RESUMO
We studied cell cultures isolated from the pulp of third molar germ of an adult human and from the skin of a human fetus on gestation day 10. Both cultures expressed similar repertoire of surface markers typical of multipotent mesenchymal cells (CD44, CD90, and CD105). Under in vitro conditions, dental pulp cells were more susceptible to factors inducing their differentiation into adipogenic, chondrogenic, and osteogenic lineage cells.
Assuntos
Adipogenia , Polpa Dentária/citologia , Células-Tronco Mesenquimais/fisiologia , Adolescente , Antígenos CD/metabolismo , Proliferação de Células , Células Cultivadas , HumanosRESUMO
A thymidine kinase UL23 gene (EC 2.7.1.145) from an acyclovir-sensitive strain L2 of herpes simplex virus type 1 was cloned and expressed in E. coli. Enzyme was purified by chromatography to a homogeneous state controlled by PAG electrophoresis. The Michaelis constants for the reactions with thymidine and an acyclovir were determined. It was found that enzyme phosphorilate some modified nucleosides such as d2T, d4T, d2C, 3TC, FLT, BVDU, GCV. A comparison of the purified enzyme properties and properties ofthymidine kinase of other strains of herpes simplex virus, previously published was carried out. It is shown that enzyme is inhibited by acyclovir H-phosphonate.
Assuntos
Aciclovir/farmacologia , Simplexvirus/enzimologia , Timidina Quinase/metabolismo , Sequência de Aminoácidos , Antivirais/metabolismo , Antivirais/farmacologia , Clonagem Molecular , Escherichia coli/genética , Expressão Gênica , Humanos , Dados de Sequência Molecular , Mutação , Análise de Sequência de DNA , Timidina Quinase/antagonistas & inibidores , Timidina Quinase/química , Timidina Quinase/isolamento & purificaçãoRESUMO
Thymosin beta4 (43 aa) is a highly conserved acidic peptide which regulates actin polymerization in mammalian cells by sequestering globular actin. Thymosin beta4 is undergoing clinical trials as a drug for the treatment of venous stasis ulcers, corneal wounds and injuries, as well as acute myocardial infarction. Currently, thymosin beta4 is produced with solid-phase chemical synthesis. Biotechnological synthesis of this peptide presents difficulties because N-terminal amino acid residue of thymosin beta4 is acetylated. In this study we propose a method for producing the recombinant precursor of thymosin beta4 and its subsequent targeted chemical acetylation. Desacetylthymosin beta4 was synthesized as a part of a hybrid protein with thioredoxin and a specific TEV (tobacco etch virus) protease cleavage site. The following scheme was developed for the purification of desacetylthymosin beta4: (i) the biosynthesis of a soluble hybrid protein (HP) in Escherichia coli; (ii) isolation of the HP by ion exchange chromatography; (iii) cleavage of the HP with TEVprotease; (iv) purification of desacetylthymosin beta4 by ultra-filtration. N-terminal acetylation of desacetylthymosin beta4 was performed with acetic anhydride under acidic conditions (pH 3). The reaction yield was 55%. Thymosin beta4 was then purified by reverse-phase high performance liquid chromatography. The proposed synthetic approach to recombinant thymosin beta4 is suitable for scale-up and can provide for the medical use of highly purified preparation with a yield of 20 mg from 1 L of culture.
Assuntos
Doenças da Córnea/tratamento farmacológico , Infarto do Miocárdio/tratamento farmacológico , Timosina/metabolismo , Timosina/uso terapêutico , Úlcera Varicosa/tratamento farmacológico , Anidridos Acéticos/química , Acetilação , Biotecnologia/métodos , Cromatografia Líquida de Alta Pressão , Humanos , Concentração de Íons de Hidrogênio , Proteínas Recombinantes de Fusão/genética , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Timosina/síntese química , Timosina/genéticaRESUMO
An artificial gene encoding thymosin alpha1 was obtained by the chemoenzymatic synthesis and cloned into Escherichia coli. An expressing recombinant plasmid containing the hybrid protein gene, which encodes amino acid sequences of thymosin alpha1 and the Saccharomyces cerevisiae intein Sce VMA, was constructed. The expression of the hybrid protein from the resulting hybrid gene in E. coli, the properties of the resulting hybrid protein, and the conditions for its nonenzymatic cleavage to thymosin alpha1 were studied. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 5; see also http: // www.maik.ru.
Assuntos
Engenharia de Proteínas/métodos , Timosina/análogos & derivados , Timosina/genética , Timosina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Escherichia coli/genética , Inteínas/genética , Dados de Sequência Molecular , Processamento de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , TimalfasinaRESUMO
Dietary components making a great contribution to the formation of human internal irradiation dose were defined. The values of soil-milk transition ratios that are ranging 0.3 to 41 E-9 MBq.kg-1.Bq-1.m2 have been obtained from the examination of 1300 milk specimens by radiochemical assay for 90Sr. The relationship of soil-milk and soil-plant transition ratios was examined to the type of soils and the distance from the NPP.