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1.
Acta Naturae ; 14(2): 16-38, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35923566

RESUMEN

1,4-Pyrazine-3-carboxamide-based antiviral compounds have been under intensive study for the last 20 years. One of these compounds, favipiravir (6-fluoro-3-hydroxypyrazine-2-carboxamide, T-705), is approved for use against the influenza infection in a number of countries. Now, favipiravir is being actively used against COVID-19. This review describes the in vivo metabolism of favipiravir, the mechanism of its antiviral activity, clinical findings, toxic properties, and the chemical synthesis routes for its production. We provide data on the synthesis and antiviral activity of structural analogs of favipiravir, including nucleosides and nucleotides based on them.

2.
Dokl Biochem Biophys ; 503(1): 52-58, 2022 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-35538278

RESUMEN

In this work, a comparative analysis of the conditions of transglycosylation reactions catalyzed by E. coli nucleoside phosphorylases was carried out, and the optimal conditions for the formation of various nucleosides were determined. Under the optimized conditions of transglycosylation reaction, fluorine-containing derivatives of N6-benzyl-2'-deoxyadenosine, potential inhibitors of replication of enteroviruses in a cell, were obtained starting from the corresponding ribonucleosides.


Asunto(s)
Escherichia coli , Flúor , Glicosilación , Nucleósidos , Purinas
3.
Acta Naturae ; 8(4): 82-90, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-28050269

RESUMEN

We propose a new approach for the synthesis of biologically important nucleotides which includes a multi-enzymatic cascade conversion of D-pentoses into purine nucleotides. The approach exploits nucleic acid exchange enzymes from thermophilic microorganisms: ribokinase, phosphoribosylpyrophosphate synthetase, and adenine phosphoribosyltransferase. We cloned the ribokinase gene from Thermus sp. 2.9, as well as two different genes of phosphoribosylpyrophosphate synthetase (PRPP-synthetase) and the adenine phosphoribosyltransferase (APR-transferase) gene from Thermus thermophilus HB27 into the expression vectors, generated high-yield E. coli producer strains, developed methods for the purification of the enzymes, and investigated enzyme substrate specificity. The enzymes were used for the conversion of D-pentoses into 5-phosphates that were further converted into 5-phospho-α-D-pentofuranose 1-pyrophosphates by means of ribokinase and PRPP-synthetases. Target nucleotides were obtained through the condensation of the pyrophosphates with adenine and its derivatives in a reaction catalyzed by APR-transferase. 2-Chloro- and 2-fluoroadenosine monophosphates were synthesized from D-ribose and appropriate heterobases in one pot using a system of thermophilic enzymes in the presence of ATP, ribokinase, PRPP-synthetase, and APR-transferase.

4.
Bull Exp Biol Med ; 158(5): 670-2, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25778656

RESUMEN

The therapeutic efficiency of recombinant thymosin ß4 (rTß4) synthesized by us was studied in vivo on spontaneous CBRB mouse model that is adequate to human chronic dermatitis. Three applications of the drug during a week significantly alleviated symptoms of the disease in female mice, and in complex with subsequent antibacterial and antifungal therapy led to a pronounced and lasting (2 months) therapeutic effect. The results attest to a possibility of using rTß4 in combination with the known treatment protocols for chronic inflammatory diseases of the skin.


Asunto(s)
Dermatitis/tratamiento farmacológico , Proteínas Recombinantes/uso terapéutico , Timosina/uso terapéutico , Animales , Enfermedad Crónica/tratamiento farmacológico , Modelos Animales de Enfermedad , Femenino , Ratones
5.
Bull Exp Biol Med ; 158(1): 164-9, 2014 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-25408529

RESUMEN

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.


Asunto(s)
Adipogénesis , Pulpa Dental/citología , Células Madre Mesenquimatosas/fisiología , Adolescente , Antígenos CD/metabolismo , Proliferación Celular , Células Cultivadas , Humanos
6.
Bioorg Khim ; 38(1): 78-85, 2012.
Artículo en Ruso | MEDLINE | ID: mdl-22792709

RESUMEN

Pigment epithelium-derived factor (PEDF), a 50 kDa secreted glycoprotein, is among the most potent endogenous inhibitors of angiogenesis. PEDF-derived fragment (44-77) possesses antiangiogenic properties of the full-sized protein and is a potential drug candidate for the treatment of ocular neovascular diseases. In this study we propose an efficient scalable biotechnological method for the production of PEDF (44-77) as part of a fusion protein with SspDnaB intein. The fusion protein was obtained in bacterial E. coli cells in the form of inclusion bodies, solubilized and subjected to autocatalytic cleavage with the release of PEDF (44-77) (yield, 77%). The target peptide was separated from the intein using tangential ultrafiltration. The final purification of PEDF (44-77) was performed by reversed-phase HPLC. The yield of the target peptide (purity, 99%) was 65 mg per 1 liter of culture. Antiangiogenic activity of the obtained peptide was studied in vitro using murine endothelial cells SVEC-4-10. PEDF (44-77) suppressed proliferation of endothelial cells by 53% and inhibited endothelial cell tube formation at the concentration of 1 nM. The ability of the recombinant PEDF (44-77) to block initial stages of angiogenesis was demonstrated using the model of rabbit corneal neovascularization.


