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Auswahl der wissenschaftlichen Literatur zum Thema „Proteins Synthesis“
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Zeitschriftenartikel zum Thema "Proteins Synthesis"
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Nilsson, Bradley L., Matthew B. Soellner und Ronald T. Raines. „Chemical Synthesis of Proteins“. Annual Review of Biophysics and Biomolecular Structure 34, Nr. 1 (Juni 2005): 91–118. http://dx.doi.org/10.1146/annurev.biophys.34.040204.144700.
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Gibney, Brian R., Francesc Rabanal und P. Leslie Dutton. „Synthesis of novel proteins“. Current Opinion in Chemical Biology 1, Nr. 4 (Dezember 1997): 537–42. http://dx.doi.org/10.1016/s1367-5931(97)80050-6.
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Mejuch, Tom, und Herbert Waldmann. „Synthesis of Lipidated Proteins“. Bioconjugate Chemistry 27, Nr. 8 (August 2016): 1771–83. http://dx.doi.org/10.1021/acs.bioconjchem.6b00261.
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Borgia, Jeffrey A., und Gregg B. Fields. „Chemical synthesis of proteins“. Trends in Biotechnology 18, Nr. 6 (Juni 2000): 243–51. http://dx.doi.org/10.1016/s0167-7799(00)01445-1.
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Hilvert, Donald. „Chemical synthesis of proteins“. Chemistry & Biology 1, Nr. 4 (Dezember 1994): 201–3. http://dx.doi.org/10.1016/1074-5521(94)90011-6.
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Plocinski, P., N. Arora, K. Sarva, E. Blaszczyk, H. Qin, N. Das, R. Plocinska et al. „Mycobacterium tuberculosis CwsA Interacts with CrgA and Wag31, and the CrgA-CwsA Complex Is Involved in Peptidoglycan Synthesis and Cell Shape Determination“. Journal of Bacteriology 194, Nr. 23 (21.09.2012): 6398–409. http://dx.doi.org/10.1128/jb.01005-12.
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ABSTRACTBacterial cell division and cell wall synthesis are highly coordinated processes involving multiple proteins. Here, we show that Rv0008c, a novel small membrane protein fromMycobacterium tuberculosis, localizes to the poles and on membranes and shows an overall punctate localization throughout the cell. Furthermore, Rv0008c interacts with two proteins, CrgA and Wag31, implicated in peptidoglycan (PG) synthesis in mycobacteria. Deletion of the Rv0008c homolog inM. smegmatis, MSMEG_0023, caused bulged cell poles, formation of rounded cells, and defects in polar localization of Wag31 and cell wall synthesis, with cell wall synthesis measured by the incorporation of the [14C]N-acetylglucosamine cell wall precursor. TheM. smegmatisMSMEG_0023crgAdouble mutant strain showed severe defects in growth, viability, cell wall synthesis, cell shape, and the localization of the FtsZ, FtsI, and Wag31 proteins. The double mutant strain also exhibited increased autolytic activity in the presence of detergents. Because CrgA and Wag31 proteins interact with FtsI individually, we believe that regulated cell wall synthesis and cell shape maintenance require the concerted actions of the CrgA, Rv0008c, FtsI, and Wag31 proteins. We propose that, together, CrgA and Rv0008c, renamed CwsA forcellwall synthesis and cellshape proteinA, play crucial roles in septal and polar PG synthesis and help coordinate these processes with the FtsZ-ring assembly in mycobacteria.
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Kohen, Amnon, Priyanka Singh und Qi Guo. „Chemoenzymatic Synthesis of Ubiquitous Biological Redox Cofactors“. Synlett 28, Nr. 10 (10.04.2017): 1151–59. http://dx.doi.org/10.1055/s-0036-1588768.
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Redox cofactors are utilized by a myriad of proteins, ranging from metabolic enzymes to those performing post-translational modifications. Labeled redox cofactors have served as a vital tool for a broad range of studies. This account describes chemoenzymatic syntheses of the isotopically labeled, biologically important redox cofactors: nicotinamide adenine dinucleotide, methylene tetrahydrofolate, and flavin nucleotides. An overview of the general strategy is presented. These examples demonstrate the utility of enzymatic synthesis.1 Introduction2 Nicotinamide Cofactors2.1 Synthesis of Remote-Labeled 14C-NADPH2.1.1 Synthesis of [Ad-14C]NADPH2.1.2 Synthesis of [Carbonyl-14C]NADPH2.2 Synthesis of S- and R-[4-3H]NADPH2.2.1 One-Step S- and Three-Step R-[4-3H]NADPH Synthesis2.2.2 One-Pot, One-Step R-[4-3H]NADPH Synthesis2.3 Synthesis of S- and R-[Ad-14C, 4-2H]NADPH2.3.1 One-Step S-, Three-Step R-[Ad-14C, 4-2H]NADPH Synthesis2.3.2 One-Pot, One-Step R-[Ad-14C, 4-2H]NADPH Synthesis3 Methylene Tetrahydrofolate4 Flavin Nucleotides5 Conclusions and Outlook
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Metanis, Norman, Reem Mousa und Post Reddy. „Chemical Protein Synthesis through Selenocysteine Chemistry“. Synlett 28, Nr. 12 (21.03.2017): 1389–93. http://dx.doi.org/10.1055/s-0036-1588762.
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Methods for the preparation of small-to-medium-sized proteins by chemical protein synthesis have matured in recent years and proven valuable for protein science. Thanks to the many recent discoveries and developments in the field, proteins up to 300 amino acids can now be prepared in the lab in a matter of days. This technology gives the scientists the flexibility to substitute any atom in the protein sequence; hence synthesis is not constrained to the 20 canonical amino acids. In this Synpacts article we briefly highlight the recent studies on selenocysteine chemistry in the field of chemical protein synthesis.1 Introduction2 Selenocysteine in Nature and in Folding Studies3 Selenocysteine in Protein Synthesis4 Selenocysteine in Natural Selenoproteins5 Outlook
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Whyte, Lyle G., und William E. Inniss. „Cold shock proteins and cold acclimation proteins in a psychrotrophic bacterium“. Canadian Journal of Microbiology 38, Nr. 12 (01.12.1992): 1281–85. http://dx.doi.org/10.1139/m92-211.
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The synthesis of proteins in the psychrotrophic bacterium Bacillus psychrophilus in response to both cold shock and continuous growth at low temperatures was examined. Cold shocks of 20 to 0, 5, or 10 °C resulted in the induction of nine, seven, and five cold shock proteins, respectively, as determined by 2-dimensional gel electrophoresis and computing scanning laser densitometry. Two cold shock proteins, with molecular masses of 61 and 34 kDa, which were induced in B. psychrophilus by cold shocks of 20 to 0 or 5 °C, were not induced in a cold-sensitive mutant of B. psychrophilus. Analysis of protein profiles of B. psychrophilus during continuous growth at 0, 5, or 10 °C revealed the synthesis of 11, 10, and 4 cold acclimation proteins, respectively. Some of these cold acclimation proteins were similar to cold shock proteins. In addition, the relative synthesis of both cold shock proteins and cold acclimation proteins increased with decreasing temperature. Thus, both types of proteins increased both in number and relative synthesis in response to cold shock and continuous growth at low temperature. Key words: cold shock proteins, cold acclimation proteins, psychrotrophic bacterium.
