the library against three viral antigens, including the H1N1 influenza virus, Hepatitis B virus, and Foot-and-mouth disease virus. Finally, the potential antibody candidates, which show K(D values between 10 and 100 nM against the target antigens, could be successfully isolated even though the library was relatively small (∼ 10(6. These results show that repeated FACS screening without regeneration of the sorted cells can be a powerful method when a rapid response to a spreading disease is required.

  1. Effect of temperature, pH, and water activity on Mucor spp. growth on synthetic medium, cheese analog and cheese.

    Science.gov (United States)

    Morin-Sardin, Stéphanie; Rigalma, Karim; Coroller, Louis; Jany, Jean-Luc; Coton, Emmanuel

    2016-06-01

    The Mucor genus includes a large number of ubiquitous fungal species. In the dairy environment, some of them play a technological role providing typical organoleptic qualities to some cheeses while others can cause spoilage. In this study, we compared the effect of relevant abiotic factors for cheese production on the growth of six strains representative of dairy technological and contaminant species as well as of a non cheese related strain (plant endophyte). Growth kinetics were determined for each strain in function of temperature, water activity and pH on synthetic Potato Dextrose Agar (PDA), and secondary models were fitted to calculate the corresponding specific cardinal values. Using these values and growth kinetics acquired at 15 °C on cheese agar medium (CA) along with three different cheese types, optimal growth rates (μopt) were estimated and consequently used to establish a predictive model. Contrarily to contaminant strains, technological strains showed higher μopt on cheese matrices than on PDA. Interestingly, lag times of the endophyte strain were strongly extended on cheese related matrices. This study offers a relevant predictive model of growth that may be used for better cheese production control but also raises the question of adaptation of some Mucor strains to the cheese. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Solution structure and dynamics of melanoma inhibitory activity protein

    International Nuclear Information System (INIS)

    Lougheed, Julie C.; Domaille, Peter J.; Handel, Tracy M.

    2002-01-01

    Melanoma inhibitory activity (MIA) is a small secreted protein that is implicated in cartilage cell maintenance and melanoma metastasis. It is representative of a recently discovered family of proteins that contain a Src Homologous 3 (SH3) subdomain. While SH3 domains are normally found in intracellular proteins and mediate protein-protein interactions via recognition of polyproline helices, MIA is single-domain extracellular protein, and it probably binds to a different class of ligands.Here we report the assignments, solution structure, and dynamics of human MIA determined by heteronuclear NMR methods. The structures were calculated in a semi-automated manner without manual assignment of NOE crosspeaks, and have a backbone rmsd of 0.38 A over the ordered regions of the protein. The structure consists of an SH3-like subdomain with N- and C-terminal extensions of approximately 20 amino acids each that together form a novel fold. The rmsd between the solution structure and our recently reported crystal structure is 0.86 A over the ordered regions of the backbone, and the main differences are localized to the most dynamic regions of the protein. The similarity between the NMR and crystal structures supports the use of automated NOE assignments and ambiguous restraints to accelerate the calculation of NMR structures

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

    Science.gov (United States)

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

    2018-03-20

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

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

    Science.gov (United States)

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

    2017-08-01

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

  5. 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.

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

    Directory of Open Access Journals (Sweden)

    Jing Zhang

    2017-12-01

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

  7. Research on the structure in solution of optically active synthetic polymers (propylene polysulphide, propylene polyoxide, tertio-butyl polysulphide)

    International Nuclear Information System (INIS)

    Sarrazin, Brigitte

    1971-03-01

    It was proposed to study the structure of sulphur-containing synthetic polymers, stereo-regular, optically active in solution and able to adopt a spiral conformation, with special reference to propylene polysulphide. Two methods were used, the first mathematical (conformational energy calculations) and the second physico-chemical, essentially spectroscopic. By conformational analysis it is possible to choose the most probable structures liable to be adopted by a given polymer in solution while the spectro-polarimetric study should, in principle, invalidate or confirm certain of these hypotheses. The conformational energy calculations showed that in fact there is no energy conformation low enough to be stable in solution. Strictly speaking however we can refer to a region of stability in which steric hindrance is low and many energy minima exist. These minima are indistinguishable both by their energy values and by their spatial localizations and are all enclosed in the region bounded by the barriers due to steric hindrance. This uncertainty does not arise from approximations made in the calculations, but from the multitude of stereochemical structure possible. Investigations into the variation of the optical rotary dispersion and the circular dichroism as a function of temperature indicated the existence of three or more equilibrium states in the dioxane. The spectra appear to be the summation of the optical activities of the numerous simultaneously possible conformations. It appears that polymers, such as propylene polysulphide or propylene polyoxide do not have stable structures in solution. These are molecules of great flexibility possessing a large number of degrees of freedom. These properties distinguish them from the natural polymers, carrying precise information, such as DNA which must consequently have stable conformations. (author) [fr

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

    Science.gov (United States)

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

    2017-07-01

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

  9. Synthetic Electric Microbial Biosensors

    Science.gov (United States)

    2017-06-10

    domains and DNA-binding domains into a single protein for deregulation of down stream genes of have been favored [10]. Initially experiments with... Germany DISTRIBUTION A. Approved for public release: distribution unlimited.   Talk title: “Synthetic biology based microbial biosensors for the...toolbox” in Heidelberg, Germany Poster title: “Anaerobic whole cell microbial biosensors” Link: http://phdsymposium.embl.org/#home   September, 2014

  10. Absolute quantification of norovirus capsid protein in food, water, and soil using synthetic peptides with electrospray and MALDI mass spectrometry

    International Nuclear Information System (INIS)

    Hartmann, Erica M.; Colquhoun, David R.; Schwab, Kellogg J.; Halden, Rolf U.

    2015-01-01

    Highlights: • Mass spectrometry-based methods for norovirus quantification are developed. • Absolute quantification is achieved using internal heavy isotope-labeled standards. • A single labeled peptide serves in two distinct detection strategies. • These methods are validated for food, water, and soil analysis. • MS-based detection limits are lowered by two orders of magnitude. - Abstract: Norovirus infections are one of the most prominent public health problems of microbial origin in the U.S. and other industrialized countries. Surveillance is necessary to prevent secondary infection, confirm successful cleanup after outbreaks, and track the causative agent. Quantitative mass spectrometry, based on absolute quantitation with stable-isotope labeled peptides, is a promising tool for norovirus monitoring because of its speed, sensitivity, and robustness in the face of environmental inhibitors. In the current study, we present two new methods for the detection of the norovirus genogroup I capsid protein using electrospray and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The peptide TLDPIEVPLEDVR was used to quantify norovirus-like particles down to 500 attomoles with electrospray and 100 attomoles with MALDI. With MALDI, we also demonstrate a detection limit of 1 femtomole and a quantitative dynamic range of 5 orders of magnitude in the presence of an environmental matrix effect. Due to the rapid processing time and applicability to a wide range of environmental sample types (bacterial lysate, produce, milk, soil, and groundwater), mass spectrometry-based absolute quantitation has a strong potential for use in public health and environmental sciences

  11. Absolute quantification of norovirus capsid protein in food, water, and soil using synthetic peptides with electrospray and MALDI mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Hartmann, Erica M. [Center for Environmental Security and Security Defense Systems Initiative, The Biodesign Institute, Arizona State University, 781 E. Terrace Mall, Tempe, AZ 85287-5904 (United States); Colquhoun, David R.; Schwab, Kellogg J. [Department of Environmental Health Sciences, The Johns Hopkins University, Bloomberg School of Public Health, 615 N. Wolfe St., Baltimore, MD 21205 (United States); Halden, Rolf U., E-mail: halden@asu.edu [Center for Environmental Security and Security Defense Systems Initiative, The Biodesign Institute, Arizona State University, 781 E. Terrace Mall, Tempe, AZ 85287-5904 (United States); Department of Environmental Health Sciences, The Johns Hopkins University, Bloomberg School of Public Health, 615 N. Wolfe St., Baltimore, MD 21205 (United States)

    2015-04-09

    Highlights: • Mass spectrometry-based methods for norovirus quantification are developed. • Absolute quantification is achieved using internal heavy isotope-labeled standards. • A single labeled peptide serves in two distinct detection strategies. • These methods are validated for food, water, and soil analysis. • MS-based detection limits are lowered by two orders of magnitude. - Abstract: Norovirus infections are one of the most prominent public health problems of microbial origin in the U.S. and other industrialized countries. Surveillance is necessary to prevent secondary infection, confirm successful cleanup after outbreaks, and track the causative agent. Quantitative mass spectrometry, based on absolute quantitation with stable-isotope labeled peptides, is a promising tool for norovirus monitoring because of its speed, sensitivity, and robustness in the face of environmental inhibitors. In the current study, we present two new methods for the detection of the norovirus genogroup I capsid protein using electrospray and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The peptide TLDPIEVPLEDVR was used to quantify norovirus-like particles down to 500 attomoles with electrospray and 100 attomoles with MALDI. With MALDI, we also demonstrate a detection limit of 1 femtomole and a quantitative dynamic range of 5 orders of magnitude in the presence of an environmental matrix effect. Due to the rapid processing time and applicability to a wide range of environmental sample types (bacterial lysate, produce, milk, soil, and groundwater), mass spectrometry-based absolute quantitation has a strong potential for use in public health and environmental sciences.

  12. Enamel matrix protein derivative plus synthetic bone substitute for the treatment of mandibular Class II furcation defects: a case series.

    Science.gov (United States)

    Queiroz, Lucas Araujo; Santamaria, Mauro; Casati, Marcio; Silverio, Karina; Nociti-Junior, Francisco; Sallum, Enilson

    2015-03-01

    The aim of this study is to report on the treatment of mandibular Class II furcation defects with enamel matrix protein derivative (EMD) combined with a βTCP/HA (β-tricalcium phosphate/hydroxyapatite) alloplastic material. Thirteen patients were selected. All patients were nonsmokers, systemically healthy, and diagnosed with chronic periodontitis; had not taken medications known to interfere with periodontal tissue health and healing; presented one Class II mandibular furcation defect with horizontal probing equal to or greater than 4 mm at buccal site. The clinical parameters evaluated were probing depth (PD), relative gingival margin position (RGMP), relative vertical clinical attachment level (RVCAL), and relative horizontal clinical attachment level (RHCAL). A paired Student t test was used to detect differences between the baseline and 6-month measurements, with the level of significance of .05. After 6 months, the treatment produced a statistically significant reduction in PD and a significant gain in RVCAL and RHCAL, but no observable change in RGMP. RVCAL ranged from 13.77 (± 1.31) at baseline to 12.15 (± 1.29) after 6 months, with a mean change of -1.62 ± 1.00 mm (P < .05). RHCAL ranged from 5.54 (± 0.75) to 2.92 (± 0.92), with a mean change of -2.62 ± 0.63 mm (P < .05). After 6 months, 76.92% of the patients improved their diagnosis to Class I furcation defects while 23.08% remained as Class II. The present study has shown that positive clinical results may be expected from the combined treatment of Class II furcation defects with EMD and βTCP/HA, especially considering the gain of horizontal attachment level. Despite this result, controlled clinical studies are needed to confirm our outcomes.

  13. 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

    and contractions. Adrenaline acts via cAMP-dependent protein kinase (PKA). The signalling mediating the effect of contractions is unknown and was explored in this study. Incubated soleus muscles from 70 g male rats were electrically stimulated to perform repeated tetanic contractions for 5 min. The contraction......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...

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

    International Nuclear Information System (INIS)

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

    1982-01-01

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

  15. Viral RNA annealing activities of human immunodeficiency virus type 1 nucleocapsid protein require only peptide domains outside the zinc fingers.

    Science.gov (United States)

    De Rocquigny, H; Gabus, C; Vincent, A; Fournié-Zaluski, M C; Roques, B; Darlix, J L

    1992-07-15

    The nucleocapsid (NC) of human immunodeficiency virus type 1 consists of a large number of NC protein molecules, probably wrapping the dimeric RNA genome within the virion inner core. NC protein is a gag-encoded product that contains two zinc fingers flanked by basic residues. In human immunodeficiency virus type 1 virions, NCp15 is ultimately processed into NCp7 and p6 proteins. During virion assembly the retroviral NC protein is necessary for core formation and genomic RNA encapsidation, which are essential for virus infectivity. In vitro NCp15 activates viral RNA dimerization, a process most probably linked in vivo to genomic RNA packaging, and replication primer tRNA(Lys,3) annealing to the initiation site of reverse transcription. To characterize the domains of human immunodeficiency virus type 1 NC protein necessary for its various functions, the 72-amino acid NCp7 and several derived peptides were synthesized in a pure form. We show here that synthetic NCp7 with or without the two zinc fingers has the RNA annealing activities of NCp15. Further deletions of the N-terminal 12 and C-terminal 8 amino acids, leading to a 27-residue peptide lacking the finger domains, have little or no effect on NC protein activity in vitro. However deletion of short sequences containing basic residues flanking the first finger leads to a complete loss of NC protein activity. It is proposed that the basic residues and the zinc fingers cooperate to select and package the genomic RNA in vivo. Inhibition of the viral RNA binding and annealing activities associated with the basic residues flanking the first zinc finger of NC protein could therefore be used as a model for the design of antiviral agents.

  16. Antimicrobial activity of human prion protein is mediated by its N-terminal region.

    Directory of Open Access Journals (Sweden)

    Mukesh Pasupuleti

    Full Text Available BACKGROUND: Cellular prion-related protein (PrP(c is a cell-surface protein that is ubiquitously expressed in the human body. The multifunctionality of PrP(c, and presence of an exposed cationic and heparin-binding N-terminus, a feature characterizing many antimicrobial peptides, made us hypothesize that PrP(c could exert antimicrobial activity. METHODOLOGY AND PRINCIPAL FINDINGS: Intact recombinant PrP exerted antibacterial and antifungal effects at normal and low pH. Studies employing recombinant PrP and N- and C-terminally truncated variants, as well as overlapping peptide 20mers, demonstrated that the antimicrobial activity is mediated by the unstructured N-terminal part of the protein. Synthetic peptides of the N-terminus of PrP killed the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, and the Gram-positive Bacillus subtilis and Staphylococcus aureus, as well as the fungus Candida parapsilosis. Fluorescence studies of peptide-treated bacteria, paired with analysis of peptide effects on liposomes, showed that the peptides exerted membrane-breaking effects similar to those seen after treatment with the "classical" human antimicrobial peptide LL-37. In contrast to LL-37, however, no marked helix induction was detected for the PrP-derived peptides in presence of negatively charged (bacteria-mimicking liposomes. PrP furthermore showed an inducible expression during wounding of human skin ex vivo and in vivo, as well as stimulation of keratinocytes with TGF-alpha in vitro. CONCLUSIONS: The demonstration of an antimicrobial activity of PrP, localisation of its activity to the N-terminal and heparin-binding region, combined with results showing an increased expression of PrP during wounding, indicate that PrPs could have a previously undisclosed role in host defense.

