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RNA, TRANSFER --- RNA, TRANSFER

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G-protein-coupled receptors (GPCRs) comprise a wide family of monomeric heptahelical glycoproteins that recognize a broad array of extracellular mediators (including cationic amines, lipids, peptides, proteins, and sensory agents).GPCRs are considered , as a dogme, like a membranous entity.However,some evidence has suggested ,the existence of an intracellular/nuclear GPCRs population (e.g. the lysophosphatidic acid receptor or also the parathormon receptor). In this study, by mean of a complementar and multidisciplinar approches, we demonstrated that B2R are located in highly purified nuclei from isolated rat hepatocytes.These nuclear receptors recognize the specific ligands of membranous receptor such as bradykinin (BK) and Hoetch 140 (HOE 140 ou icatibant). The results depicting B2R nuclear expression in isolated nuclear organelles were reproduced in situ on hepatic sections by immunogold labeling and transmission electron microscopy ; this prooving that first observation related in isolated nuclei were not an artefactual redistribution of the receptor due to cellular fractionning. Functional tests on single nucleus, by means of confocal microscopy and the calcium-sensitive probe fluo-4, showed that BK induces concentration-dependent transitory mobilization of nucleoplasmic calcium; these responses were blocked by B2R antagonist HOE 140, not by the BIR antagonist R954 and, were also found in wild-type C57/Bl6 mice, but not in B2R-KO mice.The existent scientific literature on the nuclear GPCRs signalling has been defined, in most cases, on primary cells and transfected cells, cultivated in vitro. Our results describe a new atypical nuclear localisation of RCPGs for peptidic ligand, specifically for rB2 ; and also, for the first time, the functionality of this type of receptors. By considering the participation well-established of the kallicréines / kinines system in inflammatory (e.g. trauma, infection ...), it would be interesting to determine the inter-relationship of signalling of rB2 membranaires with their nuclear counterparts. Finally, functions and potential involvements of these nuclear RCPGs, in physiology and in physiopatholog y, remain to be clarified but will be certainly very promising. Les récepteurs couplés à des protéines-G (RCPGs) comportent une large famille de protéines considérées comme entités des membranes plasmiques . Cependant, des études récentes suggèrent qu'ils puissent également se localiser au niveau des membranes nucléaires (ex. récepteurs des phospholipides, de l'endothéline et de la parathormone). Dans cette étude, en utilisant une approche pluridisciplinaire (microscopie confocale, électronique ainsi que l' immunobuvardage de type western), nous avons montré que les noyaux isolés à partir de foies de rats, expriment le récepteur B2 (rB2) qui reconnaît ses ligands spécifiques, tels la bradykinine (BK) et le HOE-140 (Icatibant). Ces résultats ont été reproduits sur des sections de tissus hépatiques intactes, par microscopie électronique en transmission, indiquant que la localisation nucléaire des rB2 n'est pas due à une redistribution artéfactuelle apparaissant lors du fractionnement cellulaire. De plus, nous avons détecté le kininogène de haut poids moléculaire (KHPM), précurseur de la BK, dans le compartiment nucléaire, suggérant qu'il existe une source de ligand disponible au sein du noyau. Nos essais fonctionnels, grâce à la microscopie confocale et à la sonde calcique, le fluo-4, ont montré que la BK induit une augmentation transitoire du calcium nucléoplasmique. Cette réponse calcique a été bloquée par l'antagoniste 82 HOE-140 et non par l'antagoniste B 1 R-954, et a été retrouvée également dans les noyaux d'hépatocytes des souris C57/Bl6 type sauvage, mais pas dans ceux des souris KO-B2 . La littérature scientifique existante sur la signalisation de RCPGs nucléaires a été principalement définie sur des cellules primaires et transfectées, cultivées in vitro. Nos résultats décrivent une nouvelle destination atypique des RCPGs pour les ligands de nature peptidique, spécifiquement pour les rB2, aux noyaux de tissus ; mais aussi, pour la première fois, la fonctionnalité de ce type de récepteurs. En considérant la participation bien établie du système kallicréines/kinines dans des désordres inflammatoires (ex. le trauma, l'infection), il serait intéressant de déterminer l'interdépendance de la signalisation des rB2 membranaires avec leurs homologues nucléaires. Enfin, les fonctions et implications potentielles de ces RCPGs nucléaires, en physiologie et en physiopathologie, restent à être élucidées mais seront certainement très prometteuses.

