66 results on '"Barbier JR"'
Search Results
2. Wastewater treatment by catalytic wet air oxidation process over Al-Fe pillared clays synthesized using microwave irradiation
- Author
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Sassi, Halima, Lafaye, Gwendoline, Ben Amor, Hédi, Gannouni, Abdelaziz, Jeday, Mohamed Razak, and Barbier, Jr, Jacques
- Published
- 2018
- Full Text
- View/download PDF
3. Catalytic wet air oxidation of phenol over metal catalyst (Ru,Pt) supported on TiO2–CeO2 oxides
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Monteros, A. Espinosa de los, Lafaye, G., Cervantes, A., Del Angel, G., Barbier Jr., J., and Torres, G.
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- 2015
- Full Text
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4. On the promoting effect of the addition of ceria to platinum based alumina catalysts for VOCs oxidation
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Sedjame, Henri-Joël, Fontaine, Céline, Lafaye, Gwendoline, and Barbier Jr, Jacques
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- 2014
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5. Nature du dépôt formé au cours de l’oxydation en voie humide catalysée du phénol
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Keav, Sylvain, Martin, Agnès, Barbier Jr, Jacques, and Duprez, Daniel
- Published
- 2010
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6. Catalytic wet air oxidation of oleic acid on ceria-supported platinum catalyst.effect of pH
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Levasseur, Benoît, Renard, Benoist, Barbier Jr., Jacques, and Duprez, Daniel
- Published
- 2006
- Full Text
- View/download PDF
7. Role of ceria-supported noble metal catalysts (Ru, Pd, Pt) in wet air oxidation of nitrogen and oxygen containing compounds
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Barbier, Jr, J., Oliviero, L., Renard, B., and Duprez, D.
- Published
- 2005
- Full Text
- View/download PDF
8. Role of the metal–support interface in the total oxidation of carboxylic acids over Ru/CeO2 catalysts
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Oliviero, L., Barbier, Jr., J., Labruquère, S., and Duprez, D.
- Published
- 1999
- Full Text
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9. Catalysts for Wet Air Oxidation Based on Ce-Zr-Pr-O Mixed Oxides Prepared by Soft Chemistry
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Mikulová, Jana, primary, Rossignol, Sylvie, additional, Barbier Jr., Jacques, additional, Kappenstein, Charles, additional, and Duprez, Daniel, additional
- Published
- 2006
- Full Text
- View/download PDF
10. Oxygen storage capacity of promoted Rh/CeC2 catalysts. Exceptional behavior of RhCu/CeO2
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Kacimi, S., Barbier, Jr., J., Taha, R., and Duprez, D.
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- 1993
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11. Wet Air Oxidation of Phenol over Ru Based Catalysts – Effects of Support
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des Los Monteros, A. E. Espinosa, Lafaye, G., Torres, G., and Barbier, Jr, J.
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- 2010
- Full Text
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12. Process intensification in phenolic wastewater treatment
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Wittine, Ozren, Keav, Sylvain, Barbier jr*, Jacques, Maduna Valkaj, Karolina, and Zrnčević, Stanka
- Subjects
wastewater treatment ,CWAO ,CWPO ,PP-CWAO ,zeolite ,phenol - Abstract
Water pollution is characterized mainly by the volume of waste to be treated and by the nature, concentration and toxicity of pollutants that form it. These parameters are all factors that have to be taken into account when choosing the treatment method to use. WAO (Wet Air Oxidation) is a phenol oxidation treatment method based on oxidation with air or oxygen, and is conducted at elevated temperatures (373-573 K) and pressures (1 - 20 MPa). With addition of homogeneous or heterogeneous catalysts, WO processes can be carried out at lower pressures and temperatures. If these processes use hydrogen peroxide as oxidizing agent, the process is called CWPO (Catalytic Wet Peroxide Oxidation), in which treatment is carried out at atmospheric pressure and temperatures below 373 K. To take advantages of the CWPO & CWAO process, and maximize the effectiveness of treatment, in this work, mixture of both oxidants (oxygen and hydrogen peroxide) was used. Activity of synthesized Cu/13X catalyst was tested in the PP-CWAO (Peroxide Promoted Catalytic Wet Air Oxidation) reaction using batch reactor. The catalysts were characterized by different methods (XRD, AAS, BET surface area). The catalytic tests were carried out in a stainless steel Parr reactor in batch operation mode at the constant catalyst mass (0.1 g / 200 cm-3), different temperatures (333 - 353K) and air pressures (2 - 20 bar). The initial concentration of phenol was 0.01 mol dm-3 while the concentration of hydrogen peroxide varied from 0.01 mol dm-3 to 0.14 mol dm-3. Based on the results it has been concluded that PP-CWAO process is more efficient than classical CWAO and CWPO processes.
- Published
- 2011
13. PHENOL OXIDATION BY A PP-CWAO TREATMENT WITH A Cu/13X CATALYST
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Wittine, Ozren, Keav, Sylvain, Jacques Barbier jr*, Jacques, Zrnčević, Stanka, Antonić Jelić, Tatjana, and Zbukoveć Logar, Nataša
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Catalytic oxidation ,hydrogen peroxide ,promoting effect ,organic aqueous wastes - Abstract
Nowadays, there are increasingly stringent regulations requiring more and more treatment of industrial effluents to generate product waters which could be easily reused or disposed of to the environment without any harmful effects. Therefore, different advanced oxidation processes were investigated as suitable precursors for the biological treatment of industrial effluents containing phenol. The promoted catalytic wet air oxidation of phenol has been investigated through the addition of hydrogen peroxide as a source of free radicals.
- Published
- 2010
14. Wet air oxidation of acetic acid over platinum catalysts supported on cerium based materials - Influence of metal and oxide particle sizes
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Mikulova, J., Barbier Jr, Jacques, Rossignol, Sylvie, Mesnard, Danielle, Duprez, Daniel, Kappenstein, Charles, Laboratoire de catalyse en chimie organique (LACCO), Université de Poitiers-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Groupe d'Etudes des Matériaux Hétérogènes (GEMH), Université de Limoges (UNILIM)-Institut des Procédés Appliqués aux Matériaux (IPAM), Université de Limoges (UNILIM)-Université de Limoges (UNILIM), Laboratoire de Catalyse en Chimie Organique (LCO), and Université de Poitiers-Centre National de la Recherche Scientifique (CNRS)
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[CHIM.CATA]Chemical Sciences/Catalysis - Published
- 2007
15. Platinum catalysts supported on Ce, Zr, Pr - oxides in catalytic wet air oxidation of acetic acid
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Mikulova, J., Rossignol, Sylvie, Barbier Jr, Jacques, Duprez, Daniel, Kappenstein, Charles, Groupe d'Etudes des Matériaux Hétérogènes (GEMH), Université de Limoges (UNILIM)-Institut des Procédés Appliqués aux Matériaux (IPAM), Université de Limoges (UNILIM)-Université de Limoges (UNILIM), Laboratoire de catalyse en chimie organique (LACCO), Université de Poitiers-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Catalyse en Chimie Organique (LCO), and Université de Poitiers-Centre National de la Recherche Scientifique (CNRS)
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[CHIM.CATA]Chemical Sciences/Catalysis - Published
- 2007
16. Mechanism of stearic acid oxidation over nanocrystalline La1-xA'xBO3 (A' = Sr, Ce; B = Co, Mn): The role of oxygen mobility
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Royer, S., Levasseur, B., Alamdari, H., Barbier Jr, Jacques, Duprez, Daniel, Kaliaguine, Serge, Department of Chemical Engineering, Laval University, Ste Foy (QC), Canada, G1K 7P4, Université Laval [Québec] (ULaval), Institut des Nanosciences de Paris (INSP), Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de catalyse en chimie organique (LACCO), Université de Poitiers-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Catalyse en Chimie Organique (LCO), Université de Poitiers-Centre National de la Recherche Scientifique (CNRS), and Department of Chemical Engineering
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[CHIM.CATA]Chemical Sciences/Catalysis ,ComputingMilieux_MISCELLANEOUS - Abstract
International audience
- Published
- 2007
17. Catalysts for Wet Air Oxidation Based on Ce-Zr-Pr-O Mixed Oxides Prepared by Soft Chemistry 11th Intern. Ceramic Congress, CIMTEC 2006, Acireale, Sicile, 4-9 juin 2006