  17. Turnover of whole body proteins and myofibrillar proteins in middle-aged active men

    International Nuclear Information System (INIS)

    Zackin, M.; Meredith, C.; Frontera, W.; Evans, W.

    1986-01-01

    Endurance-trained older men have a higher proportion of lean tissue and greater muscle cell oxidative capacity, reversing age-related trends and suggesting major changes in protein metabolism. In this study, protein turnover was determined in 6 middle-aged (52+/-1 yr) men who were well trained (VO 2 max 55.2+/-5.0 ml O 2 /kg.min) and lean (body fat 18.9+/-2.8%, muscle mass 36.6+/-0.6%). The maintained habitual exercise while consuming 0.6, 0.9 or 1.2 g protein/kg.day for 10-day periods. N flux was measured from 15 N in urea after oral 15 N-glycine administration. Myofibrillar protein breakdown was estimated from urinary 3-methyl-histidine. Dietary protein had no effect on turnover rates, even when N balance was negative. Whole body protein synthesis was 3.60+/-0.12 g/kg.day and breakdown was 3.40+/-0.14 g/kg.day for all N intakes. Whole body protein flux, synthesis and breakdown were similar to values reported for sedentary young (SY) or sedentary old (SO) men on comparable diets. 3-me-his (3.67+/-0.14 μmol/kg.day) was similar to values reported for SY but higher (p<0.01) than for SO. Myofibrillar protein breakdown per unit muscle mass (185+/-7 μmol 3-me-his/g creatinine) was higher (p<0.01) than for SY or SO. In active middle-aged men, myofibrillar proteins may account for a greater proportion of whole body protein turnover, despite an age-related reduction in muscle mass

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

    KAUST Repository

    Zourelidou, Melina; Absmanner, Birgit; Weller, Benjamin; Barbosa, Inê s CR; Willige, Bjö rn C; Fastner, Astrid; Streit, Verena; Port, Sarah A; Colcombet, Jean; de la Fuente van Bentem, Sergio; Hirt, Heribert; Kuster, Bernhard; Schulze, Waltraud X; Hammes, Ulrich Z; Schwechheimer, Claus

    2014-01-01

    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.

  19. 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.

  20. 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...... 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...

  1. VHH Activators and Inhibitors for Protein Kinase C Epsilon

    NARCIS (Netherlands)

    Summanen, M.M.I.

    2012-01-01

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

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

    Czech Academy of Sciences Publication Activity Database

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2018-04-20

    The dopamine D2 receptor (D2R) is a G protein-coupled receptor (GPCR) that is critical for many central nervous system functions. The D2R carries out these functions by signaling through two transducers: G proteins and β-arrestins (βarrs). Selectively engaging either the G protein or βarr pathway may be a way to improve drugs targeting GPCRs. The current model of GPCR signal transduction posits a chain of events where G protein activation ultimately leads to βarr recruitment. GPCR kinases (GRKs), which are regulated by G proteins and whose kinase action facilitates βarr recruitment, bridge these pathways. Therefore, βarr recruitment appears to be intimately tied to G protein activation via GRKs. Here we sought to understand how GRK2 action at the D2R would be disrupted when G protein activation is eliminated and the effect of this on βarr recruitment. We used two recently developed biased D2R mutants that can preferentially interact either with G proteins or βarrs as well as a βarr-biased D2R ligand, UNC9994. With these functionally selective tools, we investigated the mechanism whereby the βarr-preferring D2R achieves βarr pathway activation in the complete absence of G protein activation. We describe how direct, G protein-independent recruitment of GRK2 drives interactions at the βarr-preferring D2R and also contributes to βarr recruitment at the WT D2R. Additionally, we found an additive interaction between the βarr-preferring D2R mutant and UNC9994. These results reveal that the D2R can directly recruit GRK2 without G protein activation and that this mechanism may have relevance to achieving βarr-biased signaling. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Structure-Activity Relationship Studies of the Cyclic Depsipeptide Natural Product YM-254890, Targeting the Gq Protein.

    Science.gov (United States)

    Zhang, Hang; Xiong, Xiao-Feng; Boesgaard, Michael W; Underwood, Christina R; Bräuner-Osborne, Hans; Strømgaard, Kristian

    2017-06-07

    Extracellular signals perceived by G protein-coupled receptors are transmitted via G proteins, and subsequent intracellular signaling cascades result in a plethora of physiological responses. The natural product cyclic depsipeptides YM-254890 and FR900359 are the only known compounds that specifically inhibit signaling mediated by the G q subfamily. In this study we exploit a newly developed synthetic strategy for this compound class in the design, synthesis, and pharmacological evaluation of eight new analogues of YM-254890. These structure-activity relationship studies led to the discovery of three new analogues, YM-13, YM-14, and YM-18, which displayed potent and selective G q inhibitory activity. This provides pertinent information for the understanding of the G q inhibitory mechanism by this class of compounds and importantly provides a pathway for the development of labeled YM-254890 analogues. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. 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.

  6. 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

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

    Science.gov (United States)

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

    2014-01-01

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

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

    KAUST Repository

    Altawashi, Azza; Jung, Sung Yun; Liu, Dou; Su, Bing; Qin, Jun

    2012-01-01

    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

  9. Neurite outgrowth induced by a synthetic peptide ligand of neural cell adhesion molecule requires fibroblast growth factor receptor activation

    DEFF Research Database (Denmark)

    Rønn, L C; Doherty, P; Holm, A

    2000-01-01

    identified a neuritogenic ligand, termed the C3 peptide, of the first immunoglobulin (lg) module of NCAM using a combinatorial library of synthetic peptides. Here we investigate whether stimulation of neurite outgrowth by this synthetic ligand of NCAM involves FGFRs. In primary cultures of cerebellar neurons...... from wild-type mice, the C3 peptide stimulated neurite outgrowth. This response was virtually absent in cultures of cerebellar neurons from transgenic mice expressing a dominant-negative form of the FGFR1. Likewise, in PC12E2 cells transiently expressing a dominant-negative form of the mouse FGFR1...

  10. (−-Epigallocatechin 3-Gallate Synthetic Analogues Inhibit Fatty Acid Synthase and Show Anticancer Activity in Triple Negative Breast Cancer

    Directory of Open Access Journals (Sweden)

    Joan Crous-Masó

    2018-05-01

    Full Text Available (−-Epigallocatechin 3-gallate (EGCG is a natural polyphenol from green tea with reported anticancer activity and capacity to inhibit the lipogenic enzyme fatty acid synthase (FASN, which is overexpressed in several human carcinomas. To improve the pharmacological profile of EGCG, we previously developed a family of EGCG derivatives and the lead compounds G28, G37 and G56 were characterized in HER2-positive breast cancer cells overexpressing FASN. Here, diesters G28, G37 and G56 and two G28 derivatives, monoesters M1 and M2, were synthesized and assessed in vitro for their cytotoxic, FASN inhibition and apoptotic activities in MDA-MB-231 triple-negative breast cancer (TNBC cells. All compounds displayed moderate to high cytotoxicity and significantly blocked FASN activity, monoesters M1 and M2 being more potent inhibitors than diesters. Interestingly, G28, M1, and M2 also diminished FASN protein expression levels, but only monoesters M1 and M2 induced apoptosis. Our results indicate that FASN inhibition by such polyphenolic compounds could be a new strategy in TNBC treatment, and highlight the potential anticancer activities of monoesters. Thus, G28, G37, G56, and most importantly M1 and M2, are anticancer candidates (alone or in combination to be further characterized in vitro and in vivo.

  11. (-)-Epigallocatechin 3-Gallate Synthetic Analogues Inhibit Fatty Acid Synthase and Show Anticancer Activity in Triple Negative Breast Cancer.

    Science.gov (United States)

    Crous-Masó, Joan; Palomeras, Sònia; Relat, Joana; Camó, Cristina; Martínez-Garza, Úrsula; Planas, Marta; Feliu, Lidia; Puig, Teresa

    2018-05-11

    (-)-Epigallocatechin 3-gallate (EGCG) is a natural polyphenol from green tea with reported anticancer activity and capacity to inhibit the lipogenic enzyme fatty acid synthase (FASN), which is overexpressed in several human carcinomas. To improve the pharmacological profile of EGCG, we previously developed a family of EGCG derivatives and the lead compounds G28, G37 and G56 were characterized in HER2-positive breast cancer cells overexpressing FASN. Here, diesters G28, G37 and G56 and two G28 derivatives, monoesters M1 and M2, were synthesized and assessed in vitro for their cytotoxic, FASN inhibition and apoptotic activities in MDA-MB-231 triple-negative breast cancer (TNBC) cells. All compounds displayed moderate to high cytotoxicity and significantly blocked FASN activity, monoesters M1 and M2 being more potent inhibitors than diesters. Interestingly, G28, M1, and M2 also diminished FASN protein expression levels, but only monoesters M1 and M2 induced apoptosis. Our results indicate that FASN inhibition by such polyphenolic compounds could be a new strategy in TNBC treatment, and highlight the potential anticancer activities of monoesters. Thus, G28, G37, G56, and most importantly M1 and M2, are anticancer candidates (alone or in combination) to be further characterized in vitro and in vivo.

  12. Treatment of high-strength synthetic sewage in a laboratory-scale upflow anaerobic sludge bed (UASB) with aerobic activated sludge (AS) post-treatment.

    Science.gov (United States)

    Banihani, Qais H; Field, Jim A

    2013-01-01

    Performance of a combined system up-flow anaerobic sludge blanket (UASB) followed by aerobic treatment activated sludge (AS) for removal of carbonaceous and nitrogenous contaminants at an average temperature of 25°C was investigated. The combined system was fed with high strength synthetic sewage having chemical oxygen demand (COD) of 2500 mg L(-1). The organic loading rate (OLR) of the UASB reactor was increased gradually from 1.1 to 3.8 gCOD L(r) (-1) d(-1). At steady state condition, the UASB reactor achieved removal efficiency up to 83.5% of total COD (COD(tot)), 74.0% of volatile fatty acid (VFA) and 94.0% of protein. The combined system performed an excellent organic removal pushing the overall removal efficiency of COD(tot), VFA and protein to 91.0%, 99.9% and 98.2%, respectively. When the OLR of the UASB increased to 4.4 g COD L(r) (-1) d(-1), the UASB was overloaded and; thus, its effluent quality deteriorated. In respect to nitrogen removal, both partial nitrification and complete nitrification took place in aerobic post-treatment. When the dissolved oxygen (DO) concentration was >2.0 mg L(-1), complete nitrification (period B) occurred with an average nitrification efficiency of 96.2%. The partial nitrification occurred due to high OLR to AS during the overloading event (period A) and when DO concentration was <2.0 mg L(-1) (period C). The maximum accumulated nitrite concentration in periods A, B and C were 90.0, 0.9 and 75.8 mg NO(-) (2) -N L(-1), respectively. The nitrogen balance results of periods A and C indicated that there was a discrepancy between the amount of ammonium nitrogen removed and the amount of oxidized nitrogen formed. This suggests the occurrence of simultaneous nitrification/denitrification (SND) in aerobic post-treatment.

  13. Synthetic polyester-hydrolyzing enzymes from thermophilic actinomycetes.

    Science.gov (United States)

    Wei, Ren; Oeser, Thorsten; Zimmermann, Wolfgang

    2014-01-01

    Thermophilic actinomycetes produce enzymes capable of hydrolyzing synthetic polyesters such as polyethylene terephthalate (PET). In addition to carboxylesterases, which have hydrolytic activity predominantly against PET oligomers, esterases related to cutinases also hydrolyze synthetic polymers. The production of these enzymes by actinomycetes as well as their recombinant expression in heterologous hosts is described and their catalytic activity against polyester substrates is compared. Assays to analyze the enzymatic hydrolysis of synthetic polyesters are evaluated, and a kinetic model describing the enzymatic heterogeneous hydrolysis process is discussed. Structure-function and structure-stability relationships of actinomycete polyester hydrolases are compared based on molecular dynamics simulations and recently solved protein structures. In addition, recent progress in enhancing their activity and thermal stability by random or site-directed mutagenesis is presented. © 2014 Elsevier Inc. All rights reserved.

  14. Pharmacokinetics of activated protein C in guinea pigs

    International Nuclear Information System (INIS)

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

    1991-01-01

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

  15. Mitogen activated protein kinases selectively regulate palytoxin-stimulated gene expression in mouse keratinocytes

    International Nuclear Information System (INIS)

    Zeliadt, Nicholette A.; Warmka, Janel K.; Wattenberg, Elizabeth V.

    2003-01-01

    We have been investigating how the novel skin tumor promoter palytoxin transmits signals through mitogen activated protein kinases (MAPKs). Palytoxin activates three major MAPKs, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, in a keratinocyte cell line derived from initiated mouse skin (308). We previously showed that palytoxin requires ERK to increase matrix metalloproteinase-13 (MMP-13) gene expression, an enzyme implicated in carcinogenesis. Diverse stimuli require JNK and p38 to increase MMP-13 gene expression, however. We therefore used the JNK and p38 inhibitors SP 600125 and SB 202190, respectively, to investigate the role of these MAPKs in palytoxin-induced MMP-13 gene expression. Surprisingly, palytoxin does not require JNK and p38 to increase MMP-13 gene expression. Accordingly, ERK activation, independent of palytoxin and in the absence of JNK and p38 activation, is sufficient to induce MMP-13 gene expression in 308 keratinocytes. Dexamethasone, a synthetic glucocorticoid that inhibits activator protein-1 (AP-1), blocked palytoxin-stimulated MMP-13 gene expression. Therefore, the AP-1 site present in the promoter of the MMP-13 gene appears to be functional and to play a key role in palytoxin-stimulated gene expression. Previous studies showed that palytoxin simulates an ERK-dependent selective increase in the c-Fos content of AP-1 complexes that bind to the promoter of the MMP-13 gene. JNK and p38 can also modulate c-Fos. Palytoxin does not require JNK or p38 to increase c-Fos binding, however. Altogether, these studies indicate that ERK plays a distinctly essential role in transmitting palytoxin-stimulated signals to specific nuclear targets in keratinocytes derived from initiated mouse skin

  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.