Asunto(s)
Inhibidores de la Angiogénesis/biosíntesis , Inhibidores de la Angiogénesis/farmacología , Neovascularización de la Córnea/prevención & control , Proteínas del Ojo/biosíntesis , Proteínas del Ojo/farmacología , Factores de Crecimiento Nervioso/biosíntesis , Factores de Crecimiento Nervioso/farmacología , Serpinas/biosíntesis , Serpinas/farmacología , Inhibidores de la Angiogénesis/genética , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Neovascularización de la Córnea/tratamiento farmacológico , Neovascularización de la Córnea/genética , Neovascularización de la Córnea/metabolismo , Neovascularización de la Córnea/patología , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Células Endoteliales/patología , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas del Ojo/genética , Humanos , Ratones , Factores de Crecimiento Nervioso/genética , Conejos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Serpinas/genética
7.
Bioorg Khim ; 38(2): 166-76, 2012.
Artículo en Ruso | MEDLINE | ID: mdl-22792720

RESUMEN

Hirudin-1 is a highly selective inhibitor of thrombin secreted by salivary glands of the medicinal leech Hirudo medicinalis. This direct anticoagulant is used for the treatment and prevention of disorders in blood coagulation system. Apart from the existing recombinant analog of hirudin-1 (63-desulfatohirudin-1, desirudin) its modified analogs possessing higher activity and stability are of medical value. In this study artificial genes of hirudin and two its analogs (hirudin-1, [Leu1, Thr2]-hirudin-1 and [Leu1, Thr2]-hirudin-1/3) were synthesized and cloned in an expression vector pTWIN1 in frame with the gene of mini-intein SspDnaB from Synechocystis sp. Producing strains of the corresponding fusion proteins were constructed using E. coli strain ER2566. Biotechnological schemes for the production of 63-desulfatohirudin-1 and its analogs were developed. The scheme includes the following stages: isolation of the fusion protein after the desintegration of the cell biomass, refolding of the target peptide within the fusion protein, pH-inducible cleavage of the fusion protein, and chromatographic purification of the target product. Antithrombotic activity of the obtained peptides was determined by a standard amidolytic assay. The developed methods for the production of 63-desulfatohirudin-1, [Leu1, Thr2]-desulfatohirudin-1 [Leu1, Thr2]-desulfatohirudin-1/3 allowed to obtain these peptides with high yields (14, 25 and 24 mg per liter of cell culture respectively) and high activity (13423, 33333 and 19802 ATU/mg respectively).


Asunto(s)
Biotecnología/métodos , Hirudinas , Hirudo medicinalis , Animales , Escherichia coli/genética , Escherichia coli/metabolismo , Hirudinas/biosíntesis , Hirudinas/química , Hirudinas/genética , Hirudinas/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación
8.
Bioorg Khim ; 37(4): 490-5, 2011.
Artículo en Ruso | MEDLINE | ID: mdl-22096991

RESUMEN

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.


Asunto(s)
Aciclovir/farmacología , Simplexvirus/enzimología , Timidina Quinasa/metabolismo , Secuencia de Aminoácidos , Antivirales/metabolismo , Antivirales/farmacología , Clonación Molecular , Escherichia coli/genética , Expresión Génica , Humanos , Datos de Secuencia Molecular , Mutación , Análisis de Secuencia de ADN , Timidina Quinasa/antagonistas & inhibidores , Timidina Quinasa/química , Timidina Quinasa/aislamiento & purificación
9.
Bioorg Khim ; 37(2): 223-32, 2011.
Artículo en Ruso | MEDLINE | ID: mdl-21721255

RESUMEN

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.