- Author
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Mikulova, S., Rossignol, Sylvie, Barbier Jr, Jacques, Groupe d'Etudes des Matériaux Hétérogènes (GEMH), Université de Limoges (UNILIM)-Institut des Procédés Appliqués aux Matériaux (IPAM), Université de Limoges (UNILIM)-Université de Limoges (UNILIM), Laboratoire de catalyse en chimie organique (LACCO), Université de Poitiers-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), and André, Monique
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[CHIM.CATA] Chemical Sciences/Catalysis ,[CHIM.CATA]Chemical Sciences/Catalysis - Published
- 2006
18. Catalysts for wet air oxidation based on Zr-Ce-Pr-O mixed oxides prepared by soft chemistry
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Mikulova, J., Barbier Jr, Jacques, Rossignol, S., Laboratoire de catalyse en chimie organique (LACCO), Université de Poitiers-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Groupe d'Etudes des Matériaux Hétérogènes (GEMH), Université de Limoges (UNILIM)-Institut des Procédés Appliqués aux Matériaux (IPAM), Université de Limoges (UNILIM)-Université de Limoges (UNILIM), and André, Monique
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[CHIM.CATA] Chemical Sciences/Catalysis ,[CHIM.CATA]Chemical Sciences/Catalysis - Published
- 2006
19. Platinum catalysts supported on Ce, Zr, Pr - oxides in catalytic wet air oxidation of acetic acid., 1st European Conference on Environmental applications of Advanced Oxidation Processes, Chania ,Crète, 7-9 septembre 2006
- Author
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Mikulova, J., Rossignol, Sylvie, Barbier Jr, Jacques, Duprez, Daniel, Kappenstein, Charles, André, Monique, Groupe d'Etudes des Matériaux Hétérogènes (GEMH), Université de Limoges (UNILIM)-Institut des Procédés Appliqués aux Matériaux (IPAM), Université de Limoges (UNILIM)-Université de Limoges (UNILIM), Laboratoire de catalyse en chimie organique (LACCO), Université de Poitiers-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Catalyse en Chimie Organique (LCO), and Université de Poitiers-Centre National de la Recherche Scientifique (CNRS)
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[CHIM.CATA] Chemical Sciences/Catalysis ,[CHIM.CATA]Chemical Sciences/Catalysis - Published
- 2006
20. Catalytic wet air oxidation of oleic acid on ceria-supported platinum catalyst. Effect of pH.(invited paper, special issue in honor of Zoltan Paal)
- Author
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Levasseur, B., Renard, B., Barbier Jr, Jacques, Duprez, Daniel, Laboratoire de catalyse en chimie organique (LACCO), Université de Poitiers-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Catalyse en Chimie Organique (LCO), and Université de Poitiers-Centre National de la Recherche Scientifique (CNRS)
- Subjects
[CHIM.CATA]Chemical Sciences/Catalysis - Published
- 2006
21. Catalytic wet air oxidation of stearic acid on cerium oxide-supported noble metal catalysts
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Renard, B., Barbier Jr, Jacques, Duprez, Daniel, Durecu, S., Laboratoire de catalyse en chimie organique (LACCO), Université de Poitiers-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Catalyse en Chimie Organique (LCO), and Université de Poitiers-Centre National de la Recherche Scientifique (CNRS)
- Subjects
[CHIM.CATA]Chemical Sciences/Catalysis - Published
- 2005
22. Mechanism of stearic acid oxidation over nanocrystalline La1−xA′xBO3La1−xA′xBO3 (A′=Sr, Ce; B=Co, Mn): The role of oxygen mobility
- Author
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Royer, S., primary, Levasseur, B., additional, Alamdari, H., additional, Barbier Jr., J., additional, Duprez, D., additional, and Kaliaguine, S., additional
- Published
- 2008
- Full Text
- View/download PDF
23. Ruthenium and platinum catalysts supported on Ce, Zr, Pr-O mixed oxides prepared by soft chemistry for acetic acid wet air oxidation
- Author
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Mikulová, J., primary, Rossignol, S., additional, Barbier Jr., J., additional, Mesnard, D., additional, Kappenstein, C., additional, and Duprez, D., additional
- Published
- 2007
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- View/download PDF
24. Wet Air Oxidation of nitrogen-containing organic compounds and ammonia in aqueous media
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Oliviero, L., Barbier Jr., J., and Duprez, D.
- Subjects
- *
OXIDATION , *AMMONIA - Abstract
Treatment of toxic nitrogen-containing compounds is one of the major applications of the Wet Air Oxidation (WAO) processes. The aim of this paper is to review the literature dealing with the Catalytic Wet Air Oxidation (CWAO) of these nitrogenous compounds, mainly produced in chemical and pharmaceutical industries. Many studies deal with oxidation of aniline, often chosen as a model molecule of pollutant of dye industries. First, the results obtained with CWAO are compared with those obtained with other oxidation processes. Particular attention is paid to the selectivity towards organic by-products (specially, azo, nitroso and nitro compounds, phenolic compounds and carboxylic acids) as well as towards several inorganic forms of nitrogen (NH4+, N2, NO2−, NO3−). In a second part, the review focuses on the mechanism of chemical reactions that can explain the formation of the observed products. Usually, similar catalysts can be used for CWAO of oxygen-containing (phenol, carboxylic acids) and nitrogen-containing organic compounds. Ammonia is one of the most refractory by-product formed during catalytic WAO of the nitrogen-containing organic pollutants and is itself a pollutant. For this reason, recent reports about its oxidation by the CWAO process are finally reviewed. Very high selectivities to dinitrogen can be obtained on certain noble metal catalysts. As a rule, catalysts active and selective for ammonia oxidation are different (nature of active phase, support, etc.) from the solids proven to be the best catalysts for CWAO of organic compounds. Multifunctional catalysts are, thus, required for the treatment of nitrogenous organic compounds. [Copyright &y& Elsevier]
- Published
- 2003
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25. Catalytic wet air oxidation of ammonia over M/CeO2 catalysts in the treatment of nitrogen-containing pollutants
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Barbier Jr., Jacques, Oliviero, Laetitia, Renard, Benoist, and Duprez, Daniel
- Subjects
- *
OXIDATION , *AMMONIA - Abstract
Ammonia, a well-known by-product of chemical, fertiliser and metallurgy industries, is also the most refractory product of nitrogen-containing compound oxidation. Consequently,
NH4+ is a key component of waste disposal of conventional processes like anaerobic digestion or nitrification/denitrification. Catalytic wet air oxidation (CWAO) process, able to eliminate organic matter with non toxic by-product formation, was investigated for ammonium ions removal from wastewater. Oxidation of aniline and of ammonia were carried out on mono- and bimetallic noble metal catalysts (Pt, Ru, Pd, etc.) prepared by impregnation and supported on cerium oxides. In liquid phase, at high temperature (150–250 °C) and high pressure of oxygen (20 bar), a Ru/CeO2 catalyst is able to achieve the elimination of refractory nitrogenous organic products like aniline. The greatest interest of CWAO compared to the classical biological one, is that the selectivity towards molecular nitrogen is much higher (>90%). Indeed, in this process, ammonium ions give essentially N2, via hydroxylamine and below 200 °C. At higher temperatures the rate of conversion is extremely high but nitrite and nitrate ions appear in the effluent. On a RuPd/CeO2 catalyst, the optimal temperature for ammonia conversion is then 200 °C. In these conditions, the N2 selectivity is up to 90%. [Copyright &y& Elsevier]- Published
- 2002
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26. Catalysts for Wet Air Oxidation Based on Ce-Zr-Pr-O Mixed Oxides Prepared by Soft Chemistry
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Mikulová, Jana, Rossignol, Sylvie, Barbier Jr., Jacques, Kappenstein, Charles, and Duprez, Daniel
- Abstract
Sol-gel Zr0.1Ce0.9O2 and Zr0.1(Ce0.75Pr0.25)O2 mixed oxides and coprecipitated pure ceria CeO2 displaying the fluorine type structure have been used as platinum or ruthenium catalysts’ supports for catalytic wet air oxidation (CWAO) of aqueous solution of acetic acid (78 mmol.L-1). These catalysts were prepared by conventional impregnation (5 wt-%) from platinum and ruthenium precursor salts or by exchange (~2 wt-%) in the case of ruthenium. A screening of catalysts in CWAO at 200°C under 2 MPa was performed and reveals that the best platinum catalyst is supported on pure ceria displaying large surface. For ruthenium catalysts, the highest conversion after 3 hours of reaction has been reached by the Ru/Zr-Ce-O system.