  17. Activity of the Antioxidant Defense System in a Typical Bioinsecticide-and Synthetic Insecticide-treated Cowpea Storage Beetle F. (Coleoptera: Chrysomelidae

    Directory of Open Access Journals (Sweden)

    Ayodele O. Kolawole

    2014-01-01

    Full Text Available The non-enzymatic and enzymatic antioxidant defense systems play a major role in detoxification of pro-oxidant endobiotics and xenobiotics. The possible involvement of beetle non-enzymatic [α-tocopherol, glutathione (GSH, and ascorbic acid] and enzymatic [catalase (CAT, superoxide dismutase (SOD, peroxidase (POX, and polyphenol oxidase (PPO] antioxidant defense system on the insecticidal activity of synthetic insecticides (cypermethrin, 2,2-dicholorovinyl dimethyl phosphate, and λ-cyhalothrin and ethanolic plant extracts of Tithonia diversifolia, Cyperus rotundus, Hyptis suaveolens leaves , and Jatropha Curcas seeds was investigated. 2,2-Dicholorovinyl dimethyl phosphate (DDVP; 200 ppm, LC 50 = 13.24 ppm and T. diversifolia (20,000 ppm resulted in 100% beetle mortality at 96-hour post-treatment. The post-treatments significantly increased the beetle α-tocopherol and GSH contents. Activities of CAT, SOD, POX, and PPO were modulated by the synthetic insecticides and bioinsecticides to diminish the adverse effect of the chemical stresses. Quantitative and qualitative allelochemical compositions of bioinsecticides and chemical structure of synthetic insecticides possibly account and for modulation of their respective enzyme activities. Altogether, oxidative stress was enormous enough to cause maladaptation in insects. This study established that oxidative imbalance created could be the molecular basis of the efficacy of both insecticides and bio-insecticides. Two, there was development of functional but inadequate antioxidant defense mechanism in the beetle.

  18. Dynamics of synthetic activity of RNA and glycoproteins in epithel cells of endometrium in heifers after ovulation

    International Nuclear Information System (INIS)

    Pivko, J.; Grafenau, P.; Uhrin, V.; Kopecny, V.

    1998-01-01

    represented in the supranuclear parts. Intensive synthesis takes place in cells of glands of the functional zone on 3rd day, the intensity of the reaction is lower in the basal zone. The creation of glycoproteins is identical in surface as well as in deep parts of glands on 6th day, it declines in the surface parts on 9th day, whereas it remains on the same level in other parts. The intensive RNA synthesis was sustained in nuclei of epithel cells of uterus in luminal and glandular epithel. The synthetic and secretion activities of glycoproteins are intensive in the luteal phase, they decrease slightly in surface cells of glands white they are preserved in the deep cells

  19. Natural - synthetic - artificial!

    DEFF Research Database (Denmark)

    Nielsen, Peter E

    2010-01-01

    The terms "natural," "synthetic" and "artificial" are discussed in relation to synthetic and artificial chromosomes and genomes, synthetic and artificial cells and artificial life.......The terms "natural," "synthetic" and "artificial" are discussed in relation to synthetic and artificial chromosomes and genomes, synthetic and artificial cells and artificial life....

  20. Synthetic Cannabinoids.

    Science.gov (United States)

    Mills, Brooke; Yepes, Andres; Nugent, Kenneth

    2015-07-01

    Synthetic cannabinoids (SCBs), also known under the brand names of "Spice," "K2," "herbal incense," "Cloud 9," "Mojo" and many others, are becoming a large public health concern due not only to their increasing use but also to their unpredictable toxicity and abuse potential. There are many types of SCBs, each having a unique binding affinity for cannabinoid receptors. Although both Δ-tetrahydrocannabinol (THC) and SCBs stimulate the same receptors, cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2), studies have shown that SCBs are associated with higher rates of toxicity and hospital admissions than is natural cannabis. This is likely due to SCBs being direct agonists of the cannabinoid receptors, whereas THC is a partial agonist. Furthermore, the different chemical structures of SCBs found in Spice or K2 may interact in unpredictable ways to elicit previously unknown, and the commercial products may have unknown contaminants. The largest group of users is men in their 20s who participate in polydrug use. The most common reported toxicities with SCB use based on studies using Texas Poison Control records are tachycardia, agitation and irritability, drowsiness, hallucinations, delusions, hypertension, nausea, confusion, dizziness, vertigo and chest pain. Acute kidney injury has also been strongly associated with SCB use. Treatment mostly involves symptom management and supportive care. More research is needed to identify which contaminants are typically found in synthetic marijuana and to understand the interactions between different SBCs to better predict adverse health outcomes.

  1. 5' adenosine monophosphate-activated protein kinase, metabolism and exercise.

    Science.gov (United States)

    Aschenbach, William G; Sakamoto, Kei; Goodyear, Laurie J

    2004-01-01

    The 5' adenosine monophosphate-activated protein kinase (AMPK) is a member of a metabolite-sensing protein kinase family that functions as a metabolic 'fuel gauge' in skeletal muscle. AMPK is a ubiquitous heterotrimeric protein, consisting of an alpha catalytic, and beta and gamma regulatory subunits that exist in multiple isoforms and are all required for full enzymatic activity. During exercise, AMPK becomes activated in skeletal muscle in response to changes in cellular energy status (e.g. increased adenosine monophosphate [AMP]/adenosine triphosphate [ATP] and creatine/phosphocreatine ratios) in an intensity-dependent manner, and serves to inhibit ATP-consuming pathways, and activate pathways involved in carbohydrate and fatty-acid metabolism to restore ATP levels. Recent evidence shows that although AMPK plays this key metabolic role during acute bouts of exercise, it is also an important component of the adaptive response of skeletal muscles to endurance exercise training because of its ability to alter muscle fuel reserves and expression of several exercise-responsive genes. This review discusses the putative roles of AMPK in acute and chronic exercise responses, and suggests avenues for future AMPK research in exercise physiology and biochemistry.

  2. Methods of measuring Protein Disulfide Isomerase activity: a critical overview

    Science.gov (United States)

    Watanabe, Monica; Laurindo, Francisco; Fernandes, Denise

    2014-09-01

    Protein disulfide isomerase is an essential redox chaperone from the endoplasmic reticulum (ER) and is responsible for correct disulfide bond formation in nascent proteins. PDI is also found in other cellular locations in the cell, particularly the cell surface. Overall, PDI contributes to ER and global cell redox homeostasis and signaling. The knowledge about PDI structure and function progressed substantially based on in vitro studies using recombinant PDI and chimeric proteins. In these experimental scenarios, PDI reductase and chaperone activities are readily approachable. In contrast, assays to measure PDI isomerase activity, the hallmark of PDI family, are more complex. Assessment of PDI roles in cells and tissues mainly relies on gain- or loss-of-function studies. However, there is limited information regarding correlation of experimental readouts with the distinct types of PDI activities. In this mini-review, we evaluate the main methods described for measuring the different kinds of PDI activity: thiol reductase, thiol oxidase, thiol isomerase and chaperone. We emphasize the need to use appropriate controls and the role of critical interferents (e.g., detergent, presence of reducing agents). We also discuss the translation of results from in vitro studies with purified recombinant PDI to cellular and tissue samples, with critical comments on the interpretation of results.

  3. In silico study of protein to protein interaction analysis of AMP-activated protein kinase and mitochondrial activity in three different farm animal species

    Science.gov (United States)

    Prastowo, S.; Widyas, N.

    2018-03-01

    AMP-activated protein kinase (AMPK) is cellular energy censor which works based on ATP and AMP concentration. This protein interacts with mitochondria in determine its activity to generate energy for cell metabolism purposes. For that, this paper aims to compare the protein to protein interaction of AMPK and mitochondrial activity genes in the metabolism of known animal farm (domesticated) that are cattle (Bos taurus), pig (Sus scrofa) and chicken (Gallus gallus). In silico study was done using STRING V.10 as prominent protein interaction database, followed with biological function comparison in KEGG PATHWAY database. Set of genes (12 in total) were used as input analysis that are PRKAA1, PRKAA2, PRKAB1, PRKAB2, PRKAG1, PRKAG2, PRKAG3, PPARGC1, ACC, CPT1B, NRF2 and SOD. The first 7 genes belong to gene in AMPK family, while the last 5 belong to mitochondrial activity genes. The protein interaction result shows 11, 8 and 5 metabolism pathways in Bos taurus, Sus scrofa and Gallus gallus, respectively. The top pathway in Bos taurus is AMPK signaling pathway (10 genes), Sus scrofa is Adipocytokine signaling pathway (8 genes) and Gallus gallus is FoxO signaling pathway (5 genes). Moreover, the common pathways found in those 3 species are Adipocytokine signaling pathway, Insulin signaling pathway and FoxO signaling pathway. Genes clustered in Adipocytokine and Insulin signaling pathway are PRKAA2, PPARGC1A, PRKAB1 and PRKAG2. While, in FoxO signaling pathway are PRKAA2, PRKAB1, PRKAG2. According to that, we found PRKAA2, PRKAB1 and PRKAG2 are the common genes. Based on the bioinformatics analysis, we can demonstrate that protein to protein interaction shows distinct different of metabolism in different species. However, further validation is needed to give a clear explanation.

  4. Biological activity of neosergeolide and isobrucein B (and two semi-synthetic derivatives) isolated from the Amazonian medicinal plant Picrolemma sprucei (Simaroubaceae).

    Science.gov (United States)

    Silva, Ellen C C; Cavalcanti, Bruno C; Amorim, Rodrigo C N; Lucena, Jorcilene F; Quadros, Dulcimar S; Tadei, Wanderli P; Montenegro, Raquel C; Costa-Lotufo, Letícia V; Pessoa, Cláudia; Moraes, Manoel O; Nunomura, Rita C S; Nunomura, Sergio M; Melo, Marcia R S; Andrade-Neto, Valter F de; Silva, Luiz Francisco R; Vieira, Pedro Paulo R; Pohlit, Adrian M

    2009-02-01

    In the present study, in vitro techniques were used to investigate a range of biological activities of known natural quassinoids isobrucein B (1) and neosergeolide (2), known semi-synthetic derivative 1,12-diacetylisobrucein B (3), and a new semi-synthetic derivative, 12-acetylneosergeolide (4). These compounds were evaluated for general toxicity toward the brine shrimp species Artemia franciscana, cytotoxicity toward human tumour cells, larvicidal activity toward the dengue fever mosquito vector Aedes aegypti, haemolytic activity in mouse erythrocytes and antimalarial activity against the human malaria parasite Plasmodium falciparum. Compounds 1 and 2 exhibited the greatest cytotoxicity against all the tumor cells tested (IC50 = 5-27 microg/L) and against multidrug-resistant P. falciparum K1 strain (IC50 = 1.0-4.0 g/L) and 3 was only cytotoxic toward the leukaemia HL-60 strain (IC50 = 11.8 microg/L). Quassinoids 1 and 2 (LC50 = 3.2-4.4 mg/L) displayed greater lethality than derivative 4 (LC50 = 75.0 mg/L) toward A. aegypti larvae, while derivative 3 was inactive. These results suggest a novel application for these natural quassinoids as larvicides. The toxicity toward A. franciscana could be correlated with the activity in several biological models, a finding that is in agreement with the literature. Importantly, none of the studied compounds exhibited in vitro haemolytic activity, suggesting specificity of the observed cytotoxic effects. This study reveals the biological potential of quassinoids 1 and 2 and to a lesser extent their semi-synthetic derivatives for their in vitro antimalarial and cytotoxic activities.

  5. Biological activity of neosergeolide and isobrucein B (and two semi-synthetic derivatives isolated from the Amazonian medicinal plant Picrolemma sprucei (Simaroubaceae

    Directory of Open Access Journals (Sweden)

    Ellen CC Silva

    2009-02-01

    Full Text Available In the present study, in vitro techniques were used to investigate a range of biological activities of known natural quassinoids isobrucein B (1 and neosergeolide (2, known semi-synthetic derivative 1,12-diacetylisobrucein B (3, and a new semi-synthetic derivative, 12-acetylneosergeolide (4. These compounds were evaluated for general toxicity toward the brine shrimp species Artemia franciscana, cytotoxicity toward human tumour cells, larvicidal activity toward the dengue fever mosquito vector Aedes aegypti, haemolytic activity in mouse erythrocytes and antimalarial activity against the human malaria parasite Plasmodium falciparum. Compounds 1 and 2 exhibited the greatest cytotoxicity against all the tumor cells tested (IC50 = 5-27 µg/L and against multidrug-resistant P. falciparum K1 strain (IC50 = 1.0-4.0 g/L and 3 was only cytotoxic toward the leukaemia HL-60 strain (IC50 = 11.8 µg/L. Quassinoids 1 and 2 (LC50 = 3.2-4.4 mg/L displayed greater lethality than derivative 4 (LC50 = 75.0 mg/L toward A. aegypti larvae, while derivative 3 was inactive. These results suggest a novel application for these natural quassinoids as larvicides. The toxicity toward A. franciscana could be correlated with the activity in several biological models, a finding that is in agreement with the literature. Importantly, none of the studied compounds exhibited in vitro haemolytic activity, suggesting specificity of the observed cytotoxic effects. This study reveals the biological potential of quassinoids 1 and 2 and to a lesser extent their semi-synthetic derivatives for their in vitro antimalarial and cytotoxic activities.

  6. Effects of synthetic cohesin-containing scaffold protein architecture on binding dockerin-enzyme fusions on the surface of Lactococcus lactis

    Directory of Open Access Journals (Sweden)

    Wieczorek Andrew S

    2012-12-01

    Full Text Available Abstract Background The microbial synthesis of fuels, commodity chemicals, and bioactive compounds necessitates the assemblage of multiple enzyme activities to carry out sequential chemical reactions, often via substrate channeling by means of multi-domain or multi-enzyme complexes. Engineering the controlled incorporation of enzymes in recombinant protein complexes is therefore of interest. The cellulosome of Clostridium thermocellum is an extracellular enzyme complex that efficiently hydrolyzes crystalline cellulose. Enzymes interact with protein scaffolds via type 1 dockerin/cohesin interactions, while scaffolds in turn bind surface anchor proteins by means of type 2 dockerin/cohesin interactions, which demonstrate a different binding specificity than their type 1 counterparts. Recombinant chimeric scaffold proteins containing cohesins of different specificity allow binding of multiple enzymes to specific sites within an engineered complex. Results We report the successful display of engineered chimeric scaffold proteins containing both type 1 and type 2 cohesins on the surface of Lactococcus lactis cells. The chimeric scaffold proteins were able to form complexes with the Escherichia coli β-glucuronidase fused to either type 1 or type 2 dockerin, and differences in binding efficiencies were correlated with scaffold architecture. We used E. coli β-galactosidase, also fused to type 1 or type 2 dockerins, to demonstrate the targeted incorporation of two enzymes into the complexes. The simultaneous binding of enzyme pairs each containing a different dockerin resulted in bi-enzymatic complexes tethered to the cell surface. The sequential binding of the two enzymes yielded insights into parameters affecting assembly of the complex such as protein size and position within the scaffold. Conclusions The spatial organization of enzymes into complexes is an important strategy for increasing the efficiency of biochemical pathways. In this study

  7. 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...