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Kent, Stephen B. H. „Total chemical synthesis of proteins“. Chem. Soc. Rev. 38, Nr. 2 (2009): 338–51. http://dx.doi.org/10.1039/b700141j.
Der volle Inhalt der QuelleDissertationen zum Thema "Proteins Synthesis"
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Baas, Tracey Lynn. „The design, synthesis, and characterization of template assembled synthetic proteins /“. Thesis, Connect to this title online; UW restricted, 2000. http://hdl.handle.net/1773/11561.
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Draffan, Lynda Catherine. „Chemical synthesis of proteins“. Thesis, University of Edinburgh, 1996. http://hdl.handle.net/1842/13715.
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Morton, Gail Helen. „The chemical synthesis of proteins“. Thesis, University of Edinburgh, 1997. http://hdl.handle.net/1842/15437.
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The viability of extending the present methodology designed for the solid phase synthesis of peptides has been investigated. Using the catalytic domain of stromelysin (SCD. 173 residues) as the model system, a number of different factors affecting the preparation and purification of chemically synthesised proteins are examined. The purification SCD, prepared using stepwise solid phase synthesis is described. Following characterisation, it is evident that protein of the correct primary sequence has been prepared, furthermore, preliminary studies centred on the enzymatic activity of SCD indicate that active protease has been isolated. However, comparison of the conformational and biophysical properties of chemically synthesised SCD with the recombinant counterpart, suggests that there are problems associated with the folding of the synthetic SCD. The construction of chemically synthesised SCD via convergent protein synthesis is also described. Two different coupling strategies involving classical and azide fragment condensation are examined where it has been highlighted that the overall success of each fragment coupling is greatly dependent on the peptide length and sequence. As well as comparing methods for the preparation and coupling of fully and minimally protected peptides, general procedures for both solution and solid phase fragment coupling are discussed. A novel strategy for the convergent synthesis of peptides and proteins has been investigated. In this total chemical synthesis, two minimally protected peptides are joined through unique, mutually reactive functional groups, yielding a peptide analogue with a thioether replacement for the native peptide bond at the site of ligation. A general route to C-terminal sulfhydryl and N-terminal haloacetylated peptides is presented, accompanied with results of the preliminary ligation studies.
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Walker, Douglas Gordon. „Characterization of immediate-early and early proteins of murine cytomegalovirus synthesized in permissive and nonpermissive cells“. Thesis, University of British Columbia, 1985. http://hdl.handle.net/2429/25985.
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The gene products produced by murine cytomegalovirus (MCMV) in infected cells prior to viral DNA synthesis are believed to control the interaction of the virus with the cells, determining whether a permissive infection results, with virus replication, or whether further virus gene expression is inhibited, resulting in a latent or abortive infection. The aim of this study was to characterize the early viral gene products that are produced in permissive and nonpermissive cells.
The proteins produced in 3T3-L1 cells, permissively infected with MCMV, during the first six hours of infection (the period prior to viral DNA replication) were characterized by polyacrylamide gel electrophoresis. Ten of the proteins were classified as immediate-early (IE) and seven as early according to their time of synthesis and also according to their synthesis in the presence of actinomycin D following the reversal of a cycloheximide mediated block in protein synthesis. The estimated molecular weights ranged from 28K - 100K. The synthesis of a dominant IE protein of 100K was significantly increased, after the reversal of a cycloheximide block, compared to unenhanced conditions. The synthesis of two other major IE proteins of 96K and 89K were also significantly enhanced by this treatment. The 100K and 89K proteins partitioned with the nuclear, cytoplasmic and cytoskeletal fractions, while the 96K protein partitioned more strongly with the nuclei. These proteins were phosphorylated. The other IE proteins were synthesized in lesser amounts. The major early proteins, which had molecular weights of 39K and 36K, were also phosphorylated and were exclusively nucleus-associated. A number of the IE and early proteins had affinity for native and denatured DNA-cellulose.
The same major IE and early proteins were identified in nonpermissively infected J774A.1 macrophage cells. Although 0.6% of these cells became permissively infected with MCMV and the rest appeared to be nonpermissively infected, viral DNA and late protein synthesis was not detected. The major difference between the proteins produced in 3T3-L1 cells and J774A.1 cells was the affinity of the 96K protein for denatured DNA-cellulose, which was only observed when the protein was synthesized in J774A.1 cells.
The main IE and early MCMV induced proteins were also synthesized in nonpermissively infected human fibroblast cells. The only difference between the proteins produced in these cells and 3T3-L1 cells was that the 100K IE protein appeared to have a greater nuclear-affinity, when produced in the human fibroblasts, than was found when synthesized in infected 3T3-L1 cells.
In conclusion, a larger number of IE and early MCMV-induced proteins were identified in infected cells than had been previously characterized. There was no evidence of restricted MCMV gene expression occurring in two different cell types that were nonpermissively infected. This appeared to indicate that, in the nonpermissive experiments described, MCMV replication was inhibited at the stage of viral DNA synthesis.Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
Graduate
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Scott, Felicia Yi Xia. „Controlled Hybrid Material Synthesis using Synthetic Biology“. Diss., Virginia Tech, 2017. http://hdl.handle.net/10919/86147.
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The concept of creating a hybrid material is motivated by the development of an improved product with acquired properties by amalgamation of components with specific desirable traits. These new attributes can range from improvements upon existing properties, such as strength and durability, to the acquisition of new abilities, such as magnetism and conductivity. Currently, the concept of an organic-inorganic hybrid material typically describes the integration of an inorganic polymer with organically derived proteins. By building on this idea and applying the advanced technologies available today, it is possible to combine living and nonliving components to synthesize functional materials possessing unique abilities of living cells such as self-healing, evolvability, and adaptability. Furthermore, artificial gene regulation, achievable through synthetic biology, allows for an additional dimension of the control of hybrid material function.
Here, I genetically engineer E. coli with a tightly controlled artificial protein construct, allowing for inducible expression of different amounts of the surface anchored protein by addition of varying concentrations of L-arabinose. The presence of the surface protein allows the cells to bind nonliving nanoparticle substrates, effectively turning the cells into living crosslinkers. By using the living crosslinker, I was able to successfully synthesize a robust, macroscale living-nonliving hybrid material with magnetic characteristics. Furthermore, by varying the particle size and inducer concentration, the resulting material exhibited alterations in structure and function. Finally, I was able to manipulate material kinetics within a PDMS channel by applying fluctuating magnetic fields and demonstrate material durability. These results demonstrate the ability to manipulate synthesis of living-nonliving hybrid materials, which demonstrate the potential for use in promising applications in areas such as environmental monitoring and micromachining. Additionally, this work serves as a foundational step toward the integration of synthetic biology with tissue engineering by exploiting the possibility of controlling material properties with genetic engineering.Ph. D.
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Schwartz, Anne. „Characterization of normal and androgen resistant-genital skin fibroblasts using high-resolution two-dimensional gel electrophoresis“. Thesis, McGill University, 1985. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=63378.
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Zhang, Yinfeng, und 张银凤. „Protein chemical synthesis by serine and threonine ligation“. Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2014. http://hdl.handle.net/10722/202359.