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- 2006
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27. Zeolite and mixed oxide catalysts for VOCs oxidation
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Jana Gaálová, Jirátová, K., Klempa, J., Šolcová, O., Maupin, I., Mijoin, J., Magnoux, P., and Barbier Jr, J.
28. Ruthenium versus platinum on cerium materials in wet air oxidation of acetic acid.
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Gaálová J, Barbier J Jr, and Rossignol S
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- Air, Catalysis, Oxidation-Reduction, Oxides, Particle Size, Acetic Acid chemistry, Cerium, Platinum, Ruthenium
- Abstract
This study was a comparison between Ru-catalysts and similar, previously investigated, Pt-catalysts. In this paper, ruthenium catalysts for catalytic wet air oxidation are prepared, characterized and tested. Both catalysts were supported on commercial CeO2 as well as mixed oxide Zr(0.1)(Ce(0.75)Pr(0.25))(0.9)O2. The catalysts were characterized by measuring the oxygen storage capacities (OSC), BET, XRD, FTIR and chemisorption of hydrogen. In addition, the effect of sintering (treatments under H2) was compared with both of the catalysts. The comparison of the results showed that initial intrinsic activity of ruthenium is not significantly influenced by the type of the support, which is contrast to platinum. Furthermore, the particle size of Ru had an important effect on CWAO activity: the higher the particle size, the better the activity. This was different with Pt-catalysts, where the optimal particle size was smaller, having about 15% of metal dispersion., (Copyright 2010 Elsevier B.V. All rights reserved.)
- Published
- 2010
- Full Text
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29. C-terminal analogues of parathyroid hormone: effect of C-terminus function on helical structure, stability, and bioactivity.
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Potetinova Z, Barbier JR, Suen T, Dean T, Gardella TJ, and Willick GE
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- Animals, Cell Line, Chromatography, High Pressure Liquid, Circular Dichroism, Macaca mulatta, Models, Molecular, Protein Binding, Protein Structure, Secondary, Protein Structure, Tertiary, Thermodynamics, Parathyroid Hormone chemistry, Peptide Fragments chemistry
- Abstract
We have studied the effects of C-terminal group modifications (amide, methylamide, dimethylamide, aldehyde, and alcohol) on the conformation, adenylyl cyclase stimulation (AC), or binding of parathyroid hormone (hPTH) analogues, hPTH(1-28)NH(2) and hPTH(1-31)NH(2). hPTH(1-31)NH(2) has a C-terminal alpha-helix bounded by residues 17-29 [Chen, Z., et al. (2000) Biochemistry 39, 12766]. In both cases, relative to the natural analogue with a carboxyl C-terminus, the amide and methylamide had increased helix content whereas the dimethylamide forms had CD spectra more similar to the carboxyl one. Conformational effects were more pronounced with hPTH(1-28) than with hPTH(1-31), with increases in helix content of approximately 30% in contrast to 10%. Stabilization of the C-terminal helix of residues 1-28 seemed to correlate with an ability of the C-terminal function to H-bond appropriately. None of the analogues affected the AC stimulating activity significantly, but there was an up to 15-fold decrease in the level of apparent binding of the carboxyl hPTH(1-28) analogue compared to that of the methylamide and a 4-fold decrease in the level of binding of the aldehyde or dimethylamide. There was no significant change in binding activities for the 1-31 analogues. These observations are consistent with previous studies that imply the importance of a region of the hormone's C-terminal alpha-helix for tight binding to the receptor. They also show that modulation of helix stability does have an effect on the binding of the hormone, but only when the C-terminus is at the putative end of the helix. The similarity of AC stimulation even when binding changed 10-fold can be explained by assuming greater efficacy of the weaker binding PTH-receptor complexes in stimulating AC.
- Published
- 2006
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30. Backbone-methylated analogues of the principle receptor binding region of human parathyroid hormone. Evidence for binding to both the N-terminal extracellular domain and extracellular loop region.
- Author
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Barbier JR, Gardella TJ, Dean T, MacLean S, Potetinova Z, Whitfield JF, and Willick GE
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- Adenylyl Cyclases metabolism, Amino Acid Sequence, Animals, Binding Sites, Cell Line, Circular Dichroism, Humans, Methylation, Molecular Sequence Data, Parathyroid Hormone chemistry, Swine, Parathyroid Hormone metabolism
- Abstract
We have used backbone N-methylations of parathyroid hormone (PTH) to study the role of these NH groups in the C-terminal amphiphilic alpha-helix of PTH (1-31) in binding to and activating the PTH receptor (P1R). The circular dichroism (CD) spectra indicated the structure of the C-terminal alpha-helix was locally disrupted around the methylation site. The CD spectra differences were explained by assuming a helix disruption for four residues on each side of the site of methylation and taking into account the known dependence of CD on the length of an alpha-helix. Binding and adenylyl cyclase-stimulating data showed that outside of the alpha-helix, methylation of residues Asp30 and Val31 had little effect on structure or activities. Within the alpha-helix, disruption of the structure was associated with increased loss of activity, but for specific residues Val21, Leu24, Arg25, and Leu28 there was a dramatic loss of activities, thus suggesting a more direct role of these NH groups in correct P1R binding and activation. Activity analyses with P1R-delNT, a mutant with its long N-terminal region deleted, gave a different pattern of effects and implicated Ser17, Trp23, and Lys26 as important for its PTH activation. These two groups of residues are located on opposite sides of the helix. These results are compatible with the C-terminal helix binding to both the N-terminal segment and also to the looped-out extracellular region. These data thus provide direct evidence for important roles of the C-terminal domain of PTH in determining high affinity binding and activation of the P1R receptor.
- Published
- 2005
- Full Text
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31. The effects of parathyroid hormone fragments on bone formation and their lack of effects on the initiation of colon carcinogenesis in rats as indicated by preneoplastic aberrant crypt formation.
- Author
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Whitfield J, Bird RP, Morley P, Willick GE, Barbier JR, MacLean S, and Ross V
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- Animals, Azoxymethane, Carcinogens, Osteogenesis, Peptide Fragments, Rats, Rats, Sprague-Dawley, Colonic Neoplasms chemically induced, Parathyroid Hormone pharmacology, Precancerous Conditions chemically induced
- Abstract
The parathyroid hormone (PTH) and some of its fragments and analogs stimulate bone growth in various animal models and humans and one of them (hPTH-(1-34)) has been approved by the USFDA for treating osteoporosis. However, there are reports that PTH can stimulate the PI-3 kinase/mitogen-activated protein kinases-mediated proliferation of rat enterocytes and that primary hyperparathyroidism in humans is associated with an increased incidence of colon cancer. Here we have investigated the ability of two PTH fragments, hPTH-(1-34)NH(2) and [Leu(27)]cyclo(Glu(22)-Lys(26))hPTH-(1-31)NH(2) to initiate colon carcinogenesis or increase the initiatory activity of the widely used colon carcinogen azoxymethane (AOM). The initiation of colon carcinogenesis by AOM was indicated by the very early appearance of aberrant crypt foci. While both PTH peptides strongly stimulated femoral bone formation, they did not cause the appearance of ACFs or affect the number or the distribution along the colon of AOM-induced ACFs. Nor did AOM affect the PTHs' ability to stimulate bone formation. Thus, a relatively short PTH treatment that is long enough to strongly stimulate bone formation does not initiate colon carcinogenesis in rats.