  8. Protein phosphatase 5 promotes hepatocarcinogenesis through interaction with AMP-activated protein kinase.

    Science.gov (United States)

    Chen, Yao-Li; Hung, Man-Hsin; Chu, Pei-Yi; Chao, Tzu-I; Tsai, Ming-Hsien; Chen, Li-Ju; Hsiao, Yung-Jen; Shih, Chih-Ting; Hsieh, Feng-Shu; Chen, Kuen-Feng

    2017-08-15

    The serine-threonine protein phosphatase family members are known as critical regulators of various cellular functions, such as survival and transformation. Growing evidence suggests that pharmacological manipulation of phosphatase activity exhibits therapeutic benefits. Ser/Thr protein phosphatase 5 (PP5) is known to participate in glucocorticoid receptor (GR) and stress-induced signaling cascades that regulate cell growth and apoptosis, and has been shown to be overexpressed in various human malignant diseases. However, the role of PP5 in hepatocellular carcinoma (HCC) and whether PP5 may be a viable therapeutic target for HCC treatment are unknown. Here, by analyzing HCC clinical samples obtained from 215 patients, we found that overexpression of PP5 is tumor specific and associated with worse clinical outcomes. We further characterized the oncogenic properties of PP5 in HCC cells. Importantly, both silencing of PP5 with lentiviral-mediated short hairpin RNA (shRNA) and chemical inhibition of PP5 phosphatase activity using the natural compound cantharidin/norcantharidin markedly suppressed the growth of HCC cells and tumors in vitro and in vivo. Moreover, we identified AMP-activated protein kinase (AMPK) as a novel downstream target of oncogenic PP5 and demonstrated that the antitumor mechanisms underlying PP5 inhibition involve activation of AMPK signaling. Overall, our results establish a pathological function of PP5 in hepatocarcinogenesis via affecting AMPK signaling and suggest that PP5 inhibition is an attractive therapeutic approach for HCC. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    Czech Academy of Sciences Publication Activity Database

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

    2003-01-01

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

  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. Synthetic Brainbows

    KAUST Repository

    Wan, Y.

    2013-06-01

    Brainbow is a genetic engineering technique that randomly colorizes cells. Biological samples processed with this technique and imaged with confocal microscopy have distinctive colors for individual cells. Complex cellular structures can then be easily visualized. However, the complexity of the Brainbow technique limits its applications. In practice, most confocal microscopy scans use different florescence staining with typically at most three distinct cellular structures. These structures are often packed and obscure each other in rendered images making analysis difficult. In this paper, we leverage a process known as GPU framebuffer feedback loops to synthesize Brainbow-like images. In addition, we incorporate ID shuffing and Monte-Carlo sampling into our technique, so that it can be applied to single-channel confocal microscopy data. The synthesized Brainbow images are presented to domain experts with positive feedback. A user survey demonstrates that our synthetic Brainbow technique improves visualizations of volume data with complex structures for biologists.

  12. Synthetic Botany.

    Science.gov (United States)

    Boehm, Christian R; Pollak, Bernardo; Purswani, Nuri; Patron, Nicola; Haseloff, Jim

    2017-07-05

    Plants are attractive platforms for synthetic biology and metabolic engineering. Plants' modular and plastic body plans, capacity for photosynthesis, extensive secondary metabolism, and agronomic systems for large-scale production make them ideal targets for genetic reprogramming. However, efforts in this area have been constrained by slow growth, long life cycles, the requirement for specialized facilities, a paucity of efficient tools for genetic manipulation, and the complexity of multicellularity. There is a need for better experimental and theoretical frameworks to understand the way genetic networks, cellular populations, and tissue-wide physical processes interact at different scales. We highlight new approaches to the DNA-based manipulation of plants and the use of advanced quantitative imaging techniques in simple plant models such as Marchantia polymorpha. These offer the prospects of improved understanding of plant dynamics and new approaches to rational engineering of plant traits. Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.

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

    International Nuclear Information System (INIS)

    Bunnak, J.; Chaisupakitsin, M.

    2001-01-01

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

  14. 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)

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

    Directory of Open Access Journals (Sweden)

    Soumen Kahali

    2010-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-12-06

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

  17. Ionic responses rapidly elicited by activation of protein kinase C in quiescent Swiss 3T3 cells

    International Nuclear Information System (INIS)

    Vara, F.; Schneider, J.A.; Rozengurt, E.

    1985-01-01

    Diacylglycerol and phorbol esters activate protein kinase C in intact cells. The authors report here that addition of the synthetic diacylglycerol 1-oleoyl-2-acetylglycerol (OAG) to quiescent cultures of Swiss 3T3 cells caused a marked increase in the rate of ouabain-sensitive 86 Rb + uptake, a measure of the activity of the Na + /K + pump. The effect was dose-dependent and could be detected after 1 min of exposure to the diacylglycerol. OAG stimulated Na + influx via an amiloride-sensitive pathway and increased intracellular pH by 0.15 pH unit. Phorbol 12,13-dibutyrate (PBt 2 ) also enhanced ouabain sensitive 86 Rb + uptake and amiloride-sensitive 22 Na + influx. Prolonged treatment (40 hr) of 3T3 cells with PBt 2 at a saturating dose, which reduces the number of PBt 2 binding sites and protein kinase C activity, abolished the ionic response of the cells to a subsequent addition of either OAG or PBt 2 . They suggest that activation of protein kinase C elicits, either directly or indirectly, enhanced Na + /H + antiport activity, which, in turn, leads to Na + influx, intracellular pH modulation, and stimulation of the Na + /K + pump

  18. 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.

  19. 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.

  20. Selective effects of charge on G protein activation by FSH-receptor residues 551-555 and 650-653.

    Science.gov (United States)

    Grasso, P; Deziel, M R; Reichert, L E

    1995-01-01

    Two cytosolic regions of the rat testicular FSH receptor (FSHR), residues 533-555 and 645-653, have been identified as G protein-coupling domains. We localized the activity in these domains to their C-terminal sequences, residues 551-555 (KIAKR, net charge +3) and 650-653 (RKSH, net charge +3), and examined the effects of charge on G protein activation by the C-terminal peptides, using synthetic analogs containing additions, through alanine (A) linkages, of arginine (R, +), histidine (H, +) or both. RA-KIAKR (net charge +4) mimicked the effect of FSHR-(551-555) on guanine nucleotide exchange in rat testis membranes, but reduced its ability to inhibit FSH-stimulated estradiol biosynthesis in cultured rat Sertoli cells. Further increasing net charge by the addition of H (HARA-KIAKR, net charge +5) increased guanosine 5'-triphosphate (GTP) binding, but eliminated FSHR-(551-555) effects on FSH-stimulated steroidogenesis. HA-RKSH (net charge +4) significantly inhibited guanine nucleotide exchange in rat testis membranes, but stimulated basal and potentiated FSH-induced estradiol biosynthesis in cultured rat Sertoli cells. Addition of two H residues (HAHA-RKSH, net charge +5) restored GTP binding and further potentiated basal and FSH-stimulated steroidogenesis. These results suggest that positive charges in G protein-coupling domains of the FSHR play a role in modulating G protein activation and postbinding effects of FSH, such as steroidogenesis.

  1. Antimicrobial Activity of Some Synthetic Compounds on Fungi Associated with Post Harvest Rot of Red Pepper (Capsicum annum

    Directory of Open Access Journals (Sweden)

    Matthew O. KOLAWOLE

    2012-11-01

    Full Text Available Rhizopus sp, Mucor sp, Collectotrichum capsici and Geotrichum candidum were isolated but pathogenic test revealed that Collectotrichum capsici and Geotrichum candidum were the most pathogenic of all the isolates. Ni2+ + Azo has the highest inhibitory effect, closely followed by Ni2+ + PAN while Copper (II complex of Thiourea has the lowest inhibitory effect. However, 10mg/ml concentration proved to be the most effective when radial mycelial growth of the test fungi was measured. The inhibitory effects of each complex on the isolates increases with increase in incubation period. There is the feasibility of using synthetic associated with the symptoms. The three synthetic compounds, Copper (II complex of Thiourea, Ni2+ + compounds as preservatives for harvested red pepper.

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

    Science.gov (United States)

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

    2018-01-23

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

  3. 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. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Nanocarriers from GRAS Zein Proteins to Encapsulate Hydrophobic Actives.

    Science.gov (United States)

    Weissmueller, Nikolas T; Lu, Hoang D; Hurley, Amanda; Prud'homme, Robert K

    2016-11-14

    One factor limiting the expansion of nanomedicines has been the high cost of the materials and processes required for their production. We present a continuous, scalable, low cost nanoencapsulation process, Flash Nanoprecipitation (FNP) that enables the production of nanocarriers (NCs) with a narrow size distribution using zein corn proteins. Zein is a low cost, GRAS protein (having the FDA status of "Generally Regarded as Safe") currently used in food applications, which acts as an effective encapsulant for hydrophobic compounds using FNP. The four-stream FNP configuration allows the encapsulation of very hydrophobic compounds in a way that is not possible with previous precipitation processes. We present the encapsulation of several model active compounds with as high as 45 wt % drug loading with respect to zein concentration into ∼100 nm nanocarriers. Three examples are presented: (1) the pro-drug antioxidant, vitamin E-acetate, (2) an anticholera quorum-sensing modulator CAI-1 ((S)-3-hydroxytridecan-4-one; CAI-1 that reduces Vibrio cholerae virulence by modulating cellular communication), and (3) hydrophobic fluorescent dyes with a range of hydrophobicities. The specific interaction between zein and the milk protein, sodium caseinate, provides stabilization of the NCs in PBS, LB medium, and in pH 2 solutions. The stability and size changes in the three media provide information on the mechanism of assembly of the zein/active/casein NC.

  5. 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.

  6. Platelet activation by extracellular matrix proteins in haemostasis and thrombosis.

    Science.gov (United States)

    Watson, Steve P

    2009-01-01

    The prevention of excessive blood loss to avoid fatal haemorrhage is a pivotal process for all organisms possessing a circulatory system. Increased circulating blood volume and pressure, as required in larger animals, make this process all the more important and challenging. It is essential to have a powerful and rapid system to detect damage and generate an effective seal, and which is also exquisitely regulated to prevent unwanted, excessive or systemic activation so as to avoid blockage of vessels. Thus, a highly specialised and efficient haemostatic system has evolved that consists of cellular (platelets) and protein (coagulation factors) components. Importantly, this is able to support haemostasis in both the low shear environment of the venous system and the high shear environment of the arterial system. Endothelial cells, lining the entire circulation system, play a crucial role in the delicate balance between activation and inhibition of the haemostatic system. An intact and healthy endothelium supports blood flow by preventing attachment of cells and proteins which is required for initiation of coagulation and platelet activation. Endothelial cells produce and release the two powerful soluble inhibitors of platelet activation, nitric oxide and prostacyclin, and express high levels of CD39 which rapidly metabolises the major platelet feedback agonist, ADP. This antithrombotic environment however can rapidly change following activation or removal of endothelial cells through injury or rupture of atherosclerotic plaques. Loss of endothelial cells exposes the subendothelial extracellular matrix which creates strong signals for activation of the haemostatic system including powerful platelet adhesion and activation. Quantitative and qualitative changes in the composition of the subendothelial extracellular matrix influence these prothrombotic characteristics with life threatening thrombotic and bleeding complications, as illustrated by formation of

  7. Approaches to chemical synthetic biology.

    Science.gov (United States)

    Chiarabelli, Cristiano; Stano, Pasquale; Anella, Fabrizio; Carrara, Paolo; Luisi, Pier Luigi

    2012-07-16

    Synthetic biology is first represented in terms of two complementary aspects, the bio-engineering one, based on the genetic manipulation of extant microbial forms in order to obtain forms of life which do not exist in nature; and the chemical synthetic biology, an approach mostly based on chemical manipulation for the laboratory synthesis of biological structures that do not exist in nature. The paper is mostly devoted to shortly review chemical synthetic biology projects currently carried out in our laboratory. In particular, we describe: the minimal cell project, then the "Never Born Proteins" and lastly the Never Born RNAs. We describe and critically analyze the main results, emphasizing the possible relevance of chemical synthetic biology for the progress in basic science and biotechnology. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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

    NARCIS (Netherlands)

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

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

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

    NARCIS (Netherlands)

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

    1998-01-01

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

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

    Science.gov (United States)

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

    2018-04-11

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

  11. Synthetic Astrobiology

    Science.gov (United States)

    Rothschild, Lynn J.

    2017-01-01

    "Are we alone?" is one of the primary questions of astrobiology, and whose answer defines our significance in the universe. Unfortunately, this quest is hindered by the fact that we have only one confirmed example of life, that of earth. While this is enormously helpful in helping to define the minimum envelope for life, it strains credulity to imagine that life, if it arose multiple times, has not taken other routes. To help fill this gap, our lab has begun using synthetic biology - the design and construction of new biological parts and systems and the redesign of existing ones for useful purposes - as an enabling technology. One theme, the "Hell Cell" project, focuses on creating artificial extremophiles in order to push the limits for Earth life, and to understand how difficult it is for life to evolve into extreme niches. In another project, we are re-evolving biotic functions using only the most thermodynamically stable amino acids in order to understand potential capabilities of an early organism with a limited repertoire of amino acids.

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

    Science.gov (United States)

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

    2013-08-12

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

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

    NARCIS (Netherlands)

    Koppelman, S.J.

    1995-01-01

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

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

    Science.gov (United States)

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

    2017-04-15

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

  15. 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

  16. Activating human genes with zinc finger proteins, transcription activator-like effectors and CRISPR/Cas9 for gene therapy and regenerative medicine.