Asunto(s)
Enfermedades de la Córnea/tratamiento farmacológico , Infarto del Miocardio/tratamiento farmacológico , Timosina/metabolismo , Timosina/uso terapéutico , Úlcera Varicosa/tratamiento farmacológico , Anhídridos Acéticos/química , Acetilación , Biotecnología/métodos , Cromatografía Líquida de Alta Presión , Humanos , Concentración de Iones de Hidrógeno , Proteínas Recombinantes de Fusión/genética , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Timosina/síntesis química , Timosina/genética
10.
Bioorg Khim ; 33(2): 245-50, 2007.
Artículo en Ruso | MEDLINE | ID: mdl-17476985

RESUMEN

An artificial gene encoding oxyntomodulin was obtained using chemical and enzymatic methods and cloned into Escherichia coli. A recombinant plasmid was constructed containing a hybrid oxyntomodulin gene and Ssp dnaB intein from Synechocystis sp. The expression of the resulting hybrid gene in E. coli, its properties, and the conditions of its autocatalytic cleavage to oxyntomodulin were studied.


Asunto(s)
Oxintomodulina/biosíntesis , Proteínas Recombinantes/biosíntesis , Secuencia de Aminoácidos , Catálisis , AdnB Helicasas/biosíntesis , AdnB Helicasas/genética , Escherichia coli/genética , Inteínas/genética , Datos de Secuencia Molecular , Mutación , Oxintomodulina/genética , Oxintomodulina/aislamiento & purificación , Plásmidos/química , Plásmidos/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Synechocystis/enzimología , Synechocystis/genética
11.
Biochemistry (Mosc) ; 72(1): 21-8, 2007 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-17309433

RESUMEN

Substrate specificity of Escherichia coli thymidine phosphorylase to thymidine derivatives modified at 5' -, 3' -, and 2' ,3' - positions of the sugar moiety was studied. Equilibrium and kinetic constants (K(m), K(I), k(cat)) of the phosphorolysis reaction have been determined for 20 thymidine analogs. The results are compared with X-ray and molecular dynamics data. The most important hydrogen bonds in the enzyme-substrate complex are revealed.


Asunto(s)
Sustitución de Aminoácidos , Escherichia coli/enzimología , Timidina Fosforilasa/química , Timidina/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Concentración de Iones de Hidrógeno , Estructura Molecular , Nucleósidos/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato , Timidina Fosforilasa/metabolismo
13.
Bioorg Khim ; 30(6): 613-20, 2004.
Artículo en Ruso | MEDLINE | ID: mdl-15586813

RESUMEN

The biotechnological method of synthesis of ribavirin, vidarabin, and 6-azauridine by the use of immobilized recombinant enzymatic preparations of nucleoside phosphorylase was improved. The effect of ribavirin and its combinations with the other synthesized nucleosides on the reproduction of Vaccinia virus was studied using cultures of Vero cells. The combination of ribavirin and vidarabin was shown to provide an antiviral effect at lesser concentrations than when these compounds were taken separately. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 6; see also http://www.maik.ru.


Asunto(s)
Antivirales/síntesis química , Nucleósidos/síntesis química , Ribavirina/análogos & derivados , Ribavirina/síntesis química , Virus Vaccinia/efectos de los fármacos , Animales , Antivirales/farmacología , Azauridina/análogos & derivados , Azauridina/síntesis química , Azauridina/farmacología , Catálisis , Chlorocebus aethiops , Interacciones Farmacológicas , Enzimas Inmovilizadas , Nucleósidos/farmacología , Purina-Nucleósido Fosforilasa/química , Ribavirina/farmacología , Uridina Fosforilasa/química , Células Vero , Vidarabina/análogos & derivados , Vidarabina/síntesis química , Vidarabina/farmacología , Replicación Viral/efectos de los fármacos
14.
Bioorg Khim ; 30(5): 481-6, 2004.
Artículo en Ruso | MEDLINE | ID: mdl-15562968

RESUMEN

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.


Asunto(s)
Ingeniería de Proteínas/métodos , Timosina/análogos & derivados , Timosina/genética , Timosina/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Escherichia coli/genética , Inteínas/genética , Datos de Secuencia Molecular , Empalme de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Timalfasina
15.
Mol Biol (Mosk) ; 38(5): 907-13, 2004.
Artículo en Ruso | MEDLINE | ID: mdl-15554192

RESUMEN

4-Thiouridine and 4-thiothymidine were developed as efficient substrates for spectrophotometric determination of uridine phosphorylase and thymidine phosphorylase activity. 4-Thiouridine has maximum absorbance at 330 nm (pH 7.5). The change in extinction coefficient for 4-thiouridine/4-thiouracil, deltaepsilon is 3000 M(-1) x cm(-1). It appeared that 4-thiouridine is a good substrate for uridine phosphorylase with Michaelis-Menten constant 130 microM and kcat 49 s(-1). In the case of 4-thiothymidine/4-thiothymine deltaepsilon is even larger: 5000 M(-1) x cm(-1) at 336 nm.