- Published
- 2003
- Full Text
- View/download PDF
32. Structural requirements for conserved arginine of parathyroid hormone.
- Author
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Barbier JR, MacLean S, Whitfield JF, Morley P, and Willick GE
- Subjects
- Adenylyl Cyclases metabolism, Amino Acid Sequence, Amino Acid Substitution, Animals, Cell Line, Circular Dichroism, Citrulline chemistry, Enzyme Activation drug effects, Humans, Molecular Sequence Data, Norleucine chemistry, Ornithine chemistry, Parathyroid Hormone chemical synthesis, Parathyroid Hormone pharmacology, Peptide Fragments chemical synthesis, Peptide Fragments pharmacology, Protein Structure, Secondary, Rats, Structure-Activity Relationship, Arginine chemistry, Conserved Sequence, Parathyroid Hormone chemistry, Peptide Fragments chemistry
- Abstract
Arg-20 is one of two residues conserved in all peptides known to activate the parathyroid hormone (PTH) receptor. Previous studies have failed to find any naturally encoded analogues of residue 20 that had any adenylyl cyclase (AC) stimulating activity. In this work we have studied substitutions of Arg-20 with nonencoded amino acids and conformationally constrained analogues with side chains mimicking that of Arg. No analogue had more than 20% of the AC-stimulating ability of the natural Arg-20-bearing peptide. In descending order of activity, the most active analogues had (S)-4-piperidyl-(N-amidino)glycine (PipGly), norleucine (Nle), citrulline (Cit), or ornithine (Orn) at residue 20. Analogues with Arg-20 substituted with L-4-piperidyl-(N-amidino)alanine, Lys, Glu, Ala, Gln, (S)-2-amino-4-[(2-amino)pyrimidinyl]butanoic acid, or L-(4-guanidino)phenylalanine had very low or negligible activity. Low or negligible activities of Lys or Orn analogues suggested ionic interactions play a minor role in the Arg interaction with the receptor. The conformational constraints imposed by the PipGly ring had a negative effect on its ability to substitute for Arg. The side-chain H-bonding potential of the Cit ureimido group was likely an important factor in its mimicry of Arg. The increase in amphiphilicity, as demonstrated by its greater high-performance liquid chromatographic retention, and increased alpha-helix, as shown by circular dichroic spectroscopy, likely contributed to the activity of the Nle-20 analogue. The data demonstrated that specific H-bonding, hydrophobicity of the side chain, stabilization of alpha-helix, and possibly specific cation positioning were all important in the interaction of Arg-20 with receptor groups.
- Published
- 2001
- Full Text
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33. The effect of monocyclic and bicyclic analogs of human parathyroid hormone (hPTH)-(1-31)NH2 on bone formation and mechanical strength in ovariectomized rats.
- Author
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Morley P, Whitfield JF, Willick GE, Ross V, MacLean S, Barbier JR, Isaacs RJ, and Andreassen TT
- Subjects
- Adenylyl Cyclases biosynthesis, Animals, Bone Development physiology, Bone Diseases, Metabolic drug therapy, Bone Diseases, Metabolic etiology, Cyclization, Elasticity drug effects, Female, Femur drug effects, Femur pathology, Femur physiopathology, Lumbar Vertebrae drug effects, Lumbar Vertebrae pathology, Lumbar Vertebrae physiopathology, Parathyroid Hormone chemistry, Parathyroid Hormone therapeutic use, Peptide Fragments chemistry, Peptide Fragments therapeutic use, Rats, Rats, Sprague-Dawley, Stress, Mechanical, Structure-Activity Relationship, Tumor Cells, Cultured drug effects, Tumor Cells, Cultured enzymology, Weight-Bearing physiology, Bone Development drug effects, Ovariectomy, Parathyroid Hormone pharmacology, Peptide Fragments pharmacology
- Abstract
The [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-31)NH2 lactam is a stronger stimulator of adenylyl cyclase activity and a better stimulator of trabecular bone in the ovariectomized (OVX) rat model of osteopenia than hPTH-(1-31)NH2. This enhanced activity is due in large part to the stabilization of the amphiphilic receptor-binding alpha-helix in the Ser17-Gln29 region. The goal of the present study was to determine whether further cyclization could produce a more active hPTH analog. To this end, we compared the relative bioactivities of the bicyclic hPTH analog [Glu17,Leu27]cyclo(Lys13-Glu17,Glu22-Lys26)-hPTH-(1-31)NH2, made by replacing Ser17 with Glu17 and introducing a second lactam linkage between Lys13 and Glu17. The relative EC50 for adenylyl cyclase stimulation by the bicyclic hPTH analog was similar to the EC50 of the monocyclic [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-31)NH2, but the bicyclic analog was still more active than hPTH-(1-31)NH2. As expected from adenylyl cyclase stimulation being responsible for PTH's anabolic action, the bicyclic hPTH analog [Glu17, Leu27]cyclo(Lys13-Glu17, Glu22-Lys26)-hPTH-(1-31)NH2 was able to increase femoral trabecular volume and thickness and mechanical strength in OVX rats, but it was no more effective than [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-31)NH2 when injected once daily in a dose of 0.8 nmol/100 g body weight. Thus, further constraint of the conformation of hPTH-(1-31)NH2 by introducing two lactam link-ages between Lys13-Glu17 and Glu22-Lys26 did not raise the osteogenicity above that of the monocyclic analog.
- Published
- 2001
- Full Text
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34. Structure and activities of constrained analogues of human parathyroid hormone and parathyroid hormone-related peptide: implications for receptor-activating conformations of the hormones.
- Author
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Barbier JR, MacLean S, Morley P, Whitfield JF, and Willick GE
- Subjects
- Adenylyl Cyclases metabolism, Amino Acid Sequence, Animals, Circular Dichroism, Cystine chemistry, Cystine genetics, Enzyme Activation drug effects, Enzyme Activation genetics, Humans, Lactams chemistry, Lactams metabolism, Lactams pharmacology, Molecular Sequence Data, Parathyroid Hormone chemical synthesis, Parathyroid Hormone genetics, Parathyroid Hormone metabolism, Parathyroid Hormone pharmacology, Parathyroid Hormone-Related Protein, Peptide Fragments chemical synthesis, Peptide Fragments genetics, Peptide Fragments pharmacology, Peptides, Cyclic chemical synthesis, Peptides, Cyclic genetics, Peptides, Cyclic pharmacology, Protein Conformation, Proteins genetics, Proteins metabolism, Proteins pharmacology, Rats, Receptor, Parathyroid Hormone, Type 1, Structure-Activity Relationship, Tumor Cells, Cultured, Parathyroid Hormone chemistry, Proteins chemistry, Receptors, Parathyroid Hormone metabolism
- Abstract
Parathyroid hormone (PTH) has a helix-bend-helix structure in solution. Part of the C-terminal helix, residues 21-31, is amphiphilic and forms a critical receptor-binding region. Stabilization of this alpha-helix by lactam formation between residues spaced i, i + 4 on the polar face was previously reported to increase adenylyl cyclase-stimulating (AC) activity if between residues 22 and 26 but to diminish it if between residues 26 and 30 [Barbier et al. (1997) J. Med. Chem. 40, 1373-1380]. This work reports the effects of other cyclizations on the polar face, differing in ring size or position, on alpha-helix conformation, as measured by circular dichroism, and on AC-stimulating activity. All analogues cyclized between residues 22 and 26 had at least a 1. 5-fold increase in activity, suggesting an alpha-helical structure between about residues 21 and 26. Cyclization between residues 25 and 29 or residues 26 and 30 diminished activity by 20-30%, despite stabilizing alpha-helix, suggesting that residues 25-31 bind to the receptor in a helical, but not classical alpha-helical, conformation. Analogues cyclized between residues 13 and 17 had slightly increased activity. A bicyclic analogue, with lactams between residues 13 and 17 and residues 22 and 26, had about the same activity as that cyclized only between 22 and 26. Parathyroid hormone-related peptide (PTHrP) may bind in a manner similar to the common receptor, but hydrophobic moment calculations suggest that it must bind as a tighter helix in order to optimally present its hydrophobic residues to the receptor. Both PTHrP analogues cyclized between either residues 22 and 26 or residues 26 and 30 had more stable alpha-helices but reduced AC activities, consistent with this hypothesis.
- Published
- 2000
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35. Solution structure of the osteogenic 1-31 fragment of the human parathyroid hormone.