    Science.gov (United States)

    Gersbach, Charles A; Perez-Pinera, Pablo

    2014-08-01

    New technologies have recently been developed to control the expression of human genes in their native genomic context by engineering synthetic transcription factors that can be targeted to any DNA sequence. The ability to precisely regulate any gene as it occurs naturally in the genome provides a means to address a variety of diseases and disorders. This approach also circumvents some of the traditional challenges of gene therapy. In this editorial, we review the technologies that have enabled targeted human gene activation, including the engineering of transcription factors based on zinc finger proteins, transcription activator-like effectors and the CRISPR/Cas9 system. Additionally, we highlight examples in which these methods have been developed for therapeutic applications and discuss challenges and opportunities.

  17. Animal and Plant Proteins as Precursors of Peptides with ACE Inhibitory Activity – An in silico Strategy of Protein Evaluation

    Directory of Open Access Journals (Sweden)

    Anna Iwaniak

    2009-01-01

    Full Text Available This paper presents a modern in silico approach useful in the evaluation of proteins as a source of ACE inhibitors. All protein sequences analyzed were derived from the BIOPEP database. To determine the protein value, the following criteria of evaluation were applied: the profile of potential biological (ACE inhibitory activity of a protein, the frequency of the occurrence of fragments with ACE inhibitory activity (A and the potential biological activity of a protein (B. The results, based on a statistical analysis, indicate that milk proteins can be a better source of ACE inhibitors than wheat gliadins. Moreover, all analyzed gliadins possessed more potent ACE inhibitors than chicken meat proteins. No significant differences were observed when comparing A values between soy globulins and β-lactoglobulins. Although criteria such as the profile of potential biological activity of protein, as well as parameters A and B, can be suitable tools in protein evaluation, the proteolytic digestion of protein needs to be considered. Moreover, computerised methods of classifying proteins according to different algorithms are often subjective due to discretion in interpretation of the results.

  18. Mechanical properties of the compass depressors of the sea-urchin Paracentrotus lividus (Echinodermata, Echinoidea and the effects of enzymes, neurotransmitters and synthetic tensilin-like protein.

    Directory of Open Access Journals (Sweden)

    Iain C Wilkie

    Full Text Available The compass depressors (CDs of the sea-urchin lantern are ligaments consisting mainly of discontinuous collagen fibrils associated with a small population of myocytes. They are mutable collagenous structures, which can change their mechanical properties rapidly and reversibly under nervous control. The aims of this investigation were to characterise the baseline (i.e. unmanipulated static mechanical properties of the CDs of Paracentrotus lividus by means of creep tests and incremental force-extension tests, and to determine the effects on their mechanical behaviour of a range of agents. Under constant load the CDs exhibited a three-phase creep curve, the mean coefficient of viscosity being 561±365 MPa.s. The stress-strain curve showed toe, linear and yield regions; the mean strain at the toe-linear inflection was 0.86±0.61; the mean Young's modulus was 18.62±10.30 MPa; and the mean tensile strength was 8.14±5.73 MPa. Hyaluronidase from Streptomyces hyalurolyticus had no effect on creep behaviour, whilst chondroitinase ABC prolonged primary creep but had no effect on secondary creep or on any force-extension parameters; it thus appears that neither hyaluronic acid nor sulphated glycosaminoglycans have an interfibrillar load transfer function in the CD. Acetylcholine, the muscarinic agonists arecoline and methacholine, and the nicotinic agonists nicotine and 1-[1-(3,4-dimethyl-phenyl-ethyl]-piperazine produced an abrupt increase in CD viscosity; the CDs were not differentially sensitive to muscarinic or nicotinic agonists. CDs showed either no, or no consistent, response to adrenaline, L-glutamic acid, 5-hydroxytryptamine and γ-aminobutyric acid. Synthetic echinoid tensilin-like protein had a weak and inconsistent stiffening effect, indicating that, in contrast to holothurian tensilins, the echinoid molecule may not be involved in the regulation of collagenous tissue tensility. We compare in detail the mechanical behaviour of the CD with that

  19. AMP-activated protein kinase and type 2 diabetes.

    Science.gov (United States)

    Musi, Nicolas

    2006-01-01

    AMP-activated protein kinase (AMPK) is an enzyme that works as a fuel gauge, being activated in situations of high-energy phosphate depletion. Upon activation, AMPK functions to restore cellular ATP by modifying diverse metabolic pathways. AMPK is activated robustly by skeletal muscle contraction and myocardial ischemia, and may be involved in the stimulation of glucose transport and fatty acid oxidation produced by these stimuli. In liver, activation of AMPK results in enhanced fatty acid oxidation and in decreased production of glucose, cholesterol, and triglycerides. Recent studies have shown that AMPK is the cellular mediator for many of the metabolic effects of drugs such as metformin and thiazolidinediones, as well as the insulin sensitizing adipocytokines leptin and adiponectin. These data, along with evidence from studies showing that chemical activation of AMPK in vivo with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) improves blood glucose concentrations and lipid profiles, make this enzyme an attractive pharmacological target for the treatment of type 2 diabetes and other metabolic disorders.

  20. Gc protein-derived macrophage activating factor (GcMAF): isoelectric focusing pattern and tumoricidal activity.

    Science.gov (United States)

    Mohamad, Saharuddin Bin; Nagasawa, Hideko; Sasaki, Hideyuki; Uto, Yoshihiro; Nakagawa, Yoshinori; Kawashima, Ken; Hori, Hitoshi

    2003-01-01

    Gc protein is the precursor for Gc protein-derived macrophage activating factor (GcMAF), with three phenotypes: Gc1f, Gc1s and Gc2, based on its electrophoretic mobility. The difference in electrophoretic mobility is because of the difference in its posttranslational sugar moiety composition. We compared the difference between Gc protein and GcMAF electrophoretic mobility using the isoelectric focusing (IEF) method. The tumoricidal activity of GcMAF-treated macrophage was evaluated after coculture with L-929 cell. The tumoricidal mechanism was investigated using TNF bioassay and nitric oxide (NO) release. The difference in Gc protein and GcMAF electrophoretic mobility was detected. The tumoricidal activity of GcMAF-treated macrophage was detected, but no release of TNF and NO was detected. The difference of isoelectric focusing mobility in Gc protein and GcMAF would be useful to develop a GcMAF detection method. GcMAF increased macrophage tumoricidal activity but TNF and NO release were not involved in the mechanism.

  1. Synthetic Peptides Derived from Bovine Lactoferricin Exhibit Antimicrobial Activity against E. coli ATCC 11775, S. maltophilia ATCC 13636 and S. enteritidis ATCC 13076

    Directory of Open Access Journals (Sweden)

    Nataly De Jesús Huertas Méndez

    2017-03-01

    Full Text Available Linear, dimeric, tetrameric, and cyclic peptides derived from lactoferricin B–containing non-natural amino acids and the RWQWR motif were synthesized, purified, and characterized using RP-HPLC, MALDI-TOF mass spectrometry, and circular dichroism. The antibacterial activity of peptides against Escherichia coli ATCC 11775, Stenotrophomonas maltophilia ATCC 13636, and Salmonella enteritidis ATCC 13076 was evaluated. The minimum inhibitory concentration (MIC and minimum bactericidal concentration (MBC were determined. The synthetic bovine lactoferricin exhibited antibacterial activity against E. coli ATCC 11775 and S. enteritidis ATCC 13076. The dimeric peptide (RRWQWR2K-Ahx exhibited the highest antibacterial activity against the tested bacterial strain. The monomeric, cyclic, tetrameric, and palindromic peptides containing the RWQWR motif exhibited high and specific activity against E. coli ATCC 11775. The results suggest that short peptides derived from lactoferricin B could be considered as potential candidates for the development of antibacterial agents against infections caused by E. coli.

  2. Synthetic Peptides Derived from Bovine Lactoferricin Exhibit Antimicrobial Activity against E. coli ATCC 11775, S. maltophilia ATCC 13636 and S. enteritidis ATCC 13076.

    Science.gov (United States)

    Huertas Méndez, Nataly De Jesús; Vargas Casanova, Yerly; Gómez Chimbi, Anyelith Katherine; Hernández, Edith; Leal Castro, Aura Lucia; Melo Diaz, Javier Mauricio; Rivera Monroy, Zuly Jenny; García Castañeda, Javier Eduardo

    2017-03-12

    Linear, dimeric, tetrameric, and cyclic peptides derived from lactoferricin B-containing non-natural amino acids and the RWQWR motif were synthesized, purified, and characterized using RP-HPLC, MALDI-TOF mass spectrometry, and circular dichroism. The antibacterial activity of peptides against Escherichia coli ATCC 11775, Stenotrophomonas maltophilia ATCC 13636, and Salmonella enteritidis ATCC 13076 was evaluated. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined. The synthetic bovine lactoferricin exhibited antibacterial activity against E. coli ATCC 11775 and S. enteritidis ATCC 13076. The dimeric peptide (RRWQWR)₂K-Ahx exhibited the highest antibacterial activity against the tested bacterial strain. The monomeric, cyclic, tetrameric, and palindromic peptides containing the RWQWR motif exhibited high and specific activity against E. coli ATCC 11775. The results suggest that short peptides derived from lactoferricin B could be considered as potential candidates for the development of antibacterial agents against infections caused by E. coli .

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

    Directory of Open Access Journals (Sweden)

    Candra Purnawan

    2014-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Yingying Han

    2010-11-01

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

  5. Non-destructive alpha-particle activation analysis of P, Cl, K and Ca in marine macro-alga samples using synthetic multielement reference material as comparative standard

    International Nuclear Information System (INIS)

    Iwata, Y.; Naitoh, H.; Suzuki, N.

    1992-01-01

    A Synthetic Reference Material (SyRM) composed with accurately known amounts of 12 elements has been prepared. The elemental composition of the SyRM is closely similar to that of marine macro-algae sample. The elemental composition of the SyRM was regulated by the starting materials used for the synthesis. The SyRM was used as a comparative standard for non-destructive alpha-particle activation analysis of marine macro-alga samples. P, Cl, K and Ca were determined simultaneously without correction for alpha range due to difference in the elemental composition between the analytical samples and the comparative standard. (author) 19 refs.; 4 tabs

  6. Two synthetic progestins and natural progesterone are responsible for most of the progestagenic activities in municipal wastewater treatment plant effluents in the Czech and Slovak republics.

    Science.gov (United States)

    Šauer, Pavel; Stará, Alžběta; Golovko, Oksana; Valentová, Olga; Bořík, Adam; Grabic, Roman; Kroupová, Hana Kocour

    2018-06-15

    Vast numbers of xenobiotics are known still to be present in treated municipal wastewater treatment plant (WWTP) effluents. Some of these possess endocrine-disrupting potency and pose risks for exposed aquatic animals. We searched for 17 potential environmental contaminants having affinity to the progesterone receptor. Relative potency values of these progesterone receptor-active chemicals were obtained. On the basis of relative potencies and measured environmental concentrations, the contribution of progestins to measured progestagenic activities was evaluated. Wastewaters (influent and effluent) and surrounding surface waters (upstream and downstream) at six municipal WWTPs were screened using instrumental chemical analysis and in vitro reporter gene bioassay. We showed the presence of target compounds and (anti-)progestagenic activities in municipal wastewater and surface water. Nine and seven progestins were identified in influent and effluent wastewaters, respectively. Only two compounds, progesterone and medroxyprogesterone were found in surface waters. Progestagenic agonistic activities in influents were partially masked by strong anti-progestagenic activities that were detected in all influents and ranged from 2.63 to 83 ng/L of mifepristone equivalents (EQs). Progestagenic activities were detected in all effluents and ranged from 0.06 to 0.47 ng/L of reference compound ORG 2058 EQs (a synthetic progestin equivalents), thus indicating incomplete removal of progestins during wastewater treatment processing. This activity poses a continuing risk for the aquatic environment. By contrast, anti-progestagenic activities showed better removal efficiency in WWTPs compared to progestagenic agonistic activities. Anti-progestagenic activities were found in only three of six effluents and ranged from 0.26 to 2.1 ng/L mifepristone EQs. We explained most of the progestagenic activity in municipal WWTP effluents by the presence of synthetic progestins and

  7. Protein tyrosine kinase and mitogen-activated protein kinase signalling pathways contribute to differences in heterophil-mediated innate immune responsiveness between two lines of broilers

    Science.gov (United States)

    Protein tyrosine phosphorylation mediates signal transduction of cellular processes, with protein tyrosine kinases (PTKs) regulating virtually all signaling events. The mitogen-activated protein kinase (MAPK) super-family consists of three conserved pathways that convert receptor activation into ce...

  8. Analog synthetic biology.

    Science.gov (United States)

    Sarpeshkar, R

    2014-03-28

    We analyse the pros and cons of analog versus digital computation in living cells. Our analysis is based on fundamental laws of noise in gene and protein expression, which set limits on the energy, time, space, molecular count and part-count resources needed to compute at a given level of precision. We conclude that analog computation is significantly more efficient in its use of resources than deterministic digital computation even at relatively high levels of precision in the cell. Based on this analysis, we conclude that synthetic biology must use analog, collective analog, probabilistic and hybrid analog-digital computational approaches; otherwise, even relatively simple synthetic computations in cells such as addition will exceed energy and molecular-count budgets. We present schematics for efficiently representing analog DNA-protein computation in cells. Analog electronic flow in subthreshold transistors and analog molecular flux in chemical reactions obey Boltzmann exponential laws of thermodynamics and are described by astoundingly similar logarithmic electrochemical potentials. Therefore, cytomorphic circuits can help to map circuit designs between electronic and biochemical domains. We review recent work that uses positive-feedback linearization circuits to architect wide-dynamic-range logarithmic analog computation in Escherichia coli using three transcription factors, nearly two orders of magnitude more efficient in parts than prior digital implementations.

  9. A combinatorial approach to synthetic transcription factor-promoter combinations for yeast strain engineering

    DEFF Research Database (Denmark)

    Dossani, Zain Y.; Apel, Amanda Reider; Szmidt-Middleton, Heather

    2018-01-01

    regions, we have built a library of hybrid promoters that are regulated by a synthetic transcription factor. The hybrid promoters consist of native S. cerevisiae promoters, in which the operator regions have been replaced with sequences that are recognized by the bacterial LexA DNA binding protein....... Correspondingly, the synthetic transcription factor (TF) consists of the DNA binding domain of the LexA protein, fused with the human estrogen binding domain and the viral activator domain, VP16. The resulting system with a bacterial DNA binding domain avoids the transcription of native S. cerevisiae genes...... levels, using the same synthetic TF and a given estradiol. This set of promoters, in combination with our synthetic TF, has the potential to regulate numerous genes or pathways simultaneously, to multiple desired levels, in a single strain....