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Landmark advances in the field of synthetic protein chemistry have enabled the preparation of complex, homogeneous proteins, including those that carry specific posttranslational modifications (PTMs). In addition, chemical synthesis will allow one to incorporate unnatural elements to generate new biologics with altered properties and functions. Native chemical ligation (NCL) is a milestone in the chemical synthesis of proteins (Kent et al., Science, 1994, 266, 776-779), in which a C-terminal peptide thioester and an N-terminal cysteine (Cys)-containing peptide-both in side-chain unprotected forms-are selectively coupled to generate a natural peptidic linkage at the site of ligation. This method requires a cysteine at the optimal convergent ligation site. However, Cys is one of the least abundant amino acids in natural proteins. Therefore, the development of new ligation methods at other amino acids will be necessary and important in this regard.
Along these lines, our laboratory has developed a novel thiol-independent approach-serine/threonine ligation (STL). It uses the N-terminal serine or threonine of a peptide segment to chemoselectively react with another peptide segment with a C-terminal salicylaldehyde ester to form an N,O-benzylidene acetal linked product, followed by acidolysis to afford the final product at the natural Ser/Thr site. To extend the application of STL in chemical protein synthesis, we have developed a robust method for the preparation of peptide salicylaldehyde esters via Fmoc-based solid phase peptide synthesis. Furthermore, we have successfully applied this ligation method in the convergent synthesis of peptide drugs of significant therapeutic importance, including Teriparatide (Forteo), Corticorelin (oCRH), Exenatide (Byetta) and Tesamorelin (hGHRH). Of significance, we have demonstrated the effectiveness of our STL in the assembly of a more complex target of biological interest: human erythrocyte acylphosphatase (~ 11 kDa).
In summary, we have developed a new serine/threonine ligation, which can be effectively used to synthesize peptides and proteins. As there are countless serine and threonine residues in natural proteins, particularly those carrying posttranslational modifications, this method is anticipated to offer new opportunities in synthetic protein chemistry and chemical biology.published_or_final_version
Chemistry
Doctoral
Doctor of Philosophy
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Johnston, Julie Catherine. „In vitro translation of cucumber necrosis virus RNA“. Thesis, University of British Columbia, 1989. http://hdl.handle.net/2429/28969.
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The in vitro translation products directed by cucumber necrosis virus (CNV) RNA were analyzed in both rabbit reticulocyte lysate and wheat germ extract cell-free translation systems. In rabbit reticulocyte lysates, one major protein of ca. 33 Mr was produced. In wheat germ extracts, four proteins of ca. 41, 33, 21 and 20 Mr were produced. Hybrid-arrested translation (HART) studies using synthetic CNV antisense RNA corresponding to the entire CNV genome demonstrated that the four major proteins synthesized from CNV virion RNA in wheat germ extracts are virus-specific translation products. The genomic locations of the CNV in vitro translation products were determined using a number of experimental approaches including: (1) HART using antisense RNA corresponding to selected regions of the CNV genome; (2) in vitro translation of synthetic messenger-sense CNV transcripts; (3) immunoprecipitation of in vitro translation products with CNV polyclonal antisera and (4) in vitro translation of size-fractionated CNV virion RNA. Together, these experiments demonstrated that the ca. 33 Mr protein is derived from the 5' proximal coding region, the ca. 41 Mr protein is derived from an internal coding region, and that at least one but probably both of the ca. 20 and 21 Mr proteins are derived from the 3' terminal coding region(s) of the CNV genome. In addition, immunoprecipitation experiments provided further evidence that the ca. 41 Mr protein is the viral coat protein. The size, number, and genomic locations of the CNV in vitro translation products reported here are in agreement with those predicted from nucleotide sequence data (Rochon & Tremaine, 1989). The natural template for the expression of downstream cistrons in the CNV genome was investigated by in vitro translation of sucrose fractionated CNV virion RNA as well as in vitro translation of messenger-sense synthetic transcripts. These studies indicate that in vitro, both subgenomic and genomic-length CNV RNA molecules may act as templates for the synthesis of the ca. 41,21 and 20 Mr proteins as well as the ca. 33 Mr protein.Land and Food Systems, Faculty of
Graduate
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Sun, Xiaojiao. „High Affinity Synthetic Molecular Binders for Proteins : Design, Synthesis and Evaluation“. Doctoral thesis, Uppsala universitet, Fysikalisk-organisk kemi, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-183203.
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This thesis describes the design and synthesis of small molecule derivatives and their polypeptide conjugates as high affinity binders for proteins: the D-dimer protein (D-dimer), a biomarker for diagnosis of thromboembolic diseases; human myeloperoxidase (MPO), a biomarker for cardiovascular diseases; and chitinases, potential targets for asthma therapy. The interactions between the synthetic binder molecules and those proteins were evaluated by surface plasmon resonance (SPR) biosensor analysis and fluorescence spectroscopy. Competition SPR experiments or other methods proved that the small molecule components of the binder molecules were critical for binding and specifically bound to the original binding site of small molecules. The binder molecules consisted of a 42-residue helix-loop-helix polypeptide conjugated to a small molecule via aliphatic spacers of suitable length. The small molecules could be any type of moderately binding structure. In the binder development for the D-dimer, the tetrapeptide GPRP with a dissociation constant Kd of 25 μM was used and the affinity of 4C15L8GPRP obtained was estimated to be approximately 3 nM. In the binder development for MPO, salicylhydroxamic acid (SHA) with Kd of 2 μM was used and the affinity of 4C37L34C11SHA obtained was estimated to be approximately 0.4 nM. In the binder development for chitinases, a theobromine derivative (pentoxifylline) with a Kd of 43±10 μM was used and the affinity of 4C37L34-P obtained was estimated to be considerably higher than that of pentoxifylline. The binder molecules were identified from a 16-membered pool of candidates obtained by conjugating the small molecules to each member of a set of 16 designed polypeptides. The affinities were greatly enhanced by 2-3 orders of magnitude, compared to the small molecule. The polypeptides did not bind to the proteins with measurable affinities. The discovery of these new synthetic binders for protein targets can pave the way to diagnostic tests in vivo or in vitro, independent of antibodies.
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Kim, Daniel. „Characterization of the Mata pre-“. Scholarly Commons, 2009. https://scholarlycommons.pacific.edu/uop_etds/738.
Der volle Inhalt der QuelleBücher zum Thema "Proteins Synthesis"
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E, Esterhouse Toma, und Petrinos Lado B, Hrsg. Protein biosynthesis. New York: Nova Science, 2008.
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1958-, Martin Robin, Hrsg. Protein synthesis: Methods and protocols. Totowa, N.J: Humana Press, 1998.
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Fernando, Albericio, und Giralt Ernest, Hrsg. Chemical approaches to the synthesis of peptides and proteins. Boca Raton: CRC Press, 1997.
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A, Wallace Carmichael J., Hrsg. Protein engineering by semisynthesis. Boca Raton, Fla: CRC Press, 2000.
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S, Spirin A., und Swartz James R, Hrsg. Cell-free protein synthesis: Methods and protocols. Weinheim: Wiley-VCH, 2008.
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G, Spedding, Hrsg. Ribosomes and protein synthesis: A practical approach. Oxford [England]: IRL Press at Oxford University Press, 1990.
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L, Hatfield Dolph, Lee Byeong J und Pirtle Robert M, Hrsg. Transfer RNA in protein synthesis. Boca Raton: CRC Press, 1992.
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1939-, Bermek E., Hrsg. Mechanisms of protein synthesis: Structure-function relations, control mechanisms, and evolutionary aspects : Proceedings of the Symposium on Molecular Mechanisms in Protein Synthesis held at Beyaz Köşk, Emigran, Bosphorus, Istanbul. Berlin: Springer-Verlag, 1985.