- Author
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Chen Z, Xu P, Barbier JR, Willick G, and Ni F
- Subjects
- Amino Acid Sequence, Animals, Computer Simulation, Humans, Models, Molecular, Molecular Sequence Data, Nuclear Magnetic Resonance, Biomolecular, Osteogenesis, Protein Conformation, Protein Folding, Protein Structure, Secondary, Rats, Solutions, Structure-Activity Relationship, Parathyroid Hormone chemistry, Parathyroid Hormone physiology, Peptide Fragments chemistry, Peptide Fragments physiology
- Abstract
The solution conformations of a selectively osteogenic 1-31 fragment of the human parathyroid hormone (hPTH), hPTH(1-31)NH(2), have been characterized by use of very high field NMR spectroscopy at 800 MHz. The combination of the CalphaH proton and (13)Calpha chemical shifts, (3)J(NH)(alpha) coupling constants, NH proton temperature coefficients, and backbone NOEs reveals that the hPTH(1-31)NH(2) peptide has well-formed helical structures localized in two distinct segments of the polypeptide backbone. There are also many characteristic NOEs defining specific side-chain/backbone and side-chain/side-chain contacts within both helical structures. The solution structure of hPTH(1-31)NH(2) contains a short N-terminal helical segment for residues 3-11, including the helix capping residues 3 and 11 and a long C-terminal helix for residues 16-30. The two helical structures are reinforced by well-defined capping motifs and side-chain packing interactions within and at both ends of these helices. On one face of the C-terminal helix, there are side-chain pairs of Glu22-Arg25, Glu22-Lys26, and Arg25-Gln29 that can form ion-pair and/or hydrogen bonding interactions. On the opposite face of this helix, there are characteristic hydrophobic interactions involving the aromatic side chain of Trp23 packing against the aliphatic side chains of Leu15, Leu24, Lys27, and Leu28. There is also a linear array of hydrophobic residues from Val2, to Leu7, to Leu11 and continuing on to residues His14 and Leu15 in the hinge region and to Trp23 in the C-terminal helix. Capping and hydrophobic interactions at the end of the N-terminal and at the beginning of the C-terminal helix appear to consolidate the helical structures into a V-shaped overall conformation for at least the folded population of the hPTH(1-31)NH(2) peptide. Stabilization of well-folded conformations in this linear 1-31 peptide fragment and possibly other analogues of human PTH may have a significant impact on the biological activities of the PTH peptides in general and specifically for the osteogenic/anabolic activities of bone-building PTH analogues.
- Published
- 2000
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36. Lactam formation increases receptor binding, adenylyl cyclase stimulation and bone growth stimulation by human parathyroid hormone (hPTH)(1-28)NH2.
- Author
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Whitfield JF, Morley P, Willick GE, Isaacs RJ, MacLean S, Ross V, Barbier JR, Divieti P, and Bringhurst FR
- Subjects
- Animals, Cell Line, Enzyme Activation, Humans, Protein Binding, Rats, Rats, Sprague-Dawley, Swine, Teriparatide pharmacology, Adenylyl Cyclases metabolism, Bone Development drug effects, Lactams metabolism, Peptide Fragments pharmacology, Receptors, Parathyroid Hormone metabolism, Teriparatide analogs & derivatives
- Abstract
Human parathyroid hormone (1-28)NH2 [hPTH(1-28)NH2] is the smallest of the PTH fragments that can fully stimulate adenylyl cyclase in ROS 17/2 rat osteoblast-like osteosarcoma cells. This fragment has an IC50 of 110 nM for displacing 125I-[Nle8,18,Tyr34]bovine PTH(1-34)NH2 from HKRK B7 porcine kidney cells, which stably express 950,000 human type 1 PTH/PTH-related protein (PTHrP) receptors (PTH1Rs) per cell. It also has an EC50 of 23.9 nM for stimulating adenylyl cyclase in ROS 17/2 cells. Increasing the amphiphilicity of the alpha-helix in the residue 17-28 region by replacing Lys27 with Leu and stabilizing the helix by forming a lactam between Glu22 and Lys26 to produce the [Leu27]cyclo(Glu22-Lys26)hPTH(1-28)NH2 analog dramatically reduced the IC50 for displacing 125I-[Nle8,18,Tyr34]bPTH(1-34)NH2 from hPTH1Rs from 110 to 6 nM and dropped the EC50 for adenylyl cyclase stimulation in ROS 17/2 cells from 23.9 to 9.6 nM. These modifications also increased the osteogenic potency of hPTH(1-28)NH2. Thus, hPTH(1-28)NH2 did not significantly stimulate either femoral or vertebral trabecular bone growth in rats when injected daily at a dose of 5 nmol/100 g body weight for 6 weeks, beginning 2 weeks after ovariectomy (OVX), but it strongly stimulated the growth of trabeculae in the cancellous bone of the distal femurs and L5 vertebrae when injected at 25 nmol/100 g body weight. By contrast [Leu27]cyclo(Glu22-Lys26)hPTH(1-28)NH2 significantly stimulated trabecular bone growth when injected at 5 nmol/100 g of body weight. Thus, these modifications have brought the bone anabolic potency of hPTH(1-28)NH2 considerably closer to the potencies of the larger PTH peptides and analogs.
- Published
- 2000
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37. The stimulation of vertebral and tibial bone growth by the parathyroid hormone fragments, hPTH-(1-31)NH2, [Leu27]cyclo(Glu22-Lys26)hPTH-(1-31)NH2, and hPTH-(1-30)NH2.
- Author
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Whitfield JF, Morley P, Fraher L, Hodsman AB, Holdsworth DW, Watson PH, Willick GE, Barbier JR, Gulam M, Isaacs RJ, MacLean S, and Ross V
- Subjects
- Animals, Female, Osteogenesis, Ovariectomy, Peptide Fragments pharmacology, Rats, Spine growth & development, Tibia growth & development, Time Factors, Bone Development drug effects, Parathyroid Hormone pharmacology, Spine drug effects, Tibia drug effects
- Abstract
The native human parathyroid hormone, hPTH-(1-84), and certain carboxyl truncated analogs such as hPTH-(1-34) and even smaller fragments such as hPTH-(1-31)NH2, [Leu27]cyclo(Glu22-Lys26)hPTH-(1-31)NH2, and hPTH-(1-30)NH2 stimulate femoral trabecular and cortical bone growth in ovariectomized (OVX) rats. Here we show that when injected once daily for 6 weeks starting 2 weeks after OVX in doses of 1 or 2 nmol/100 g of body weight, hPTH-(1-31)NH2, [Leu27] cyclo(Glu22-Lys26)hPTH-(1-31)NH2, and hPTH-(1-34)NH2 prevented the loss of trabecular volume in the L5 vertebrae induced by OVX. In fact, by the end of the sixth week of injections (i.e., the eighth week after OVX) the fragments had increased the volume and trabecular thickness significantly above the values in vehicle-injected sham-operated rats. hPTH-(1-30)NH2 can stimulate vertebral bone growth as much as the larger fragments, but 10-25 times more of it was needed to do so. The same daily doses of hPTH-(1-31)NH2, [Leu27]cyclo(Glu22-Lys26)hPTH-(1-31)NH2, and hPTH-(1-34)NH2 also raised the trabecular volume and thickness in the L5 vertebrae of rats well above the values in vehicle-treated animals when the injections were started 9 weeks after OVX. This restoration of trabecular bone in the L5 vertebrae in estrogen-deprived animals was accompanied by a significant increase in the bone mineral density (BMD) of the L1-L4 vertebrae and tibias. However, there was no significant drop in the pelvic BMD in the estrogen-deprived animals and the effects of hPTH-(1-31)NH2, [Leu27]cyclo(Glu22-(Lys) hPTH-(1-31)NH2, and hPTH-(1-34)NH2 on the pelvic BMD were equivocal.
- Published
- 2000
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38. High-yield expression of fully bioactive N-terminal parathyroid hormone analog in Escherichia coli.
- Author
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Sung WL, Chan BS, Luk CK, Zahab DM, Willick GE, Barbier JR, Isaacs R, Maclean S, Ross V, Morley P, and Whitfield JF
- Subjects
- Adenylyl Cyclases metabolism, Amino Acid Sequence, Animals, Base Sequence, Cloning, Molecular, Escherichia coli, Female, Humans, Molecular Sequence Data, Osteosarcoma, Ovariectomy, Plasmids, Proinsulin genetics, Rats, Rats, Sprague-Dawley, Recombinant Fusion Proteins biosynthesis, Recombinant Proteins biosynthesis, Recombinant Proteins isolation & purification, Recombinant Proteins pharmacology, Sequence Deletion, Teriparatide chemistry, Teriparatide isolation & purification, Tumor Cells, Cultured, Osteoporosis prevention & control, Teriparatide pharmacology
- Abstract
A fully active analog of human parathyroid hormone (hPTH) has been produced by recombinant expression in Escherichia coli. Initially, a nucleotide sequence encoding hPTH(1-34)-Asp-Pro was ligated to a proinsulin gene in the plasmid pUC8, for the eventual expression of a fusion protein of 137 amino acids. Unexpectedly, the proinsulin gene and 340 bp downstream were deleted by an unknown mechanism during transformation of the E. coli. This resulted in a new plasmid encoding a small (72-amino acid) fusion product of hPTH(1-34)-Asp35-Pro36-X, where X is a 36-residue "arbitrary" downstream sequence of pUC8. The fusion product was efficiently expressed and the hPTH analog, [Asp35]hPTH-(1-35), was readily released by acid cleavage, with a yield of 100 mg/L. This analog had an effective concentration for half-maximal adenylyl cyclase stimulation (EC50) in rat osteosarcoma cells of 14 nM, which was identical to that for hPTH-(1-34). In the ovariectomized rat model of osteoporosis, [Asp35]hPTH-(1-35) was fully active as a bone anabolic agent.