  10. Toxoplasma gondii-derived synthetic peptides containing B- and T-cell epitopes from GRA2 protein are able to enhance mice survival in a model of experimental toxoplasmosis

    Directory of Open Access Journals (Sweden)

    Luciana Machado Bastos

    2016-06-01

    Full Text Available Toxoplasmosis is a zoonosis distributed all over the world, which the etiologic agent is an intracellular protozoan parasite, Toxoplasma gondii. This disease may cause abortions and severe diseases in many warm-blood hosts, including humans, particularly the immunocompromised patients. The parasite specialized secretory organelles, as micronemes, rhoptries and dense granules, are critical for the successful parasitism. The dense granule protein 2 (GRA2 is a parasite immunogenic protein secreted during infections and previous studies have been shown that this parasite component is crucial for the formation of intravacuolar membranous nanotubular network (MNN, as well as for secretion into the vacuole and spatial organization of the parasites within the vacuole. In the present study, we produced a monoclonal antibody to GRA2 (C3C5 mAb, isotype IgG2b, mapped the immunodominant epitope of the protein by phage display and built GRA2 synthetic epitopes to evaluate their ability to protect mice in a model of experimental infection. Our results showed that synthetic peptides for B- and T-cell epitopes are able to improve survival of immunized animals. In contrast with non-immunized animals, the immunized mice with both B- and T-cell epitopes had a better balance of cytokines and demonstrated higher levels of IL-10, IL-4 and IL-17 production, though similar levels of TNF-alpha and IL-6 were observed. The immunization with both B- and T-cell epitopes resulted in survival rate higher than 85% of the challenged mice. Overall, these results demonstrate that immunization with synthetic epitopes for both B- and T-cells from GRA2 protein can be more effective to protect against infection by T. gondii.

  11. Natural coagulation inhibitors and active protein c resistance in preeclampsia

    Directory of Open Access Journals (Sweden)

    Cengiz Demir

    2010-01-01

    Full Text Available INTRODUCTION: The etiology of preeclampsia is not fully established. A few studies have shown a relationship between natural coagulation inhibitors and preeclampsia. OBJECTIVES: The purpose of this study was to investigate the status of natural coagulation inhibitors and active protein C resistance (APC-R in preeclampsia. PATIENTS AND METHODS: We studied 70 women with preeclampsia recruited consecutively and 70 healthy pregnant and 70 nonpregnant women as controls. Plasma protein C (PC, free protein S (fPS, antithrombin III (ATIII and APC-R were evaluated. RESULTS: ATIII values were found to be significantly lower in preeclamptic patients than in the control groups (p< 0.001. Nevertheless, there was no significant difference between the healthy pregnant and nonpregnant women groups (p=0.141. The fPS values of the preeclamptic and healthy pregnant groups were lower than that of the nonpregnant group (p< 0.001, and the fPS value of the preeclamptic pregnant women was lower than that of healthy pregnant women (p<0.001. The PC value of the preeclamptic pregnant women was lower than that of the control groups (p< 0.001. The PC value of the healthy pregnant women was lower than that of the nonpregnant women (p< 0.001. The mean APC activity values were lower in the preeclamptic patients than that of the control groups (p< 0.001, p< 0.001. The APC-R positivity rates of the preeclamptic groups were higher than that of the control groups (p<0.001. CONCLUSIONS: This study demonstrated that ATIII, fPS, PC values and APC resistance were lower and APC-R positivity was higher in preeclamptic women than in normal pregnant and nonpregnant women.

  12. Stromal serine protein kinase activity in spinach chloroplasts

    International Nuclear Information System (INIS)

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

    1987-01-01

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

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

    Science.gov (United States)

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

    2016-08-01

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

  14. The Staphyloccous aureus Eap protein activates expression of proinflammatory cytokines.

    Science.gov (United States)

    Scriba, Thomas J; Sierro, Sophie; Brown, Eric L; Phillips, Rodney E; Sewell, Andrew K; Massey, Ruth C

    2008-05-01

    The extracellular adhesion protein (Eap) secreted by the major human pathogen Staphylococcus aureus is known to have several effects on human immunity. We have recently added to knowledge of these roles by demonstrating that Eap enhances interactions between major histocompatibility complex molecules and human leukocytes. Several studies have indicated that Eap can induce cytokine production by human peripheral blood mononuclear cells (PBMCs). To date, there has been no rigorous attempt to identify the breadth of cytokines produced by Eap stimulation or to identify the cell subsets that respond. Here, we demonstrate that Eap induces the secretion of the proinflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) by CD14(+) leukocytes (monocytes and macrophages) within direct ex vivo PBMC populations (note that granulocytes are also CD14(+) but are largely depleted from PBMC preparations). Anti-intercellular adhesion molecule 1 (CD54) antibodies inhibited this induction and implicated a role for this known Eap binding protein in cellular activation. IL-6 and TNF-alpha secretion by murine cells exposed to Eap was also observed. The activation of CD14(+) cells by Eap suggests that it could play a significant role in both septic shock and fever, two of the major pathological features of S. aureus infections.

  15. Indole and synthetic derivative activate chaperone expression to reduce polyQ aggregation in SCA17 neuronal cell and slice culture models

    Directory of Open Access Journals (Sweden)

    Kung PJ

    2014-10-01

    Full Text Available Pin-Jui Kung,1,* Yu-Chen Tao,1,* Ho-Chiang Hsu,1 Wan-Ling Chen,1 Te-Hsien Lin,1 Donala Janreddy,2 Ching-Fa Yao,2 Kuo-Hsuan Chang,3 Jung-Yaw Lin,1 Ming-Tsan Su,1 Chung-Hsin Wu,1 Guey-Jen Lee-Chen,1 Hsiu-Mei Hsieh-Li1 1Department of Life Science, 2Department of Chemistry, National Taiwan Normal University, Taipei, Taiwan; 3Department of Neurology, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taipei, Taiwan *These authors contributed equally to this work Abstract: In spinocerebellar ataxia type 17 (SCA17, the expansion of a translated CAG repeat in the TATA box binding protein (TBP gene results in a long polyglutamine (polyQ tract in the TBP protein, leading to intracellular accumulation of aggregated TBP and cell death. The molecular chaperones act in preventing protein aggregation to ameliorate downstream harmful events. In this study, we used Tet-On SH-SY5Y cells with inducible SCA17 TBP/Q79-green fluorescent protein (GFP expression to test indole and synthetic derivative NC001-8 for neuroprotection. We found that indole and NC001-8 up-regulated chaperone expression to reduce polyQ aggregation in neuronal differentiated TBP/Q79 cells. The effects on promoting neurite outgrowth and on reduction of aggregation on Purkinje cells were also confirmed with cerebellar primary and slice cultures of SCA17 transgenic mice. Our results demonstrate how indole and derivative NC001-8 reduce polyQ aggregation to support their therapeutic potentials in SCA17 treatment. Keywords: spinocerebellar ataxia type 17, TATA box binding protein, polyQ aggregation, indole and derivative, therapeutics

  16. Semi-synthetic preparation of 1-O-[1'-14C]hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) using plant cell cultures

    International Nuclear Information System (INIS)

    Weber, N.; Mangold, H.K.

    1985-01-01

    Incubation of photomixotrophic cell suspension cultures of rape (Brassica napus) and heterotrophic cell suspension cultures of soya (Glycine max) with 1-O-[1'- 14 C]hexadecyl-sn-glycerol or rac-1-O-[1'- 14 C]hexadecylglycerol leads in high yield (up to 78%) to labeled 1-O-hexadecyl-2-acyl-sn-glycero-3-phosphocholines. Alkaline hydrolysis of the choline glycerophospholipids yields pure 1-O-[1'- 14 C]hexadecyl-sn-glycero-3-phosphocholine. 1-O-[1'-14C]Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) is obtained by acetylating the lyso compound. The semi-synthetic preparation described leads to labeled platelet activating factor in an overall yield of 50-60% without loss of specific activity

  17. Synthetic approaches to uniform polymers.

    Science.gov (United States)

    Ali, Monzur; Brocchini, Steve

    2006-12-30

    Uniform polymers are characterised by a narrow molecular weight distribution (MWD). Uniformity is also defined by chemical structure in respect of (1) monomer orientation, sequence and stereo-regularity, (2) polymer shape and morphology and (3) chemical functionality. The function of natural polymers such as polypeptides and polynucleotides is related to their conformational structure (e.g. folded tertiary structure). This is only possible because of their high degree of uniformity. While completely uniform synthetic polymers are rare, polymers with broad structure and MWD are widely used in medicine and the biomedical sciences. They are integral components in final dosage forms, drug delivery systems (DDS) and in implantable devices. Increasingly uniform polymers are being used to develop more complex medicines (e.g. delivery of biopharmaceuticals, enhanced formulations or DDS's for existing actives). In addition to the function imparted by any new polymer it will be required to meet stringent specifications in terms of cost containment, scalability, biocompatibility and performance. Synthetic polymers with therapeutic activity are also being developed to exploit their polyvalent properties, which is not possible with low molecular weight molecules. There is need to utilise uniform polymers for applications where the polymer may interact with the systemic circulation, tissues or cellular environment. There are also potential applications (e.g. stimuli responsive coatings) where uniform polymers may be used for their more defined property profile. While it is not yet practical to prepare synthetic polymers to the same high degree of uniformity as proteins, nature also effectively utilises many polymers with lower degrees of uniformity (e.g. polysaccharides, poly(amino acids), polyhydroxyalkanoates). In recent years it has become possible to prepare with practical experimental protocols sufficient quantities of polymers that display many aspects of uniformity. This

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

    Directory of Open Access Journals (Sweden)

    Bravo Maria

    2012-05-01

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

  19. Post-Stroke Depression Modulation and in Vivo Antioxidant Activity of Gallic Acid and Its Synthetic Derivatives in a Murine Model System.

    Science.gov (United States)

    Nabavi, Seyed Fazel; Habtemariam, Solomon; Di Lorenzo, Arianna; Sureda, Antoni; Khanjani, Sedigheh; Nabavi, Seyed Mohammad; Daglia, Maria

    2016-04-28

    Gallic acid (3,4,5-trihydroxybenzoic acid, GA) is a plant secondary metabolite, which shows antioxidant activity and is commonly found in many plant-based foods and beverages. Recent evidence suggests that oxidative stress contributes to the development of many human chronic diseases, including cardiovascular and neurodegenerative pathologies, metabolic syndrome, type 2 diabetes and cancer. GA and its derivative, methyl-3-O-methyl gallate (M3OMG), possess physiological and pharmacological activities closely related to their antioxidant properties. This paper describes the antidepressive-like effects of intraperitoneal administration of GA and two synthetic analogues, M3OMG and P3OMG (propyl-3-O-methylgallate), in balb/c mice with post-stroke depression, a secondary form of depression that could be due to oxidative stress occurring during cerebral ischemia and the following reperfusion. Moreover, this study determined the in vivo antioxidant activity of these compounds through the evaluation of superoxide dismutase (SOD) and catalase (Cat) activity, thiobarbituric acid-reactive substances (TBARS) and reduced glutathione (GSH) levels in mouse brain. GA and its synthetic analogues were found to be active (at doses of 25 and 50 mg/kg) in the modulation of depressive symptoms and the reduction of oxidative stress, restoring normal behavior and, at least in part, antioxidant endogenous defenses, with M3OMG being the most active of these compounds. SOD, TBARS, and GSH all showed strong correlation with behavioral parameters, suggesting that oxidative stress is tightly linked to the pathological processes involved in stroke and PSD. As a whole, the obtained results show that the administration of GA, M3OMG and P3OMG induce a reduction in depressive symptoms and oxidative stress.

  20. Post-Stroke Depression Modulation and in Vivo Antioxidant Activity of Gallic Acid and Its Synthetic Derivatives in a Murine Model System

    Directory of Open Access Journals (Sweden)

    Seyed Fazel Nabavi

    2016-04-01

    Full Text Available Gallic acid (3,4,5-trihydroxybenzoic acid, GA is a plant secondary metabolite, which shows antioxidant activity and is commonly found in many plant-based foods and beverages. Recent evidence suggests that oxidative stress contributes to the development of many human chronic diseases, including cardiovascular and neurodegenerative pathologies, metabolic syndrome, type 2 diabetes and cancer. GA and its derivative, methyl-3-O-methyl gallate (M3OMG, possess physiological and pharmacological activities closely related to their antioxidant properties. This paper describes the antidepressive-like effects of intraperitoneal administration of GA and two synthetic analogues, M3OMG and P3OMG (propyl-3-O-methylgallate, in balb/c mice with post-stroke depression, a secondary form of depression that could be due to oxidative stress occurring during cerebral ischemia and the following reperfusion. Moreover, this study determined the in vivo antioxidant activity of these compounds through the evaluation of superoxide dismutase (SOD and catalase (Cat activity, thiobarbituric acid-reactive substances (TBARS and reduced glutathione (GSH levels in mouse brain. GA and its synthetic analogues were found to be active (at doses of 25 and 50 mg/kg in the modulation of depressive symptoms and the reduction of oxidative stress, restoring normal behavior and, at least in part, antioxidant endogenous defenses, with M3OMG being the most active of these compounds. SOD, TBARS, and GSH all showed strong correlation with behavioral parameters, suggesting that oxidative stress is tightly linked to the pathological processes involved in stroke and PSD. As a whole, the obtained results show that the administration of GA, M3OMG and P3OMG induce a reduction in depressive symptoms and oxidative stress.

  1. 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...

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

    Czech Academy of Sciences Publication Activity Database

    Procházka, Radek; Blaha, Milan

    2015-01-01

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

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

    International Nuclear Information System (INIS)

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

    2012-01-01

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

  4. 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.

  5. 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.

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

    Science.gov (United States)

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

    2012-12-01

    DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecX Hs) can interact with the H. 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.

  7. Protein kinase activity associated with the corticosteroid binder IB

    International Nuclear Information System (INIS)

    Vujicic, M.; Djordjevic-Markovic, R.; Radic, O.; Krstic, M.; Kanazir, D.