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Arnold, Revzin, Hrsg. The Biology of nonspecific DNA-protein interactions. Boca Raton, Fla: CRC Press, 1990.
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Hengge-Aronis, Regine. Studies of secretion of periplasmic proteins in Escherichia coli. Konstanz: Hartung-Gorre, 1986.
Den vollen Inhalt der Quelle findenBuchteile zum Thema "Proteins Synthesis"
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Buxbaum, Engelbert. „Synthesis of Peptides“. In Biophysical Chemistry of Proteins, 289–90. Boston, MA: Springer US, 2010. http://dx.doi.org/10.1007/978-1-4419-7251-4_31.
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Kermode, Allison R., und J. Derek Bewley. „Synthesis, processing and deposition of seed proteins: The pathway of protein synthesis and deposition in the cell“. In Seed Proteins, 807–41. Dordrecht: Springer Netherlands, 1999. http://dx.doi.org/10.1007/978-94-011-4431-5_34.
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Houghten, Richard A., Sarah R. Hoffmann, Mairead K. Bray, Nicole Frizzell, J. M. Ostresh, Suzanne M. Pratt und John Sitarik. „Simultaneous Multiple Peptide Synthesis: The Rapid Preparation of Large Numbers of Peptides“. In Proteins, 463–70. Boston, MA: Springer US, 1987. http://dx.doi.org/10.1007/978-1-4613-1787-6_46.
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Rivier, J., R. Galyean, W. Woo, D. Karr, T. Richmond und J. Spiess. „The Symposium Test Peptide (STP): Synthesis and Characterization of a Model Peptide“. In Proteins, 789–95. Boston, MA: Springer US, 1987. http://dx.doi.org/10.1007/978-1-4613-1787-6_81.
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Liu, Lei, Sam Danishefsky und David Crich. „Chemical Synthesis of Proteins“. In Organic Chemistry - Breakthroughs and Perspectives, 221–45. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co. KGaA, 2012. http://dx.doi.org/10.1002/9783527664801.ch6.
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Frigerio, Lorenzo, und Lynne M. Roberts. „The Synthesis of Ricinus communis Lectins“. In Toxic Plant Proteins, 191–205. Berlin, Heidelberg: Springer Berlin Heidelberg, 2010. http://dx.doi.org/10.1007/978-3-642-12176-0_10.
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Buntru, Matthias, Simon Vogel, Ricarda Finnern und Stefan Schillberg. „Plant-Based Cell-Free Transcription and Translation of Recombinant Proteins“. In Recombinant Proteins in Plants, 113–24. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2241-4_8.
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AbstractPlant cell-free lysates contain all the cellular components of the protein biosynthesis machinery, providing an alternative to intact plant cells, tissues, and whole plants for the production of recombinant proteins. Cell-free lysates achieve rapid protein production (within hours or days) and allow the synthesis of proteins that are cytotoxic or unstable in living cells. The open nature of cell-free lysates and their homogeneous and reproducible performance is ideal for protein production, especially for screening applications, allowing the direct addition of nucleic acid templates encoding proteins of interest, as well as other components such as enzyme substrates, chaperones, artificial amino acids, or labeling molecules. Here we describe procedures for the production of recombinant proteins in the ALiCE (Almost Living Cell-free Expression) system, a lysate derived from tobacco cell suspension cultures that can be used to manufacture protein products for molecular and biochemical analysis as well as applications in the pharmaceutical industry.
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Tanoue, Eiichiro. „Proteins in the Sea — Synthesis“. In Dynamics and Characterization of Marine Organic Matter, 383–463. Dordrecht: Springer Netherlands, 2000. http://dx.doi.org/10.1007/978-94-017-1319-1_18.
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Fernández-Tejada, Alberto, John Brailsford, Qiang Zhang, Jae-Hung Shieh, Malcolm A. S. Moore und Samuel J. Danishefsky. „Total Synthesis of Glycosylated Proteins“. In Protein Ligation and Total Synthesis I, 1–26. Cham: Springer International Publishing, 2014. http://dx.doi.org/10.1007/128_2014_622.
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Halvorson, Harlyn O. „The Induced Synthesis of Proteins“. In Advances in Enzymology - and Related Areas of Molecular Biology, 99–156. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2006. http://dx.doi.org/10.1002/9780470122679.ch3.
Der volle Inhalt der QuelleKonferenzberichte zum Thema "Proteins Synthesis"
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Terwilliger, Thomas C., und Joel Berendzen. „Protein Crystallography: From X-ray diffraction spots to a three-dimensional image“. In Signal Recovery and Synthesis. Washington, D.C.: Optica Publishing Group, 1998. http://dx.doi.org/10.1364/srs.1998.swa.1.
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Proteins are remarkable molecular machines that are essential for life. They can do many things ranging from the precise control of blood clotting to synthesizing complex organic compounds. Pictures of protein molecules are in high demand in biotechnology because they are important for applications such as drug discovery and for engineering enzymes for commercial use.
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Jackson, C. W., N. K. Hutson und S. A. Steward. „CHANGES IN PROTEIN SYNTHESIS PROFILES OF MEGAKARYOCYTES (MK) DURING MATURATION“. In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643545.
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Several key differentiation events occur within the recognizable MK compartment; however, little is known about the macromolecular changes responsible for these events. In this study, protein synthesis profiles of morphologically immature and mature guinea pig MK populations have been analyzed by twodimensional gel electrophoresis after in vivo labeling with 35S-methionine. MK were enriched by a bovine plasma aggregation enrichment procedure (Blood 69:173, 1987) and then fractionated into immature and mature populations based on differences in their respective buoyant densities (Brit. J. Haematol. 64:33, 1986). With this protocol, immature and mature MK populations were obtained in which MK constituted 95% of the cell mass. Ninety percent of the MK in the immature population had basophilic, immature morphology while ≥90% of those in the mature population had acidophilic, mature staining characteristics after Wright's staining. Protease inhibitors were used throughout the isolation procedure. The cells were solubilized and proteins subjected to two-dimensional electrophoresis according to O'Farrell (J. Biol. Chem. 250:4007, 1975). To examine basic proteins, proteins were electrophoresed in the first dimension under nonequilibrium conditions in a pH gradient as described by O'Farrell et al. (Cell 12:1133, 1977). Analyses of fluorograms revealed both qualitative and quantitative differences in synthesis profiles between these two MK populations. Among acidic proteins whose synthesis was readily detected in immature but not mature MK were ones whose MW and pi were respectively: 120K, 6.4; 7OK, 5.9; 70K, 6.9; 65K, 6.8; 55K, 6.2; 55K, 6.0; 53K, 5.8; 53K, 6.5; 52K, 6.7; 50K, 6.8; 41K, 5.5 and 33K, 6.7. Acidic and neutral proteins prominently synthesized in mature but not immature MK were found at MW and PI of: 110K, 5.7; 110K, 5.8 and 80K, 7.2. Basic proteins prominently synthesized in immature but not mature MK were found at MWs of: 110K; 70K; 52K; 48K; 39K and 18K. Basic proteins actively synthesized by mature but not immature MK had MWs of: 83K; 43K and 17K. These findings demonstrate that differences in protein synthesis patterns can be readily detected between immature and mature MK and provide baseline data with which to explore the role of these proteins in MK differentiation
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Kornilov, F. D., E. A. Ananiev, E. V. Vasilieva, K. S. Mineev, S. A. Goncharuk und M. V. Goncharuk. „CO-TRANSLATIONAL FOLDING OF MEMBRANE PROTEINS DURING CELL FREE SYNTHESIS“. In X Международная конференция молодых ученых: биоинформатиков, биотехнологов, биофизиков, вирусологов и молекулярных биологов — 2023. Novosibirsk State University, 2023. http://dx.doi.org/10.25205/978-5-4437-1526-1-337.