- Published
- 2000
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39. Prolonged low-dose infusion of human parathyroid hormone does not increase femoral cancellous bone volume in ovariectomized rats.
- Author
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Morley P, Whitfield JF, Willick GE, Ross V, MacLean S, Isaacs RJ, Mendoza E, and Barbier JR
- Subjects
- Animals, Bone Density, Female, Femur, Humans, Rats, Rats, Sprague-Dawley, Bone and Bones drug effects, Osteoporosis prevention & control, Ovariectomy, Parathyroid Hormone administration & dosage, Peptide Fragments administration & dosage
- Abstract
Objective: Daily injections of human parathyroid hormone (hPTH) increase bone volume in various animal species and in osteoporotic women. For hPTH to be widely accepted as an anabolic therapy for treating postmenopausal osteoporosis alternative delivery options need to be explored to replace the need for daily patient subcutaneous self-injection. Among these are inhalation, oral delivery and the use of programmable implanted minipumps to deliver the peptide. While infusion of high doses of PTH causes bone loss and hypercalcemia, no studies have assessed the effects of prolonged infusion of low doses of PTH on bone growth., Design and Methods: [Leu(27)]-cyclo(Glu(22)-Lys(26))-hPTH-(1--31)NH(2) was delivered by Alzet minipumps to ovariectomized rats for 6 weeks after which histomorphometric indices (cancellous bone volume, trabecular thickness, mean trabecular number) of bone formation were measured in distal femurs., Results: Infusing low doses (0.05 and 0.1 nmole/100g body weight/day) of the hPTH analog, [Leu(27)]-cyclo(Glu(22)-Lys(26))-hPTH-(1--31)NH(2), for 6 weeks does not prevent the ovariectomy-induced loss of rat femoral cancellous bone volume, trabecular thickness or trabecular number., Conclusion: These results support the absolute requirement of daily injections for the osteogenic action of hPTH on bone.
- Published
- 1999
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40. Stimulation of membrane-associated protein kinase-C activity in spleen lymphocytes by hPTH-(1-31)NH2, its lactam derivative, [Leu27]-cyclo(Glu22-Lys26)-hPTH-(1-31)NH2, and hPTH-(1-30)NH2.
- Author
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Whitfield JF, Isaacs R, MacLean S, Morley P, Barbier JR, and Willick GE
- Subjects
- Animals, Dose-Response Relationship, Drug, Female, Humans, Osteosarcoma metabolism, Rats, Rats, Sprague-Dawley, Lymphocytes metabolism, Parathyroid Hormone metabolism, Peptide Fragments metabolism, Protein Kinase C metabolism, Spleen metabolism
- Abstract
Human parathyroid hormone, hPTH-(1-34), stimulates adenylyl cyclase and phosphatidylinositol-bisphosphate-specific phospholipase-C (PIP2-PLC), as indicated by increased membrane-associated protein kinase C (PKC) activity in ROS 17/2 rat osteosarcoma cells. The C-terminally truncated hPTH-(1-31)NH2 stimulates adenylyl cyclase as strongly as hPTH-(1-34) in these cells, but it does not stimulate PKC activity. Even [Leu27]-cyclo(Glu22-Lys26)-hPTH-(1-31)NH2, a 6-fold stronger adenylyl cyclase stimulator than hPTH-(1-34), cannot stimulate PKC activity in ROS cells. Therefore PTH required its 32-34 region to stimulate PIP2-PLC/PKCs in this osteosarcoma line. In contrast, hPTH-(1-31)NH2 [Leu27]-cyclo(Glu22-Lys26)-hPTH-(1-31)NH2 and even hPTH-(1-30)NH2 can stimulate PKC activity in freshly isolated rat spleen lymphocytes as strongly as hPTH-(1-34)NH2. The difference in the ability of membrane-associated PKC activity in spleen lymphocytes, but not in ROS cells, to be stimulated by C-terminally truncated PTH fragments might be due to different receptor densities or to the lymphocyte's atypical PTH/PTHrP receptor.
- Published
- 1999
- Full Text
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41. Comparison of the abilities of human parathyroid hormone (hPTH)-(1-34) and [Leu27]-cyclo(Glu22-Lys26)-hPTH-(1-31)NH2 to stimulate femoral trabecular bone growth in ovariectomized rats.
- Author
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Whitfield JF, Morley P, Willick G, MacLean S, Ross V, Isaacs RJ, and Barbier JR
- Subjects
- Adenylyl Cyclases metabolism, Animals, Bone Density drug effects, Bone Development drug effects, Cell Line, Female, Humans, Osteoblasts enzymology, Rats, Bone Density physiology, Bone Development physiology, Ovariectomy, Parathyroid Hormone pharmacology, Peptide Fragments pharmacology, Teriparatide pharmacology
- Abstract
hPTH-(1-31)NH2, so far the smallest of the potently anabolic N-terminal fragments of the human parathyroid hormone, stimulates trabecular growth in the distal femurs of ovariectomized (OVX) rats as strongly as hPTH-(1-34) when injected at a high daily dose such as 1 nmol/100 g of body weight, but it is only about 70% as effective as hPTH-(1-34) when injected at the suboptimal 0.6 nmol/100 g of body weight. A lactam derivative of hPTH-(1-31)-NH2, [Leu27]-cyclo(Glu22-Lys26)-hPTH-(1-31)NH2, is a much more effective stimulator of adenylyl cyclase in ROS 17/2 rat osteoblast-like cells and a significantly more effective stimulator of femoral trabecular growth in OVX rats than hPTH-(1-31)NH2. We have now shown that [Leu27]-cyclo(Glu22-Lys26)-hPTH-(1-31)NH2 prevents the OVX-induced loss of femoral trabeculae significantly more effectively than hPTH-(1-34) and stimulates the thickening of the trabeculae remaining in severely depleted femoral trabecular bone of OVX rats as effectively as hPTH-(1-34) when injected at 0.6 nmol/100 g of body weight.
- Published
- 1998
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42. Cyclization by a specific lactam increases the ability of human parathyroid hormone (hPTH)-(1-31)NH2 to stimulate bone growth in ovariectomized rats.
- Author
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Whitfield JF, Morley P, Willick G, Langille R, Ross V, MacLean S, and Barbier JR
- Subjects
- Adenylyl Cyclases metabolism, Analysis of Variance, Animals, Bone Neoplasms pathology, Disease Models, Animal, Female, Femur physiology, Humans, Molecular Weight, Osteoporosis, Postmenopausal prevention & control, Osteosarcoma pathology, Ovariectomy, Parathyroid Hormone chemistry, Parathyroid Hormone therapeutic use, Peptide Fragments chemical synthesis, Peptide Fragments chemistry, Peptide Fragments therapeutic use, Rats, Serine metabolism, Structure-Activity Relationship, Tumor Cells, Cultured, Valine metabolism, Bone Development drug effects, Femur drug effects, Lactams chemistry, Osteoporosis, Postmenopausal drug therapy, Parathyroid Hormone pharmacology, Peptide Fragments pharmacology
- Abstract
Human parathyroid hormone (hPTH)-(1-31)NH2 (Ostabolin), which only stimulates adenylyl cyclase (AC) instead of AC and phospholipase-C as do hPTH(1-84) and hPTH(1-34), strongly stimulates femoral cortical and trabecular bone growth in ovariectomized (OVX) rats. Two side-chain lactams have been introduced in the hydrophilic face of the receptor-binding region of the fragment's Ser17-Val31 amphiphilic alpha-helix in an attempt to develop improved analogs for the treatment of osteoporosis. Replacing the polar Lys27 with an apolar Leu on the hydrophobic face of this alpha-helix and stabilizing the helix with a lactam between Glu22 and Lys26 produced a fragment, [Leu27]-cyclo(Glu22-Lys26)-hPTH(1-31)NH2, which had six times the AC-stimulating ability of hPTH(1-31)NH2 in ROS 17/2 rat osteosarcoma cells, but the other helix-stabilizing lactam derivative [Leu27]-cyclo(Lys26-Arg30)-hPTH(1-31)NH2 did not have a greater AC-stimulating ability than hPTH(1-31)NH2, to stimulate AC in ROS 17/2 rat osteosarcoma cells. As expected from AC stimulation being responsible for PTH's anabolic action, [Leu27]-cyclo(Glu22-Lys26)-hPTH(1-31)NH2 was, depending on the experimental design, a 1.4 to 2 times better stimulator of trabecular bone growth in the OVX rat model than either hPTH(1-31)NH2 or [Leu27]-cyclo(Lys26-Arg30)-hPTH(1-31)NH2. Thus, there is now a more potently anabolic derivative of hPTH(1-31)NH2, [Leu27]-cyclo(Glu22-Lys26)-hPTH(1-31)NH2, which might ultimately prove to be one of the more effective therapeutics for osteoporosis.