Immunization --- RNA, Transfer --- Mice --- Promoter Regions, Genetic --- Skin --- Luciferases --- Gene Expression

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Peptide Elongation Factors --- Eukaryotic Initiation Factor-1 --- Amino Acid Sequence --- RNA, Transfer, Amino Acyl --- metabolism --- metabolism --- metabolism

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Site-specific mutagenesis of DNA, developed some thirty years ago, has proven to be one of the most important advances in biology. By allowing the site-specific replacement of any amino acid in a protein with one of the other nineteen amino acids, it ushered in the new era of "Protein Engineering". The field of protein engineering has, however, evolved rapidly since then and the last fifteen years have witnessed remarkable advances through the use of new chemical, biochemical and molecular biological tools towards the synthesis and manipulation of proteins. The chapters included in this book reflect the rapid evolution of protein engineering and its many applications in basic research, biotechnology, material sciences and therapy. This book will provide the reader with an introduction to state-of the-art concepts and methods and will be of use to anyone interested in the study of proteins, in academia as well as in industry.

Protein engineering. --- RNA, Transfer -- chemical synthesis. --- Protein engineering --- Protein Engineering --- RNA, Transfer --- Genetic Engineering --- RNA --- Genetic Techniques --- Nucleic Acids --- Nucleic Acids, Nucleotides, and Nucleosides --- Investigative Techniques --- Chemicals and Drugs --- Analytical, Diagnostic and Therapeutic Techniques and Equipment --- Biomedical Engineering --- Animal Biochemistry --- Cytology --- Biology --- Health & Biological Sciences --- Human Anatomy & Physiology --- Biochemical engineering. --- Engineering, Protein --- Protein design --- Proteins --- Bio-process engineering --- Bioprocess engineering --- Design --- Life sciences. --- Biochemistry. --- Cell biology. --- Biophysics. --- Biological physics. --- Materials science. --- Life Sciences. --- Cell Biology. --- Biochemistry, general. --- Biochemical Engineering. --- Biophysics and Biological Physics. --- Materials Science, general. --- Biochemistry --- Biotechnology --- Chemical engineering --- Biochemical engineering --- Genetic engineering --- Cytology. --- Materials. --- Biological and Medical Physics, Biophysics. --- Engineering --- Engineering materials --- Industrial materials --- Engineering design --- Manufacturing processes --- Biological chemistry --- Chemical composition of organisms --- Organisms --- Physiological chemistry --- Chemistry --- Medical sciences --- Cell biology --- Cellular biology --- Cells --- Cytologists --- Materials --- Composition --- Material science --- Physical sciences --- Biological physics --- Physics

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RNA Modification provides a useful examination of the science and its role in biological regulation, the current frontier of life science research, and includes various RNA modications and their role in gene expression. It represents the most up-to-date knowledge and protocols available today.

• Dynamic RNA modifications and their roles in biological regulation are the current frontier of life science research
• This volume of Methods in Enzymology represents up to date knowledge and protocols

RNA Processing, Post-Transcriptional --- Pseudouridine --- RNA, Transfer --- Gene Expression Regulation --- Uridine --- Biochemical Processes --- RNA --- Metabolism --- Genetic Processes --- Ribonucleosides --- Pyrimidine Nucleosides --- Biochemical Phenomena --- Chemical Processes --- Nucleic Acids --- Metabolic Phenomena --- Nucleosides --- Genetic Phenomena --- Nucleic Acids, Nucleotides, and Nucleosides --- Phenomena and Processes --- Chemical Phenomena --- Pyrimidines --- Chemicals and Drugs --- Heterocyclic Compounds, 1-Ring --- Heterocyclic Compounds --- Animal Biochemistry --- Human Anatomy & Physiology --- Health & Biological Sciences --- RNA editing. --- ARN --- pharmacokinetics --- Edition --- Editing, RNA --- Messenger RNA editing --- mRNA editing --- RNA Processing, Post-Transcriptional. --- pharmacokinetics. --- Genetic regulation