    1997-01-01

    The physiological effects elicited by glucocorticoids are mediated via glucocorticoid receptors (GR). Analysis of specific glucocorticoid binding to radioactively labelled [ 3 H] triamcinolone acetonide in rat liver cytosol and analysis by ion exchange chromatography have revealed the presence of two distinct molecular species. The major form, designated as binder II appears to correspond to the well characterized glucocorticoid receptor by virtue of its size, charge, steroid binding characteristics and ability to bind to DNA.The second form, designated as corticosteroid binder IB, is a minor binding component in the liver. The binder IB differs from the binder II receptor by virtue of its lower molecular weight and its elution in the pre gradient of DEAE-Sephadex A-50 column which retains the un activated binder II receptor complexes. We examined the kinase activity of partially purified corticosteroid binder IB. Using (γ 3 2 P) ATP we detected kinase activity associated with the IB fraction from the rat liver. This kinase phosphorylate mixed histones and and dose not phosphorylate IB protein in vitro. The kinase activity is completely inhibited by the addition of Mg 2 + ions and is partially inhibited by the addition of Ca 2 +ions. (author)

  8. A new approach to the modification of cell membrane glycosphingolipids: Ganglioside composition of JTC-12 P3 cells altered by feeding with galactose as a sole carbohydrate source in protein- and lipid-free synthetic medium

    International Nuclear Information System (INIS)

    Kawaguchi, Tatsuya; Takaoka, Toshiko; Yoshida, Eiko; Iwamori, Masao; Nagai, Yoshitaka; Takatsuki, Kiyoshi

    1988-01-01

    A significant difference in the glycosphingolipid composition of JTC-12 P3 cells established from monkey kidney tissue was observed when cells cultured in a protein- and lipid-free synthetic medium containing glucose (DM-160) as a sole carbohydrate source were transferred and cultured in the same medium containing galactose and pyruvic acid (DM-170) in place of glucose. In particular, the amounts of gangliosides GM3, GM2, and GD3 in the cells cultured in DM-170 were 5.3-, 17.8-, and more than 8-fold those in the cells cultured in DM-160, respectively, indicating that anabolism of gangliosides is greatly enhanced in cells cultured in the presence of galactose and pyruvic acid, as compared with cells cultured in the presence of glucose. In fact, after cultivation of cells in the medium with N-acetyl-D-[ 14 C]mannosamine for 96 h, the radioactivity incorporated into the gangliosides of the cells in DM-170 was 10-fold that of the cells in DM-160. Among the gangliosides of the cells in DM-170, highly sialylated molecules such as GD3, GD1a, GD1b, and GT1b were preferentially labeled, indicating that the sialytransferases responsible for the synthesis of gangliosides are significantly more activated in cells cultured in DM-170 than in DM-160. These observations reveal that the glycosphingolipid composition of the plasma membrane can be modified epigenetically under well-defined conditions and provide important clues for clarifying the roles of glycosphingolipids associated with particular cell functions

  9. Synthetic Biology and Personalized Medicine

    Science.gov (United States)

    Jain, K.K.

    2013-01-01

    Synthetic biology, application of synthetic chemistry to biology, is a broad term that covers the engineering of biological systems with structures and functions not found in nature to process information, manipulate chemicals, produce energy, maintain cell environment and enhance human health. Synthetic biology devices contribute not only to improve our understanding of disease mechanisms, but also provide novel diagnostic tools. Methods based on synthetic biology enable the design of novel strategies for the treatment of cancer, immune diseases metabolic disorders and infectious diseases as well as the production of cheap drugs. The potential of synthetic genome, using an expanded genetic code that is designed for specific drug synthesis as well as delivery and activation of the drug in vivo by a pathological signal, was already pointed out during a lecture delivered at Kuwait University in 2005. Of two approaches to synthetic biology, top-down and bottom-up, the latter is more relevant to the development of personalized medicines as it provides more flexibility in constructing a partially synthetic cell from basic building blocks for a desired task. PMID:22907209

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

    Directory of Open Access Journals (Sweden)

    Galo L. Mejia

    2016-07-01

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

  11. Adenosine monophosphate-activated protein kinase modulates the activated phenotype of hepatic stellate cells.

    Science.gov (United States)

    Caligiuri, Alessandra; Bertolani, Cristiana; Guerra, Cristina Tosti; Aleffi, Sara; Galastri, Sara; Trappoliere, Marco; Vizzutti, Francesco; Gelmini, Stefania; Laffi, Giacomo; Pinzani, Massimo; Marra, Fabio

    2008-02-01

    Adiponectin limits the development of liver fibrosis and activates adenosine monophosphate-activated protein kinase (AMPK). AMPK is a sensor of the cellular energy status, but its possible modulation of the fibrogenic properties of hepatic stellate cells (HSCs) has not been established. In this study, we investigated the role of AMPK activation in the biology of activated human HSCs. A time-dependent activation of AMPK was observed in response to a number of stimuli, including globular adiponectin, 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), or metformin. All these compounds significantly inhibited platelet-derived growth factor (PDGF)-stimulated proliferation and migration of human HSCs and reduced the secretion of monocyte chemoattractant protein-1. In addition, AICAR limited the secretion of type I procollagen. Knockdown of AMPK by gene silencing increased the mitogenic effects of PDGF, confirming the negative modulation exerted by this pathway on HSCs. AMPK activation did not reduce PDGF-dependent activation of extracellular signal-regulated kinase (ERK) or Akt at early time points, whereas a marked inhibition was observed 24 hours after addition of PDGF, reflecting a block in cell cycle progression. In contrast, AICAR blocked short-term phosphorylation of ribosomal S6 kinase (p70(S6K)) and 4E binding protein-1 (4EBP1), 2 downstream effectors of the mammalian target of rapamycin (mTOR) pathway, by PDGF. The ability of interleukin-a (IL-1) to activate nuclear factor kappa B (NF-kappaB) was also reduced by AICAR. Activation of AMPK negatively modulates the activated phenotype of HSCs.

  12. Synthetic Oligodeoxynucleotides Containing Multiple Telemeric TTAGGG Motifs Suppress Inflammasome Activity in Macrophages Subjected to Oxygen and Glucose Deprivation and Reduce Ischemic Brain Injury in Stroke-Prone Spontaneously Hypertensive Rats.

    Directory of Open Access Journals (Sweden)

    Jing Zhao

    Full Text Available The immune system plays a fundamental role in both the development and pathobiology of stroke. Inflammasomes are multiprotein complexes that have come to be recognized as critical players in the inflammation that ultimately contributes to stroke severity. Inflammasomes recognize microbial and host-derived danger signals and activate caspase-1, which in turn controls the production of the pro-inflammatory cytokine IL-1β. We have shown that A151, a synthetic oligodeoxynucleotide containing multiple telemeric TTAGGG motifs, reduces IL-1β production by activated bone marrow derived macrophages that have been subjected to oxygen-glucose deprivation and LPS stimulation. Further, we demonstrate that A151 reduces the maturation of caspase-1 and IL-1β, the levels of both the iNOS and NLRP3 proteins, and the depolarization of mitochondrial membrane potential within such cells. In addition, we have demonstrated that A151 reduces ischemic brain damage and NLRP3 mRNA levels in SHR-SP rats that have undergone permanent middle cerebral artery occlusion. These findings clearly suggest that the modulation of inflammasome activity via A151 may contribute to a reduction in pro-inflammatory cytokine production by macrophages subjected to conditions that model brain ischemia and modulate ischemic brain damage in an animal model of stroke. Therefore, modulation of ischemic pathobiology by A151 may have a role in the development of novel stroke prevention and therapeutic strategies.

  13. Mammalian protein secretion without signal peptide removal. Biosynthesis of plasminogen activator inhibitor-2 in U-937 cells

    International Nuclear Information System (INIS)

    Ye, R.D.; Wun, T.C.; Sadler, J.E.

    1988-01-01

    Plasminogen activator inhibitor-2 (PAI-2) is a serine protease inhibitor that regulates plasmin generation by inhibiting urokinase and tissue plasminogen activator. The primary structure of PAI-2 suggests that it may be secreted without cleavage of a single peptide. To confirm this hypothesis we have studied the glycosylation and secretion of PAI-2 in human monocytic U-937 cells by metabolic labeling, immunoprecipitation, glycosidase digestion, and protein sequencing. PAI-2 is variably glycosylated on asparagine residues to yield intracellular intermediates with zero, one, two, or three high mannose-type oligosaccharide units. Secretion of the N-glycosylated species began by 1 h of chase and the secreted molecules contained both complex-type N-linked and O-linked oligosaccharides. Enzymatically deglycosylated PAI-2 had an electrophoretic mobility identical to that of the nonglycosylated precursor and also to that of PAI-2 synthesized in vitro in a rabbit reticulocyte lysate from synthetic mRNA derived from full length PAI-2 cDNA. The amino-terminal protein sequence of secreted PAI-2 began with the initiator methionine residue. These results indicate that PAI-2 is glycosylated and secreted efficiently without the cleavage of a signal peptide. PAI-2 shares this property with its nearest homologue in the serine protease inhibitor family, chicken ovalbumin, and appears to be the first well characterized example of this phenomenon among natural mammalian proteins

  14. Synthetic Phage for Tissue Regeneration

    Directory of Open Access Journals (Sweden)

    So Young Yoo

    2014-01-01

    Full Text Available Controlling structural organization and signaling motif display is of great importance to design the functional tissue regenerating materials. Synthetic phage, genetically engineered M13 bacteriophage has been recently introduced as novel tissue regeneration materials to display a high density of cell-signaling peptides on their major coat proteins for tissue regeneration purposes. Structural advantages of their long-rod shape and monodispersity can be taken together to construct nanofibrous scaffolds which support cell proliferation and differentiation as well as direct orientation of their growth in two or three dimensions. This review demonstrated how functional synthetic phage is designed and subsequently utilized for tissue regeneration that offers potential cell therapy.

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

    Science.gov (United States)

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

    2017-08-01

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

  16. Selective functional activity measurement of a PEGylated protein with a modification-dependent activity assay.

    Science.gov (United States)

    Weber, Alfred; Engelmaier, Andrea; Mohr, Gabriele; Haindl, Sonja; Schwarz, Hans Peter; Turecek, Peter L

    2017-01-05

    BAX 855 (ADYNOVATE) is a PEGylated recombinant factor VIII (rFVIII) that showed prolonged circulatory half-life compared to unmodified rFVIII in hemophilic patients. Here, the development and validation of a novel assay is described that selectively measures the activity of BAX 855 as cofactor for the serine protease factor IX, which actives factor X. This method type, termed modification-dependent activity assay, is based on PEG-specific capture of BAX 855 by an anti-PEG IgG preparation, followed by a chromogenic FVIII activity assay. The assay principle enabled sensitive measurement of the FVIII cofactor activity of BAX 855 down to the pM-range without interference by non-PEGylated FVIII. The selectivity of the capture step, shown by competition studies to primarily target the terminal methoxy group of PEG, also allowed assessment of the intactness of the attached PEG chains. Altogether, the modification-dependent activity not only enriches, but complements the group of methods to selectively, accurately, and precisely measure a PEGylated drug in complex biological matrices. In contrast to all other methods described so far, it allows measurement of the biological activity of the PEGylated protein. Data obtained demonstrate that this new method principle can be extended to protein modifications other than PEGylation and to a variety of functional activity assays. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. 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.

  18. Functional relevance of G-protein-coupled-receptor-associated proteins, exemplified by receptor-activity-modifying proteins (RAMPs).

    Science.gov (United States)

    Fischer, J A; Muff, R; Born, W

    2002-08-01

    The calcitonin (CT) receptor (CTR) and the CTR-like receptor (CRLR) are close relatives within the type II family of G-protein-coupled receptors, demonstrating sequence identity of 50%. Unlike the interaction between CT and CTR, receptors for the related hormones and neuropeptides amylin, CT-gene-related peptide (CGRP) and adrenomedullin (AM) require one of three accessory receptor-activity-modifying proteins (RAMPs) for ligand recognition. An amylin/CGRP receptor is revealed when CTR is co-expressed with RAMP1. When complexed with RAMP3, CTR interacts with amylin alone. CRLR, initially classed as an orphan receptor, is a CGRP receptor when co-expressed with RAMP1. The same receptor is specific for AM in the presence of RAMP2. Together with human RAMP3, CRLR defines an AM receptor, and with mouse RAMP3 it is a low-affinity CGRP/AM receptor. CTR-RAMP1, antagonized preferentially by salmon CT-(8-32) and not by CGRP-(8-37), and CRLR-RAMP1, antagonized by CGRP-(8-37), are two CGRP receptor isotypes. Thus amylin and CGRP interact specifically with heterodimeric complexes between CTR and RAMP1 or RAMP3, and CGRP and AM interact with complexes between CRLR and RAMP1, RAMP2 or RAMP3.

  19. Activation of purified calcium channels by stoichiometric protein phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Nunoki, K.; Florio, V.; Catterall, W.A. (Univ. of Washington, Seattle (USA))

    1989-09-01

    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 {sup 45}Ca{sup 2+} uptake into vesicles containing reconstituted calcium channels were increased severalfold after incubation with ATP and PK-A. The degree of stimulation of {sup 45}Ca{sup 2+} 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, Cd{sup 2+}, Ni{sup 2+}, and Mg{sup 2+}. 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.

  20. Activation of purified calcium channels by stoichiometric protein phosphorylation

    International Nuclear Information System (INIS)

    Nunoki, K.; Florio, V.; Catterall, W.A.