- Published
- 1997
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43. Bioactivities and secondary structures of constrained analogues of human parathyroid hormone: cyclic lactams of the receptor binding region.
- Author
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Barbier JR, Neugebauer W, Morley P, Ross V, Soska M, Whitfield JF, and Willick G
- Subjects
- Amino Acid Sequence, Amino Acids analysis, Animals, Antihypertensive Agents chemical synthesis, Antihypertensive Agents metabolism, Antihypertensive Agents pharmacology, Blood Pressure drug effects, Circular Dichroism, Enzyme Activation, Humans, Lactams chemical synthesis, Lactams metabolism, Lactams pharmacology, Models, Molecular, Molecular Sequence Data, Osteoblasts enzymology, Parathyroid Hormone metabolism, Parathyroid Hormone pharmacology, Peptide Fragments chemical synthesis, Peptide Fragments pharmacology, Protein Conformation, Protein Structure, Secondary, Rats, Tumor Cells, Cultured, Adenylyl Cyclases metabolism, Antihypertensive Agents chemistry, Lactams chemistry, Parathyroid Hormone chemistry, Receptors, Parathyroid Hormone metabolism
- Abstract
In a search for analogues of human parathyroid hormone (hPTH) with improved activities and bioavailabilities, we have prepared the following three lactam analogues of hPTH-(1-31)-NH2 (1) or [Leu27]hPTH-(1-31)-NH2 (2): [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-31)-NH2 (3), [Leu27]cyclo(Lys26-Asp30)-hPTH-(1-31)-NH2 (4), and cyclo(Lys27-Asp30)-hPTH-(1-31)-NH2 (5). Analogues 1, 2, and 5 had seven or eight residues of alpha-helix, as estimated from their circular dichroism (CD) spectra, in contrast to 12 residues in cyclic analogues 3 and 4. Thus, lactams 3 and 4 stabilized a helix previously shown to exist within residues 17-29. The adenylyl cyclase activity (EC50), measured in rat osteosarcoma 17/2 cells, of 5 (40.3 +/- 2.3 nM) was half that of its linear form 1 (19.9 +/- 3.9 nM). The linear Leu27 mutant 2 was twice as active (11.5 +/- 5.2) as analogue 1, and lactam analogue 3 was 6-fold more active (3.3 +/- 0.3 nM). Lactam analogue 4 had less activity (16.9 +/- 3.3 nM) than 2, its linear form. Peptides hPTH-(1-30)-NH2 (6), [Leu27]hPTH-(1-30)-NH2 (7), and [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-30)-NH2 (8) all had AC-stimulating activities similar to that of 1. When injected intravenously, with a dose of 0.8 nmol/100 g of analogue in acid saline, hypotensive effects paralleled their adenylyl cyclase activities. They behaved quite differently when applied subcutaneously. Analogues 1, 5, and 6, the weakest, showed about half the drop in blood pressure observed with 3 and 4, the most active. In contrast, the time required to reach a maximum drop in blood pressure of 4-8, after subcutaneous administration, was 2-4 times that of the other analogues. Thus, the bioavailabilities of the lactam analogues, unlike their adenylyl cyclase-stimulating activities, were highly dependent on the presence or conformation of Val31.
- Published
- 1997
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44. The hypotensive actions of osteogenic and nonosteogenic parathyroid hormone fragments.
- Author
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Whitfield JF, Morley P, Ross V, Preston E, Soska M, Barbier JR, Isaacs RJ, Maclean S, Ohannessian-Barry L, and Willick GE
- Subjects
- Adenylyl Cyclases biosynthesis, Adenylyl Cyclases drug effects, Animals, Bone Development drug effects, Disease Models, Animal, Female, Femur drug effects, Femur pathology, Injections, Intravenous, Injections, Subcutaneous, Ovariectomy, Rats, Rats, Sprague-Dawley, Blood Pressure drug effects, Hypertension drug therapy, Osteogenesis drug effects, Parathyroid Hormone pharmacology, Peptide Fragments pharmacology
- Abstract
Parathyroid hormone (PTH), hPTH-(1-84), and its hPTH-(1-34), hPTH-(1-31)NH2, and hPTH-(1-30)NH2 fragments reduced the tail artery pressure in anesthetized female Sprague-Dawley rats by 42.4-67.1% within about 1 minute after injection into a femoral vein, but reduced the pressure by only 8.5-36.2% 2-19 minutes after subcutaneous injection. hPTH-(1-84) and hPTH-(1-34) stimulate both adenylyl cyclase and phospholipase-C in their target cells, but the hypotensive action must have been stimulated specifically by adenylyl cyclase activation, because hPTH-(1-30)NH2 and hPTH-(1-31)NH2, which can only stimulate adenylyl cyclase, were potently hypotensive when injected intravenously whereas hPTH-(7-84), which can only stimulate phospholipase-C, was not significantly hypotensive when injected intravenously. Since PTH's osteogenic action is also mediated by adenylyl cyclase stimulation, it was expected that the hypotensive response might be used to screen new PTH constructs for possible osteogenicity. Indeed, the osteogenic activities of subcutaneously injected hPTH-(1-31)NH2, hPTH-(1-34), and hPTH-(1-84) correlated closely to their hypotensive activities, with hPTH-(1-34) being much more hypotensive and significantly more osteogenic than the other two molecules. hPTH-(1-31)NH2 and hPTH-(1-84) were equally osteogenic and hypotensive. However, this correlation broke down with hPTH-(1-30)NH2 which does not stimulate bone formation, but in the present study it stimulated adenylyl cyclase and reduced tail artery pressure almost as much as hPTH-(1-31)NH2 and hPTH-(1-34). Nevertheless, the ability to significantly reduce arterial pressure is a common property of osteogenic PTH and PTH fragments and is thus a rapidly determinable preliminary indicator of in vivo bioactivity of PTH fragments.
- Published
- 1997
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45. Comparison of the ability of recombinant human parathyroid hormone, rhPTH-(1-84), and hPTH-(1-31)NH2 to stimulate femoral trabecular bone growth in ovariectomized rats.
- Author
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Whitfield JF, Morley P, Willick GE, Ross V, MacLean S, Barbier JR, Isaacs RJ, and Ohannessian-Barry L
- Subjects
- Animals, Female, Humans, Osteoporosis physiopathology, Ovariectomy, Rats, Recombinant Proteins administration & dosage, Signal Transduction drug effects, Bone Remodeling drug effects, Femur pathology, Osteoporosis drug therapy, Parathyroid Hormone administration & dosage, Parathyroid Hormone-Related Protein, Peptide Fragments administration & dosage, Proteins administration & dosage
- Abstract
A recombinant human parathyroid hormone, rhPTH-(1-84), which is currently in Phase II clinical trial, and hPTH-(1-31)NH2 (Ostabolin) are promising anabolic agents for treating osteoporosis because they can stimulate cortical and trabecular bone growth in osteopenic, ovariectomized (OVX) rats and in osteoporotic, postmenopausal women when injected subcutaneously and intermittently at low doses. We have now found that, despite their different sizes and signaling properties (rhPTH-(1-84) stimulates adenylyl cyclase and phospholipase C; hPTH-(1-31)NH2 only stimulates adenylyl cyclase), they are equally osteogenic in OVX rats. Thus daily subcutaneous injections of 0.6 nmol/100 g of body weight of rhPTH-(1-84) or hPTH-(1-31)NH2 into 3-month-old OVX rats for 6 weeks starting 2 weeks after OVX equally reduced the otherwise large OVX-triggered loss of femoral trabecular bone. Daily subcutaneous injections of 0. 4 or 0.8 nmol/100 g of body weight of the two agents for 6 weeks also equally increased the mean thickness of the remaining femoral trabeculae in 3-month-old and 1-year-old OVX rats to 20 to 80% above the value in normal animals when started 9 weeks after ovariectomy.
- Published
- 1997
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46. Peptide synthesis on chitin.