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The biochemical obtaining of natively folded membrane proteins (MPs) is an extremely important task of modern structural biology. We have studied the efficiency of co-translational incorporation of MPs into various membrane-mimicking media — liposomes, micelles, and bicelles during the cell-free synthesis. The retinal protein of Exiguobacterium sibiricum (ESR) was chosen as an object of study.
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Garcia, A., G. Liman, K. Fluke, S. Davidson, T. Santangelo, G. Chadwick und P. Welander. „Identification of H-Gdgt Synthesis Proteins“. In IMOG 2023. European Association of Geoscientists & Engineers, 2023. http://dx.doi.org/10.3997/2214-4609.202333298.
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Mattern-Schain, Samuel I., Mary-Anne Nguyen, Tayler M. Schimel, James Manuel, Joshua Maraj, Donald Leo, Eric Freeman, Scott Lenaghan und Stephen A. Sarles. „Totipotent Cellularly-Inspired Materials“. In ASME 2019 Conference on Smart Materials, Adaptive Structures and Intelligent Systems. American Society of Mechanical Engineers, 2019. http://dx.doi.org/10.1115/smasis2019-5745.
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Abstract This work draws inspiration from totipotent cellular systems to design smart materials whose compositions and properties can be learned or evolved. Totipotency refers to the inherent genetic potential of a single cell to adapt and produce all types of differentiated cells within an organism. To study this principal and apply it synthetically, tissue-like compartmentalized assemblies are constructed via lipid membrane-separated aqueous droplets in a hydrophobic medium through the droplet interface bilayer (DIB) method. Within our droplets, we explore synthetic totipotency via cell-free reactions including actin polymerization and cell free protein synthesis (CFPS). The transcription and translation of our CFPS reactions are controlled by stimuli-responsive riboswitches (RS). Via this scheme, adaptable material properties and functions are achieved in vitro via protein production from cell-free machinery administered through RS governance. Here, we present thermally or chemically-triggered riboswitches for orthogonal production of representative fluorescent protein products, as well functional proteins. To characterize the material properties of target proteins, we study the formation of polymerized actin shells to stabilize organically-encased droplets and span DIBs. We present a modified protocol for chemically-triggered actin polymerization as well as a thermally triggered actin RS. We characterize theophylline (TP)-triggered production of alpha hemolysin (α-HL) through CFPS and synthesized an organic-soluble trigger that can be sensed from the oil phase by a RS in an aqueous bioreactor droplet. We also demonstrate increased droplet conductivity when CFPS α-HL products are incorporated in DIBs. This interdisciplinary work involves cell culture, gene expression, organic synthesis, vesicle formation, protein quantification, tensiometry, droplet aspiration, microplate fluorescence/absorption experiments, fluorescent microscopy, and electrophysiology. This project is an essential design analysis for creating smart, soft materials using synthetic biology and provides motivation for artificial tissues capable of adapting in response to external stimuli.
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Agard, David A., Yashushi Hiraoka und John W. Sedat. „Three-dimensional Optical Microscopy of Biological Specimens“. In Signal Recovery and Synthesis. Washington, D.C.: Optica Publishing Group, 1986. http://dx.doi.org/10.1364/srs.1986.thd1.
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The ability to analyze biological specimens in three dimensions represents one of the major achievements of modern structural biology. For all but the simplest repeating structures, three-dimensional analysis is a crucial prerequisite for understanding complex biological assemblies. Near atomic resolution analysis of of crystalline proteins, nucleic acids, and viruses by X-ray crystallography approaches the routine. The current frontier focuses on the three dimensional analysis of non-crystalline, non-symmetric biological structures of cellular dimension. Electron microscope tomography and three dimensional optical microscopy are perhaps the most powerful methods for these studies.
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Rao, Jiajia. „Tuning plant protein for improved functionality and flavor profile: From field to application“. In 2022 AOCS Annual Meeting & Expo. American Oil Chemists' Society (AOCS), 2022. http://dx.doi.org/10.21748/xqxj4886.
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Recently, plant proteins are gaining in popularity as consumers are looking to rebalance their diet with more plant-based options. As such, there is a need to understand the potential of these plant proteins to deliver nutrition and functionality in various food products. However, plant proteins are still under-utilization in food and beverage industry due to their characteristic beany and off flavor, lower technical functionality, such as limited solubility, and some negative taste attributes. In general, plant protein can be obtained from pulses, cereals and oilseeds. Each plant has a unique protein structure and composition, thus protein extracted from different plant has different functional properties and performance in food. In general, plant contain non-protein components including starch, fiber and oil. High purity of plant protein are often extracted to by removing all abovementioned non-protein components through extraction processing. One would expect different extraction methods can influence protein functionality through denaturation, modification, hydrolysis and cross-linking. This talk begins with the discussion of how different plant variety and processing impact on protein functionality using hemp protein as an example. Selective physical and chemical modification methods (e.g., soluble complexes, pH shift, maillard reaction, phosphorylation) for generating novel protein constructs with preferable solubility, foaming, and emulsification properties, thermal stability, and flavor profiles will be comprehensively discussed by using pea protein isolate (PPI) as a representative. In general, protein solubility could be improved by means of abovementioned three methods. Maillard-driven synthesis of the cross-linked PPI-gum arabic conjugates greatly improved the flavor profile and functionality of PPI. In terms of phosphorylation, sodium hexametaphosphate (SHMP) is a good candidate to form phosphorylated PPI with enhanced functionalities including foaming, emulsifying properties and thermal stability. Our results suggested that protein structure-function researches are valuable in tailoring proteins for specific functional outcomes and expanding the availability of plant proteins.
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Waart, P. v. d., K. T. Preissner, U. Delvos und G. Müller-Berghaus. „SIMULTANEOUS PRODUCTION OF ENDOTHELIAL CELL-DERIVED PROTEIN S AND FACTOR V, AND INACTIVATION OF FACTOR Va AT THE ENDOTHELIAL CELL SURFACE“. In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644737.
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Several proteins synthesized and expressed by endothelial cells are involved in the regulation of coagulation. The synthesis and expression of factor V and protein S has been demonstrated in independent studies. The present work evaluates the simultaneous synthesis and expression of bovine factor V and protein S and the effect of endothelial protein S on the inactivation of endothelial factor Va by activated protein C. The accumulation of both proteins in conditioned medium was detected by SDS-PAGE followed by immunoblotting, and their activities were tested by functional assays. The synthesis of protein S and factor V per 105 cells over 24 h amounted up to 2 ng protein S and 440 ng factor V, respectively. The addition of thrombin did not increase the yield of synthesized cofactors. Thrombin did neither proteolyse protein S on endothelial cells nor in a purified system in the presence of thrombomodulin and calcium ions. Factor V was secreted partly in its activated form as evidenced by the appearance of active intermediates with M = 220,000-280,000 on immunoblots as well as by only a three-Fold further activation of factor V/Va following addition of thrombin. The rate constant for the inactivation of factor Va by activated protein C was only two-fold higher for factor Va derived from cells cultured in the presence of vitamin K as compared in the presence of warfarin. For the inactivation of comparable factor Va concentrations in conditioned medium a 10-fold higher and on endothelial cells a 40-fold higher concentration of activated protein C was required to obtain similar inactivation rates of factor Va as compared to a purified system. These results suggest that resting endothelial cells contain a factor V activator, and that a regulatory mechanism is operative on the endothelial cell surface that suppresses the inactivation potential of activated protein C/ protein S.