    1989-01-01

    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 45 Ca 2+ uptake into vesicles containing reconstituted calcium channels were increased severalfold after incubation with ATP and PK-A. The degree of stimulation of 45 Ca 2+ 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, Cd 2+ , Ni 2+ , and Mg 2+ . 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

  1. Prion Protein Promotes Kidney Iron Uptake via Its Ferrireductase Activity*

    Science.gov (United States)

    Haldar, Swati; Tripathi, Ajai; Qian, Juan; Beserra, Amber; Suda, Srinivas; McElwee, Matthew; Turner, Jerrold; Hopfer, Ulrich; Singh, Neena

    2015-01-01

    Brain iron-dyshomeostasis is an important cause of neurotoxicity in prion disorders, a group of neurodegenerative conditions associated with the conversion of prion protein (PrPC) from its normal conformation to an aggregated, PrP-scrapie (PrPSc) isoform. Alteration of iron homeostasis is believed to result from impaired function of PrPC in neuronal iron uptake via its ferrireductase activity. However, unequivocal evidence supporting the ferrireductase activity of PrPC is lacking. Kidney provides a relevant model for this evaluation because PrPC is expressed in the kidney, and ∼370 μg of iron are reabsorbed daily from the glomerular filtrate by kidney proximal tubule cells (PT), requiring ferrireductase activity. Here, we report that PrPC promotes the uptake of transferrin (Tf) and non-Tf-bound iron (NTBI) by the kidney in vivo and mainly NTBI by PT cells in vitro. Thus, uptake of 59Fe administered by gastric gavage, intravenously, or intraperitoneally was significantly lower in PrP-knock-out (PrP−/−) mouse kidney relative to PrP+/+ controls. Selective in vivo radiolabeling of plasma NTBI with 59Fe revealed similar results. Expression of exogenous PrPC in immortalized PT cells showed localization on the plasma membrane and intracellular vesicles and increased transepithelial transport of 59Fe-NTBI and to a smaller extent 59Fe-Tf from the apical to the basolateral domain. Notably, the ferrireductase-deficient mutant of PrP (PrPΔ51–89) lacked this activity. Furthermore, excess NTBI and hemin caused aggregation of PrPC to a detergent-insoluble form, limiting iron uptake. Together, these observations suggest that PrPC promotes retrieval of iron from the glomerular filtrate via its ferrireductase activity and modulates kidney iron metabolism. PMID:25572394

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

    Science.gov (United States)

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

    2014-01-01

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

  3. Cardiac imaging in RASopathies/mitogen activated protein kinase syndromes

    Directory of Open Access Journals (Sweden)

    Rita Gravino

    2014-07-01

    Full Text Available RASopathies include a spectrum of disorders due to dysregulation of RAS/mitogen activated protein kinase pathway that plays an essential role in the control of the cell cycle and differentiation. As a consequence, its dysregulation has profound developmental consequences, in particular cardiac malformations. RASopathies with cardiac features are: Noonan syndrome, multiple lentigines syndrome, cardio-faciocutaneous syndrome, Costello syndrome, neurofibromatosis- 1, Legius syndrome, neurofibromatosis- Noonan syndrome. The former syndromes are associated with a high rate of cardiac involvement (60-85% and 12 genes: PTPN11, SOS1, RAF1, KRAS, HRAS, BRAF, MEK1/MAP2K1, MEK2/MAP2K2, NRAS, SHOC2, CBL and SPRED1. Although the majority of these diseases are readily distinguishable in clinical terms, an integrated imaging study of the cardiac condition associated to RASopathies helps to better define risk assessment, surveillance, and management of these patients.

  4. 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.

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

    KAUST Repository

    Altawashi, Azza

    2012-02-28

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

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

    International Nuclear Information System (INIS)

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

    2008-01-01

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

  7. The endothelial protein C receptor and activated protein C play a limited role in host defense during experimental tuberculosis

    NARCIS (Netherlands)

    Kager, Liesbeth M.; Roelofs, Joris J. T. H.; de Vos, Alex F.; Wieland, Catharina W.; Schouten, Marcel; Meijers, Joost C. M.; Isermann, Berend; van't Veer, Cornelis; Esmon, Charles T.; van der Poll, Tom

    2013-01-01

    The protein C (PC) system is an important regulator of both coagulation and inflammation. Activated PC (APC), together with its receptor the endothelial protein C receptor (EPCR), has anticoagulant and anti-inflammatory properties. During tuberculosis (TB), a devastating chronic pulmonary disease

  8. 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 w...

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

    Science.gov (United States)

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

    2009-02-24

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-10-10

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

  11. 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

  12. Bio-templated CdSe quantum dots green synthesis in the functional protein, lysozyme, and biological activity investigation

    International Nuclear Information System (INIS)

    Wang, Qisui; Li, Song; Liu, Peng; Min, Xinmin

    2012-01-01

    Bifunctional fluorescence (CdSe Quantum Dots) – protein (Lysozyme) nanocomposites were synthesized at room temperature by a protein-directed, solution-phase, green-synthetic method. Fluorescence (FL) and absorption spectra showed that CdSe QDs were prepared successfully with Lyz. The average particle size and crystalline structure of QDs were investigated by high-resolution transmission electron microscopy (HRTEM) and X-ray diffraction (XRD), respectively. With attenuated total reflection-fourier transform infrared (ATR-FTIR) spectra and thermogravimetric (TG) analysis, it was confirmed that there is interaction between QDs and amide I, amide II groups in Lyz. FL polarization was measured and FL imaging was done to monitor whether QDs could be responsible for possible changes in the conformation and activity of Lyz. Interestingly, the results showed Lyz still retain the biological activity after formation of QDs, but the secondary structure of the Lyz was changed. And the advantage of this synthesis method is producing excellent fluorescent QDs with specifically biological function. -- Highlights: ► Lysozyme-directed green synthesis of CdSe quantum dots. ► Lysozyme still retain the biological activity after formation of CdSe. ► The method is the production of fluorescent QDs with highly specific and functions.

  13. Impact of Transgenic Brassica napus Harboring the Antifungal Synthetic Chitinase (NiC Gene on Rhizosphere Microbial Diversity and Enzyme Activities

    Directory of Open Access Journals (Sweden)

    Mohammad S. Khan

    2017-07-01

    Full Text Available Transgenic Brassica napus harboring the synthetic chitinase (NiC gene exhibits broad-spectrum antifungal resistance. As the rhizosphere microorganisms play an important role in element cycling and nutrient transformation, therefore, biosafety assessment of NiC containing transgenic plants on soil ecosystem is a regulatory requirement. The current study is designed to evaluate the impact of NiC gene on the rhizosphere enzyme activities and microbial community structure. The transgenic lines with the synthetic chitinase gene (NiC showed resistance to Alternaria brassicicola, a common disease causing fungal pathogen. The rhizosphere enzyme analysis showed no significant difference in the activities of fivesoil enzymes: alkalyine phosphomonoestarase, arylsulphatase, β-glucosidase, urease and sucrase between the transgenic and non-transgenic lines of B. napus varieties, Durr-e-NIFA (DN and Abasyne-95 (AB-95. However, varietal differences were observed based on the analysis of molecular variance. Some individual enzymes were significantly different in the transgenic lines from those of non-transgenic but the results were not reproducible in the second trail and thus were considered as environmental effect. Genotypic diversity of soil microbes through 16S–23S rRNA intergenic spacer region amplification was conducted to evaluate the potential impact of the transgene. No significant diversity (4% for bacteria and 12% for fungal between soil microbes of NiC B. napus and the non-transgenic lines was found. However, significant varietal differences were observed between DN and AB-95 with 79% for bacterial and 54% for fungal diversity. We conclude that the NiC B. napus lines may not affect the microbial enzyme activities and community structure of the rhizosphere soil. Varietal differences might be responsible for minor changes in the tested parameters.

  14. Nitric oxide stress and activation of AMP-activated protein kinase impair β-cell sarcoendoplasmic reticulum calcium ATPase 2b activity and protein stability.

    Science.gov (United States)

    Tong, X; Kono, T; Evans-Molina, C

    2015-06-18

    The sarcoendoplasmic reticulum Ca(2+) ATPase 2b (SERCA2b) pump maintains a steep Ca(2+) concentration gradient between the cytosol and ER lumen in the pancreatic β-cell, and the integrity of this gradient has a central role in regulated insulin production and secretion, maintenance of ER function and β-cell survival. We have previously demonstrated loss of β-cell SERCA2b expression under diabetic conditions. To define the mechanisms underlying this, INS-1 cells and rat islets were treated with the proinflammatory cytokine interleukin-1β (IL-1β) combined with or without cycloheximide or actinomycin D. IL-1β treatment led to increased inducible nitric oxide synthase (iNOS) gene and protein expression, which occurred concurrently with the activation of AMP-activated protein kinase (AMPK). IL-1β led to decreased SERCA2b mRNA and protein expression, whereas time-course experiments revealed a reduction in protein half-life with no change in mRNA stability. Moreover, SERCA2b protein but not mRNA levels were rescued by treatment with the NOS inhibitor l-NMMA (NG-monomethyl L-arginine), whereas the NO donor SNAP (S-nitroso-N-acetyl-D,L-penicillamine) and the AMPK activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) recapitulated the effects of IL-1β on SERCA2b protein stability. Similarly, IL-1β-induced reductions in SERCA2b expression were rescued by pharmacological inhibition of AMPK with compound C or by transduction of a dominant-negative form of AMPK, whereas β-cell death was prevented in parallel. Finally, to determine a functional relationship between NO and AMPK signaling and SERCA2b activity, fura-2/AM (fura-2-acetoxymethylester) Ca(2+) imaging experiments were performed in INS-1 cells. Consistent with observed changes in SERCA2b expression, IL-1β, SNAP and AICAR increased cytosolic Ca(2+) and decreased ER Ca(2+) levels, suggesting congruent modulation of SERCA activity under these conditions. In aggregate, these results show that SERCA2b

  15. Multifaceted effects of synthetic TLR2 ligand and Legionella pneumophilia on Treg-mediated suppression of T cell activation

    Directory of Open Access Journals (Sweden)

    Sutmuller Roger PM

    2011-03-01

    Full Text Available Abstract Background Regulatory T cells (Treg play a crucial role in maintaining immune homeostasis and self-tolerance. The immune suppressive effects of Tregs should however be limited in case effective immunity is required against pathogens or cancer cells. We previously found that the Toll-like receptor 2 (TLR2 agonist, Pam3CysSK4, directly stimulated Tregs to expand and temporarily abrogate their suppressive capabilities. In this study, we evaluate the effect of Pam3CysSK4 and Legionella pneumophila, a natural TLR2 containing infectious agent, on effector T (Teff cells and dendritic cells (DCs individually and in co-cultures with Tregs. Results TLR2 agonists can directly provide a co-stimulatory signal inducing enhanced proliferation and cytokine production of naive CD4+ Teff cells. With respect to cytokine production, DCs appear to be most sensitive to low amounts of TLR agonists. Using wild type and TLR2-deficient cells in Treg suppression assays, we accordingly show that all cells (e.g. Treg, Teff cells and DCs contributed to overcome Treg-mediated suppression of Teff cell proliferation. Furthermore, while TLR2-stimulated Tregs readily lost their ability to suppress Teff cell proliferation, cytokine production by Teff cells was still suppressed. Similar results were obtained upon stimulation with TLR2 ligand containing bacteria, Legionella pneumophila. Conclusions These findings indicate that both synthetic and natural TLR2 agonists affect DCs, Teff cells and Treg directly, resulting in multi-modal modulation of Treg-mediated suppression of Teff cells. Moreover, Treg-mediated suppression of Teff cell proliferation is functionally distinct from suppression of cytokine secretion.

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

    African Journals Online (AJOL)

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

  17. A Synthetic Biology Framework for Programming Eukaryotic Transcription Functions

    Science.gov (United States)

    Khalil, Ahmad S.; Lu, Timothy K.; Bashor, Caleb J.; Ramirez, Cherie L.; Pyenson, Nora C.; Joung, J. Keith; Collins, James J.

    2013-01-01

    SUMMARY Eukaryotic transcription factors (TFs) perform complex and combinatorial functions within transcriptional networks. Here, we present a synthetic framework for systematically constructing eukaryotic transcription functions using artificial zinc fingers, modular DNA-binding domains found within many eukaryotic TFs. Utilizing this platform, we construct a library of orthogonal synthetic transcription factors (sTFs) and use these to wire synthetic transcriptional circuits in yeast. We engineer complex functions, such as tunable output strength and transcriptional cooperativity, by rationally adjusting a decomposed set of key component properties, e.g., DNA specificity, affinity, promoter design, protein-protein interactions. We show that subtle perturbations to these properties can transform an individual sTF between distinct roles (activator, cooperative factor, inhibitory factor) within a transcriptional complex, thus drastically altering the signal processing behavior of multi-input systems. This platform provides new genetic components for synthetic biology and enables bottom-up approaches to understanding the design principles of eukaryotic transcriptional complexes and networks. PMID:22863014

  18. Synthetic (+)-antroquinonol exhibits dual actions against insulin resistance by triggering AMP kinase and inhibiting dipeptidyl peptidase IV activities.

    Science.gov (United States)

    Hsu, C Y; Sulake, R S; Huang, P-K; Shih, H-Y; Sie, H-W; Lai, Y-K; Chen, C; Weng, C F

    2015-01-01

    The fungal product (+)-antroquinonol activates AMP kinase (AMPK) activity in cancer cell lines. The present study was conducted to examine whether chemically synthesized (+)-antroquinonol exhibited beneficial metabolic effects in insulin-resistant states by activating AMPK and inhibiting dipeptidyl peptidase IV (DPP IV) activity. Effects of (+)-antroquinonol on DPP IV activity were measured with a DPPIV Assay Kit and effects on GLP-1-induced PKA were measured in AR42J cells. Translocation of the glucose transporter 4, GLUT4, induced either by insulin-dependent PI3K/AKT signalling or by insulin-independent AMPK activation, was assayed in differentiated myotubes. Glucose uptake and GLUT4 translocation were assayed in L6 myocytes. Mice with diet-induced obesity were used to assess effects of acute and chronic treatment with (+)-antroquinonol on glycaemic control in vivo. The results showed that of (+)-antroquinonol (100 μM ) inhibited the DPP IV activity as effectively as the clinically used inhibitor, sitagliptin. The phosphorylation of AMPK Thr(172) in differentiated myotubes was significantly increased by (+)-antroquinonol. In cells simultaneously treated with S961 (insulin receptor antagonist), insulin and (+)-antroquinonol, the combination of (+)-antroquinonol plus insulin still increased both GLUT4 translocation and glucose uptake. Further, (+)-antroquinonol and sitagliptin reduced blood glucose, when given acutely or chronically to DIO mice. Chemically synthesized (+)-antroquinonol exhibits dual effects to ameliorate insulin resistance, by increasing AMPK activity and GLUT4 translocation, along with inhibiting DPP IV activity. © 2014 The British Pharmacological Society.

  19. Calcium-Oxidant Signaling Network Regulates AMP-activated Protein Kinase (AMPK) Activation upon Matrix Deprivation*

    Science.gov (United States)

    Sundararaman, Ananthalakshmy; Amirtham, Usha; Rangarajan, Annapoorni

    2016-01-01

    The AMP-activated protein kinase (AMPK) has recently been implicated in anoikis resistance. However, the molecular mechanisms that activate AMPK upon matrix detachment remain unexplored. In this study, we show that AMPK activation is a rapid and sustained phenomenon upon matrix deprivation, whereas re-attachment to the matrix leads to its dephosphorylation and inactivation. Because matrix detachment leads to loss of integrin signaling, we investigated whether integrin signaling negatively regulates AMPK activation. However, modulation of focal adhesion kinase or Src, the major downstream components of integrin signaling, failed to cause a corresponding change in AMPK signaling. Further investigations revealed that the upstream AMPK kinases liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) contribute to AMPK