- Author
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Neugebauer W, Williams RE, Barbier JR, Brzezinski R, and Willick G
- Subjects
- Amino Acid Sequence, Chitin chemistry, Molecular Sequence Data, Peptides chemistry, Chitin chemical synthesis, Peptides chemical synthesis
- Abstract
The use of chitin as a support for solid-phase peptide synthesis is described and illustrated by synthesis of four peptides, varying in length from 10 to 29 residues. Syntheses were performed in a continuous-flow peptide synthesizer, using Fmoc chemistry. A cleavable linker, p-[(R,S)-alpha-[1-(9H-fluoren-9-yl)-methoxyformamido]-2,4-di methoxybenzyl]- phenoxyacetic acid, was attached to chitosan at the desired substitution level, and the complex acetylated to yield a linker substituted chitin. The effects of temperature, solvents and degree of linker substitution on the syntheses were studied. Acyl carrier peptide (ACP) synthesis studies indicated that temperature was the single most important parameter. Increasing the temperature of the synthesis from 20 to 55 degrees C resulted in an enormous improvement of this synthesis, with about 90% of the crude product being the correct peptide. Denaturing solvents, such as DMSO, could be used without significant effect on the flow properties of the support. The synthesis of one peptide was mainly improved by lowering the degree of substitution from 0.3 to 0.1 mmol/g, suggesting peptide aggregation was a problem in this case. The results of three syntheses on chitin were comparable with those obtained with a commonly used commercial support. This work shows that, under appropriate conditions, chitin can be utilized directly as a support for peptide synthesis.
- Published
- 1996
- Full Text
- View/download PDF
47. Stimulation of the growth of femoral trabecular bone in ovariectomized rats by the novel parathyroid hormone fragment, hPTH-(1-31)NH2 (Ostabolin).
- Author
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Whitfield JF, Morley P, Willick GE, Ross V, Barbier JR, Isaacs RJ, and Ohannessian-Barry L
- Subjects
- Animals, Bone Density drug effects, Calcium analysis, Disease Models, Animal, Dose-Response Relationship, Drug, Female, Femur chemistry, Femur drug effects, Femur growth & development, Femur ultrastructure, Injections, Subcutaneous, Microscopy, Electron, Scanning, Rats, Rats, Sprague-Dawley, Bone Development drug effects, Ovariectomy, Parathyroid Hormone pharmacology, Peptide Fragments pharmacology
- Abstract
The human parathyroid hormone, hPTH-(1-84), and its hPTH-(1-34) fragment are promising anabolic agents for treating osteoporosis because they can strongly stimulate the production of biomechanically effective cortical and trabecular bone in osteopenic ovariectomized (OVX) rats and trabecular bone in osteoporotic postmenopausal humans. The ideal PTH fragment for treating osteoporosis would be the smallest and functionally simplest fragment that activates only one signal mechanism and still strongly stimulates trabecular bone growth. A new PTH fragment, hPTH-(1-31)NH2, which only stimulates adenylyl cyclase instead of stimulating both adenylyl cyclase and phospholipase-C as do hPTH-(1-84) and hPTH-(1-34), is this minimum, high-potency anabolic fragment. hPTH-(1-31)NH2 (which we have named Ostabolin) can greatly thicken trabeculae and increase the dry weight and calcium content of trabecular bone in the distal femurs of osteopenic, young, sexually mature OVX Sprague-Dawley rats when injected subcutaneously each day for 6 weeks at doses between 0.4 and 1.6 nmole/100 g of body weight.
- Published
- 1996
- Full Text
- View/download PDF
48. Solution structure and adenylyl cyclase stimulating activities of C-terminal truncated human parathyroid hormone analogues.
- Author
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Neugebauer W, Barbier JR, Sung WL, Whitfield JF, and Willick GE
- Subjects
- Animals, Circular Dichroism, Enzyme Activation, Humans, Parathyroid Hormone chemistry, Peptide Fragments chemistry, Peptide Fragments physiology, Protein Conformation, Protein Structure, Secondary, Rats, Solutions, Tumor Cells, Cultured, Adenylyl Cyclases metabolism, Parathyroid Hormone physiology
- Abstract
Analogues of human parathyroid hormone (hPTH) truncated at the C-terminal end have been studied for adenylyl cyclase (AC) activity and for solution conformation by circular dichroism (CD) spectroscopy. Analogues of hPTH-(1-34)-NH2, containing the first 28-31 residues, had only a slightly diminished ability to stimulate AC in rat osteosarcoma (ROS) cells as compared to that of the parent analogue. CD data on hPTH-(16-34)-NH2 and C-terminal deletion mutants of hPTH-(1-34)-NH2 supported the presence of a partially stable alpha-helix over residues 17-28. A carboxyl-terminal mutant, hPTH-(1-30)-OH, showed both reduced helix and greatly reduced AC-stimulating activity as compared to the corresponding amide analogue. In contrast, both of these analogues, in the presence of palmitoyloleoylphosphatidylserine (POPS) vesicles, showed an equal stabilization of alpha-helix. All other analogues showed at least some enhancement of alpha-helix in the presence of POPS. However, both in neutral, aqueous buffer and in POPS, the relative amount of alpha-helix decreased greatly as the peptide was shortened below the 1-28 sequence. These data provide additional support for an amphiphilic alpha-helix over residues 21-28 being the conformation for receptor binding of hPTH for stimulation of AC activity. Modeling human parathyroid hormone-related peptide as an alpha-helix over this same region, and comparison to hPTH, suggests that both may bind via the hydrophobic face to the receptor.
- Published
- 1995
- Full Text
- View/download PDF
49. Internal ribosome-binding site directs expression of parathyroid hormone analogue (8-84) in Escherichia coli.
- Author
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Sung WL, Luk CK, Zahab DM, Barbier JR, Lafontaine M, and Willick GE
- Subjects
- Binding Sites, Cloning, Molecular, Codon, Genes, Humans, Oligodeoxyribonucleotides, Plasmids, Escherichia coli genetics, Genes, Synthetic, Parathyroid Hormone genetics, Peptide Fragments genetics, Ribosomes metabolism
- Abstract
Expression of the human parathyroid hormone (PTH) gene in E. coli yielded intact PTH and PTH-(8-84). To determine if PTH-(8-84) is the result of a competing translation initiated from methionine codon-8 or degradation of the intact PTH, twelve new gene constructs with or without an internal ribosome-binding site (iRBS) in the PTH-(1-5) region were prepared via substitution with degenerate codons. Expression of constructs without iRBS produced only intact PTH. Constructs with weak iRBS, including one that resembles the cDNA sequence, yielded PTH-(8-84) as a minor product. In contrast, constructs with strong iRBS produced predominantly or exclusively this shorter analogue.
- Published
- 1991
- Full Text
- View/download PDF
50. Specific degenerate codons enhanced selective expression of human parathyroid hormone in Escherichia coli.
- Author
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Sung WL, Zahab DM, Barbier JR, Watson D, Yaguchi M, Neugebauer W, and Willick GE
- Subjects
- Amino Acid Sequence, Base Sequence, Blotting, Western, Chromatography, Ion Exchange, Codon, Electrophoresis, Polyacrylamide Gel, Escherichia coli genetics, Gene Expression Regulation, Bacterial, Genes, Bacterial, Humans, Molecular Sequence Data, Plasmids, Parathyroid Hormone genetics
- Abstract
Specific degenerate codons in the amino-terminal region of a synthetic human parathyroid hormone (PTH) gene exerted dramatic effects on both products and yield of expression of this 84-amino acid polypeptide in Escherichia coli. With adenine-rich degenerate codons constituting the PTH-(1-5) region, intact PTH has been expressed as the only PTH product at 6.5 mg/liter. In contrast, with guanine-rich degenerate codons, the predominent product was analogue PTH-(8-84). Use of cytosine- or thymine-rich degenerate codons generated only a small amount of immunoreactive product (0.2 mg/l). With the amino terminal region reconstituted with adenine-rich degenerate codons, the mid and carboxyl regions of the synthetic gene were also reconstructed to imitate the E. coli-favored codon degeneracy. Expression yielded the intact PTH at 20 mg/liter. Gel electrophoresis and Western blots, with antibodies specific to the amino or carboxyl terminus of PTH, indicated only a single PTH-related polypeptide, with the same mobility as a synthetic intact PTH sample. Amino acid sequencing, composition analysis, mass spectrometry, and the adenylate cyclase bioassays confirmed the purified product as the processed intact PTH.
- Published
- 1991
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