Asunto(s)
Pentosiltransferasa/metabolismo , Tiouridina/química , Timidina/análogos & derivados , Timidina/química , Espectroscopía de Resonancia Magnética , Pentosiltransferasa/química , Pirimidina Fosforilasas , Espectrofotometría , Especificidad por Sustrato , Tiouridina/metabolismo , Timidina/metabolismo
16.
Bioorg Khim ; 29(6): 616-22, 2003.
Artículo en Ruso | MEDLINE | ID: mdl-14743536

RESUMEN

New methods of chemical and chemoenzymatic synthesis of nucleoside 5'-thiophosphates and 5'-alpha-thiotriphosphates are developed. The 5'-alpha-thiotriphosphates are used as substrates both in template-dependent enzymatic PCR synthesis and in a T7-RNA transcription polymerase system. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.


Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Nucleósidos/síntesis química , Compuestos Organofosforados/química , Compuestos de Sulfhidrilo/química , Secuencia de Bases , Electroforesis en Gel de Agar , Resonancia Magnética Nuclear Biomolecular , Nucleósidos/química , Nucleósidos/metabolismo , Especificidad por Sustrato , Proteínas Virales
17.
Bioorg Khim ; 27(1): 40-4, 2001.
Artículo en Ruso | MEDLINE | ID: mdl-11255641

RESUMEN

Renaturation of recombinant human interleukin-3 produced as inclusion bodies in the transformed cells of Escherichia coli was studied and optimized. Importance was shown of removing from the protein solution the hydrophobic cellular components causing irreversible aggregation of the protein under renaturation conditions. An effect of pH on the secondary structure of the denatured protein was revealed by CD spectroscopy. It was thereby found that at pH 8.5, which is the optimal value for denaturation, the protein has the secondary structure most close to the native one. The isolation according to the scheme proposed allows preparation of interleukin-3 in 50% yield with 99% purity and biological activity 2 x 10(7) U/mg.


Asunto(s)
Interleucina-3/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Escherichia coli , Humanos , Interleucina-3/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación
18.
Bioorg Khim ; 25(7): 548-53, 1999 Jul.
Artículo en Ruso | MEDLINE | ID: mdl-10563208

RESUMEN

To evaluate the effect on translation of distal regions of the encoding mRNA part capable of the complementary binding to the ribosome binding site (RBS), a series of plasmids were constructed containing fragments inserted into the il3 gene and determining secondary interactions in mRNA. A comparison of the levels of the in vivo gene expression showed that the complementary interactions of the translation initiation region (TIR) with distal regions of the mRNA encoding part affect translation. The effectiveness of these interactions decreased with an increase in the distance between the RBS and the complementary mRNA region, whereas the secondary structure formed by the TIR and the adjacent mRNA region was more stable despite the presence of regions in mRNA capable of forming energetically more favorable structures involving these elements.


Asunto(s)
Codón Iniciador/genética , Escherichia coli/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , Conformación de Ácido Nucleico , Plásmidos/genética , ARN Mensajero/química , Ribosomas/genética
19.
Bioorg Khim ; 23(6): 492-6, 1997 Jun.
Artículo en Ruso | MEDLINE | ID: mdl-9265471

RESUMEN

Artificial genes were synthesized by the PCR method. Single-stranded DNA contained in an unpurified mixture of oligodeoxynucleotides after automated synthesis was used as a template. The features of this approach were studied.


Asunto(s)
Genes Sintéticos , Reacción en Cadena de la Polimerasa/métodos , Moldes Genéticos , Animales , Escherichia coli , Plasmodium falciparum/genética
20.
Bioorg Khim ; 23(12): 949-52, 1997 Dec.
Artículo en Ruso | MEDLINE | ID: mdl-9499370

RESUMEN

Artificial genes for chains A and B of ectatomin, an Ectatomma tuberculatum ant toxin, were obtained by chemical and enzymic synthesis and cloned into new plasmid vectors. Expression plasmids with the genes of hybrid proteins were constructed containing human interleukin-3 or its terminal 63-mer fragment as well as chains A and B of ectatomin, which are linked via a region containing the cleavage site of specific protease, enterokinase (hybrid proteins IL3ETOXA, IL3ETOXB, ILETOXA, and ILETOXB). Escherichia coli producer strains providing a high yield of IL3ETOXA and IL3ETOXB proteins as inclusion bodies were obtained.


Asunto(s)
Venenos de Hormiga/biosíntesis , Secuencia de Aminoácidos , Venenos de Hormiga/química , Venenos de Hormiga/genética , Secuencia de Bases , Electroforesis , Electroforesis en Gel de Poliacrilamida , Enteropeptidasa/química , Escherichia coli/genética , Vectores Genéticos , Humanos , Interleucina-3/genética , Datos de Secuencia Molecular , Plásmidos , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
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