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TAM, James P. „Design and Synthesis of Peptide Biologics by Deconstruction of Proteins“. In The Twenty-Third American and the Sixth International Peptide Symposium. Prompt Scientific Publishing, 2013. http://dx.doi.org/10.17952/23aps.2013.001.
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Macmillan, Derek, und Alison M. Daines. „SEMI-SYNTHESIS OF GLYCOSYLATED PROTEINS AND MIMICS USING CHEMOSELECTIVE LIGATIONS“. In XXIst International Carbohydrate Symposium 2002. TheScientificWorld Ltd, 2002. http://dx.doi.org/10.1100/tsw.2002.438.
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Desai, Holly E. Synthesis and Structural Characterization of Reflectin Proteins. Fort Belvoir, VA: Defense Technical Information Center, Februar 2012. http://dx.doi.org/10.21236/ada563720.
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Singh, Anjali. Amino Acids: Building Blocks of Proteins. ConductScience, Juni 2022. http://dx.doi.org/10.55157/cs20220612.
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Amino acids are essential organic compounds serving as protein building blocks. Recognized for their biological roles, they underpin proteins' structure and interactions. Classified by polarity and nutritional necessity, essential amino acids, not synthesized by the body, include histidine, leucine, lysine, and more, while non-essential ones are produced internally. These molecules exhibit diverse functions, from neurotransmitter precursor synthesis to immune support. Industries leverage amino acids in animal feed, artificial sweeteners, flavor enhancers, and drug manufacturing, highlighting their vital role in various applications beyond biological systems.
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Barash, Itamar, und Robert E. Rhoads. Translational Mechanisms that Govern Milk Protein Levels and Composition. United States Department of Agriculture, November 2004. http://dx.doi.org/10.32747/2004.7586474.bard.
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Original objectives: The long term objective of the project is to achieve higher content of protein in the milk of ruminants by modulating the translational machinery in the mammary gland. The first specific aim of the BARD proposal was to characterize responsiveness of various experimental systems to combination of lactogenic hormones and amino acids with particular emphasis on discrimination between the control of total protein synthesis and milk protein synthesis. Based on the results, we planned to proceed by characterizing the stage of protein synthesis in which the stimulation by lactogenic hormones and amino acid occur and finally we proposed to identify which components of the translation machinery are modified. Background to the topic: Milk protein is the most valuable component in milk, both for direct human consumption and for manufacturing cheese and other protein-based products. Attempts to augment protein content by the traditional methods of genetic selection and improved nutritional regimes have failed. The proposal was based on recent results suggesting that the limiting factor for augmenting protein synthesis in the bovine mammary gland is the efficiency of converting amino acids to milk proteins. Major conclusions, solutions, achievements: Insulin and prolactin synergistically stimulate â-casein mRNA translation by cytoplasmatic polyadenylation. The interaction between insulin and prolactin was demonstrated two decades ago as crucial for milk-protein synthesis, but the molecular mechanisms involved were not elucidated. We found in differentiated CID 9 mouse mammary epithelial cells line that insulin and prolactin synergistically increases the rate of milk protein mRNA translation. We focused on â-casein, the major milk protein, and found that the increase in â-casein mRNA translation was reflected in a shift to larger polysomes, indicating an effect on translational initiation. Inhibitors of the PI3K, mTOR, and MAPK pathways blocked insulin-stimulated total protein and â-casein synthesis but not the synergistic stimulation. Conversely, cordycepin, a polyadenylation inhibitor, abolished synergistic stimulation of protein synthesis without affecting insulin-stimulated translation. The poly(A) tract of â-casein mRNA progressively increased over 30 min of treatment with insulin plus prolactin. The 3’-untranslated region of â-casein mRNA was found to contain a cytoplasmic polyadenylation element (CPE), and in reporter constructs, this was sufficient for the translational enhancement and mRNA-specific polyadenylation. Furthermore, insulin and prolactin stimulated phosphorylation of cytoplasmic polyadenylation element binding protein (CPEB) but did not increase cytoplasmic polyadenylation.
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Barash, Itamar, und Robert Rhoads. Translational Mechanisms Governing Milk Protein Levels and Composition. United States Department of Agriculture, 2006. http://dx.doi.org/10.32747/2006.7696526.bard.
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Original objectives: The long-term goal of the research is to achieve higher protein content in the milk of ruminants by modulating the translational apparatus of the mammary gland genetically, nutritionally, or pharmacologically. The short-term objectives are to obtain a better understanding of 1) the role of amino acids (AA) as regulators of translation in bovine and mouse mammary epithelial cells and 2) the mechanism responsible for the synergistic enhancement of milk-protein mRNA polyadenylation by insulin and prolactin. Background of the topic: In many cell types and tissues, individual AA affect a signaling pathway which parallels the insulin pathway to modulate rates and levels of protein synthesis. Diverse nutritional and hormonal conditions are funneled to mTOR, a multidomain serine/threonine kinase that regulates a number of components in the initiation and elongation stages of translation. The mechanism by which AA signal mTOR is largely unknown. During the current grant period, we have studied the effect of essential AA on mechanisms involved in protein synthesis in differentiated mammary epithelial cells cultured under lactogenic conditions. We also studied lactogenic hormone regulation of milk protein synthesis in differentiated mammary epithelial cells. In the first BARD grant (2000-03), we discovered a novel mechanism for mRNA-specific hormone-regulated translation, namely, that the combination of insulin plus prolactin causes cytoplasmic polyadenylation of milk protein mRNAs, which leads to their efficient translation. In the current BARD grant, we have pursued the signaling pathways of this novel hormone action. Major conclusions/solutions/achievements: The positive and negative signaling from AA to the mTOR pathway, combined with modulation of insulin sensitization, mediates the synthesis rates of total and specific milk proteins in mammary epithelial cells. The current in vitro study revealed cryptic negative effects of Lys, His, and Thr on cellular mechanisms regulating translation initiation and protein synthesis in mammary epithelial cells that could not be detected by conventional in vivo analyses. We also showed that a signaling pathway involving Jak2 and Stat5, previously shown to lead from the prolactin receptor to transcription of milk protein genes, is also used for cytoplasmic polyadenylation of milk protein mRNAs, thereby stabilizing these mRNAs and activating them for translation. Implications: In vivo, plasma AA levels are affected by nutritional and hormonal effects as well as by conditions of exercise and stress. The amplitude in plasma AA levels resembles that applied in the current in vitro study. Thus, by changing plasma AA levels in the epithelial cell microenvironment or by sensitizing the mTOR pathway to their presence, it should be possible to modulate the rate of milk protein synthesis. Furthermore, knowledge that phosphorylation of Stat5 is required for enhanced milk protein synthesis in response to lactogenic opens the possibility for pharmacologic approaches to increase the phosphorylation of Stat5 and, thereby, milk protein production.
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Christopher, David A., und Avihai Danon. Plant Adaptation to Light Stress: Genetic Regulatory Mechanisms. United States Department of Agriculture, Mai 2004. http://dx.doi.org/10.32747/2004.7586534.bard.
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Original Objectives: 1. Purify and biochemically characterize RB60 orthologs in higher plant chloroplasts; 2. Clone the gene(s) encoding plant RB60 orthologs and determine their structure and expression; 3. Manipulate the expression of RB60; 4. Assay the effects of altered RB60 expression on thylakoid biogenesis and photosynthetic function in plants exposed to different light conditions. In addition, we also examined the gene structure and expression of RB60 orthologs in the non-vascular plant, Physcomitrella patens and cloned the poly(A)-binding protein orthologue (43 kDa RB47-like protein). This protein is believed to a partner that interacts with RB60 to bind to the psbA5' UTR. Thus, to obtain a comprehensive view of RB60 function requires analysis of its biochemical partners such as RB43. Background & Achievements: High levels of sunlight reduce photosynthesis in plants by damaging the photo system II reaction center (PSII) subunits, such as D1 (encoded by the chloroplast tpsbAgene). When the rate of D1 synthesis is less than the rate of photo damage, photo inhibition occurs and plant growth is decreased. Plants use light-activated translation and enhanced psbAmRNA stability to maintain D1 synthesis and replace the photo damaged 01. Despite the importance to photosynthetic capacity, these mechanisms are poorly understood in plants. One intriguing model derived from the algal chloroplast system, Chlamydomonas, implicates the role of three proteins (RB60, RB47, RB38) that bind to the psbAmRNA 5' untranslated leader (5' UTR) in the light to activate translation or enhance mRNA stability. RB60 is the key enzyme, protein D1sulfide isomerase (Pill), that regulates the psbA-RN :Binding proteins (RB's) by way of light-mediated redox potentials generated by the photosystems. However, proteins with these functions have not been described from higher plants. We provided compelling evidence for the existence of RB60, RB47 and RB38 orthologs in the vascular plant, Arabidopsis. Using gel mobility shift, Rnase protection and UV-crosslinking assays, we have shown that a dithiol redox mechanism which resembles a Pill (RB60) activity regulates the interaction of 43- and 30-kDa proteins with a thermolabile stem-loop in the 5' UTR of the psbAmRNA from Arabidopsis. We discovered, in Arabidopsis, the PD1 gene family consists of II members that differ in polypeptide length from 361 to 566 amino acids, presence of signal peptides, KDEL motifs, and the number and positions of thioredoxin domains. PD1's catalyze the reversible formation an disomerization of disulfide bonds necessary for the proper folding, assembly, activity, and secretion of numerous enzymes and structural proteins. PD1's have also evolved novel cellular redox functions, as single enzymes and as subunits of protein complexes in organelles. We provide evidence that at least one Pill is localized to the chloroplast. We have used PDI-specific polyclonal and monoclonal antisera to characterize the PD1 (55 kDa) in the chloroplast that is unevenly distributed between the stroma and pellet (containing membranes, DNA, polysomes, starch), being three-fold more abundant in the pellet phase. PD1-55 levels increase with light intensity and it assembles into a high molecular weight complex of ~230 kDa as determined on native blue gels. In vitro translation of all 11 different Pill's followed by microsomal membrane processing reactions were used to differentiate among PD1's localized in the endoplasmic reticulum or other organelles. These results will provide.1e insights into redox regulatory mechanisms involved in adaptation of the photosynthetic apparatus to light stress. Elucidating the genetic mechanisms and factors regulating chloroplast photosynthetic genes is important for developing strategies to improve photosynthetic efficiency, crop productivity and adaptation to high light environments.
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Lapidot, Moshe, und Vitaly Citovsky. molecular mechanism for the Tomato yellow leaf curl virus resistance at the ty-5 locus. United States Department of Agriculture, Januar 2016. http://dx.doi.org/10.32747/2016.7604274.bard.
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Tomato yellow leaf curl virus (TYLCV) is a major pathogen of tomato that causes extensive crop loss worldwide, including the US and Israel. Genetic resistance in the host plant is considered highly effective in the defense against viral infection in the field. Thus, the best way to reduce yield losses due to TYLCV is by breeding tomatoes resistant or tolerant to the virus. To date, only six major TYLCV-resistance loci, termed Ty-1 to Ty-6, have been characterized and mapped to the tomato genome. Among tomato TYLCV-resistant lines containing these loci, we have identified a major recessive quantitative trait locus (QTL) that was mapped to chromosome 4 and designated ty-5. Recently, we identified the gene responsible for the TYLCV resistance at the ty-5 locus as the tomato homolog of the gene encoding messenger RNA surveillance factor Pelota (Pelo). A single amino acid change in the protein is responsible for the resistant phenotype. Pelo is known to participate in the ribosome-recycling phase of protein biosynthesis. Our hypothesis was that the resistant allele of Pelo is a “loss-of-function” mutant, and inhibits or slows-down ribosome recycling. This will negatively affect viral (as well as host-plant) protein synthesis, which may result in slower infection progression. Hence we have proposed the following research objectives: Aim 1: The effect of Pelota on translation of TYLCV proteins: The goal of this objective is to test the effect Pelota may or may not have upon translation of TYLCV proteins following infection of a resistant host. Aim 2: Identify and characterize Pelota cellular localization and interaction with TYLCV proteins: The goal of this objective is to characterize the cellular localization of both Pelota alleles, the TYLCV-resistant and the susceptible allele, to see whether this localization changes following TYLCV infection, and to find out which TYLCV protein interacts with Pelota. Our results demonstrate that upon TYLCV-infection the resistant allele of pelota has a negative effect on viral replication and RNA transcription. It is also shown that pelota interacts with the viral C1 protein, which is the only viral protein essential for TYLCV replication. Following subcellular localization of C1 and Pelota it was found that both protein localize to the same subcellular compartments. This research is innovative and potentially transformative because the role of Peloin plant virus resistance is novel, and understanding its mechanism will lay the foundation for designing new antiviral protection strategies that target translation of viral proteins. BARD Report - Project 4953 Page 2
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Grafi, Gideon, und Brian Larkins. Endoreduplication in Maize Endosperm: An Approach for Increasing Crop Productivity. United States Department of Agriculture, September 2000. http://dx.doi.org/10.32747/2000.7575285.bard.
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The focus of this research project is to investigate the role of endoreduplication in maize endosperm development and the extent to which this process contributes to high levels of starch and storage protein synthesis. Although endoreduplication has been widely observed in many cells and tissues, especially those with high levels of metabolic activity, the molecular mechanisms through which the cell cycle is altered to produce consecutive cycles of S-phase without an intervening M-phase are unknown. Our previous research has shown that changes in the expression of several cell cycle regulatory genes coincide with the onset of endoreduplication. During this process, there is a sharp reduction in the activity of the mitotic cyclin-dependent kinase (CDK) and activation of the S-phase CDK. It appears the M-phase CDK is stable, but its activity is blocked by a proteinaceous inhibitor. Coincidentally, the S-phase checkpoint protein, retinoblastoma (ZmRb), becomes phosphorylated, presumably releasing an E2F-type transcriptional regulator which promotes the expression of genes responsible for DNA synthesis. To investigate the role of these cell cycle proteins in endoreduplication, we have created transgenic maize plants that express various genes in an endosperm-specific manner using a storage protein (g-zein) promoter. During the first year of the grant, we constructed point mutations of the maize M-phase kinase, p34cdc2. One alteration replaced aspartic acid at position 146 with asparagine (p3630-CdcD146N), while another changed threonine 161 to alanine (p3630-CdcT161A). These mutations abolish the activity of the CDK. We hypothesized that expression of the mutant forms of p34cdc2 in endoreduplicating endosperm, compared to a control p34cdc2, would lead to extra cycles of DNA synthesis. We also fused the gene encoding the regulatory subunit of the M- phase kinase, cyclin B, under the g-zein promoter. Normally, cyclin B is expected to be destroyed prior to the onset of endoreduplication. By producing high levels of this protein in developing endosperm, we hypothesized that the M-phase would be extended, potentially reducing the number of cycles of endoreduplication. Finally, we genetically engineered the wheat dwarf virus RepA protein for endosperm-specific expression. RepA binds to the maize retinoblastoma protein and presumably releases E2F-like transcription factors that activate DNA synthesis. We anticipated that inactivation of ZmRb by RepA would lead to additional cycles of DNA synthesis.
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Amir, Rachel, David J. Oliver, Gad Galili und Jacline V. Shanks. The Role of Cysteine Partitioning into Glutathione and Methionine Synthesis During Normal and Stress Conditions. United States Department of Agriculture, Januar 2013. http://dx.doi.org/10.32747/2013.7699850.bard.
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The objective of this research is to study the nature of the competition for cysteine (Cys), the first organic sulfur-containing compound, between its two main metabolites, glutathione (GSH) and methionine (Met). GSH plays a central role in protecting plants during various stresses, while Met, an essential amino acid, regulates essential processes and metabolites in plant cells through its metabolite S-adenosyl-Met. Our results, which are based on flux analysis and measurements of Met- metabolites, show that the flux towards Met synthesis is high during non-stress conditions, however the flux is significantly reduced under stress conditions, when there is high synthesis of GSH. Under oxidative stress the expression level of the regulatory enzyme of Met synthesis, cystathionine g-synthase (CGS) was reduced. By using three different systems, we have found that that GSH down regulates the expression level of CGS, thus reducing Met synthesis. We have found that this regulation occurs at the post-transcriptional level, and further studies have shown that it occurs at post-translationaly. To reveal how oxidative stress affects the flux towards Met and GSH, flux analysis was performed. We have found that the level of Met is significantly reduced, while the level of glutathione significantly increases during stress. Under stress conditions most of the glutathione is converted from GSH to GSSG (the oxidised form of glutathione). These results suggest that under normal growth conditions, Cys is channelled towards both pathways to support GSH accumulation and the synthesis of growth-essential Met metabolites. However, during oxidative stress, when a high level of GSH is required to protect the plants, the levels of GSH increase while those of CGS are reduced. This reduction leaves more Cys available for GSH synthesis under stress conditions. In addition we have also studied the effects of high GSH level on the transcriptome profile. The analysis revealed that GSH affects the expression level of many major genes coding to enzymes or proteins associated with photosynthesis, starch degradation, hormone metabolism (especially genes associated with jasmonate), biotic stress (especially genes associated with PR-proteins), cytochrome P450 genes, regulation of transcription and signaling (especially genes associated with receptor kinases and calcium). These results suggest that indeed GSH levels affect different pathways and metabolites in plants.
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Wang, X. F., und M. Schuldiner. Systems biology approaches to dissect virus-host interactions to develop crops with broad-spectrum virus resistance. Israel: United States-Israel Binational Agricultural Research and Development Fund, 2020. http://dx.doi.org/10.32747/2020.8134163.bard.
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More than 60% of plant viruses are positive-strand RNA viruses that cause billion-dollar losses annually and pose a major threat to stable agricultural production, including cucumber mosaic virus (CMV) that infects numerous vegetables and ornamental trees. A highly conserved feature among these viruses is that they form viral replication complexes (VRCs) to multiply their genomes by hijacking host proteins and remodeling host intracellular membranes. As a conserved and indispensable process, VRC assembly also represents an excellent target for the development of antiviral strategies that can be used to control a wide-range of viruses. Using CMV and a model virus, brome mosaic virus (BMV), and relying on genomic tools and tailor-made large-scale resources specific for the project, our original objectives were to: 1) Identify host proteins that are required for viral replication complex assembly. 2) Dissect host requirements that determine viral host range. 3) Provide proof-of-concept evidence of a viral control strategy by blocking the viral replication complex-localized phospholipid synthesis. We expect to provide new ways and new concepts to control multiple viruses by targeting a conserved feature among positive-strand RNA viruses based on our results. Our work is going according to the expected timeline and we are progressing well on all aims. For Objective 1, among ~6,000 yeast genes, we have identified 96 hits that were possibly play critical roles in viral replication. These hits are involved in cellular pathways of 1) Phospholipid synthesis; 2) Membrane-shaping; 3) Sterol synthesis and transport; 4) Protein transport; 5) Protein modification, among many others. We are pursuing several genes involved in lipid metabolism and transport because cellular membranes are primarily composed of lipids and lipid compositional changes affect VRC formation and functions. For Objective 2, we have found that CPR5 proteins from monocotyledon plants promoted BMV replication while those from dicotyledon plants inhibited it, providing direct evidence that CPR5 protein determines the host range of BMV. We are currently examining the mechanisms by which dicot CPR5 genes inhibit BMV replication and expressing the dicot CPR5 genes in monocot plants to control BMV infection. For Objective 3, we have demonstrated that substitutions in a host gene involved in lipid synthesis, CHO2, prevented the VRC formation by directing BMV replication protein 1a (BMV 1a), which remodels the nuclear membrane to form VRCs, away from the nuclear membrane, and thus, no VRCs were formed. This has been reported in Journal of Biological Chemistry. Based on the results from Objective 3, we have extended our plan to demonstrate that an amphipathic alpha-helix in BMV 1a is necessary and sufficient to target BMV 1a to the nuclear membrane. We further found that the counterparts of the BMV 1a helix from a group of viruses in the alphavirus-like superfamily, such as CMV, hepatitis E virus, and Rubella virus, are sufficient to target VRCs to the designated membranes, revealing a conserved feature among the superfamily. A joint manuscript describing these exciting results and authored by the two labs will be submitted shortly. We have also successfully set up systems in tomato plants: 1) to efficiently knock down gene expression via virus-induced gene silencing so we could test effects of lacking a host gene(s) on CMV replication; 2) to overexpress any gene transiently from a mild virus (potato virus X) so we could test effects of the overexpressed gene(s) on CMV replication. In summary, we have made promising progress in all three Objectives. We have identified multiple new host proteins that are involved in VRC formation and may serve as good targets to develop antiviral strategies; have confirmed that CPR5 from dicot plants inhibited viral infection and are generating BMV-resistance rice and wheat crops by overexpressing dicot CPR5 genes; have demonstrated to block viral replication by preventing viral replication protein from targeting to the designated organelle membranes for the VRC formation and this concept can be further employed for virus control. We are grateful to BARD funding and are excited to carry on this project in collaboration.
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Deutsch, Christopher. Discovery and Characterization of the Proteins Involved in the Synthesis of N⁶-Threonylcarbamoyl Adenosine, a Nucleoside Modification of tRNA. Portland State University Library, Januar 2000. http://dx.doi.org/10.15760/etd.3075.
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