RESUMEN
The interaction of "core" 2'-5'-oligoadenylates (2-5A) and their analogues with proteins albumin, interferon and immunoglobulin G was studied by fluorescence spectroscopy methods. Strong quenching of protein fluorescence (up to 67%) was observed upon interaction of oligoadenylates in concentration of 5 x 10(-5) M with albumin. Investigated compounds quenched the emission of interferon to a lesser extent, whereas no significant fluorescence changes occurred upon interaction with immunoglobulin under the same conditions. The level of quenching depended on the structure of 2-5A compounds and decreased with the decrease of their concentration. These data suggest that 2-5A actively bind to albumin and less efficiently to interferon, and practically do not interact with immunoglobulin. Taking into consideration different efficiency of quenching of the fluorescence excited at 280 and 296 nm, the assumption has been made about a possible role of tyrosine and tryptophan in the binding of a given compound to proteins. Possible mechanisms of interaction of oligoadenylates with proteins are discussed.
Asunto(s)
Nucleótidos de Adenina/química , Inmunoglobulina G/química , Interferón-alfa/química , Oligorribonucleótidos/química , Albúmina Sérica/química , Relación Dosis-Respuesta a Droga , Humanos , Oligopéptidos , Unión Proteica , Espectrometría de FluorescenciaRESUMEN
Pegylated interferon alpha-2b (PEG-IFN alpha-2b) is a domestic preparation of a modified recombinant interferon alpha-2b with prolonged effect. The preparation was obtained by N-terminal pegylation of IFN alpha-2b with polyethylene glycol (PEG). This paper presents the method of PEG-IFN alpha-2b synthesis and characteristics of the obtained product. PAAG electrophoresis, Western blot analysis and MALDI-TOF mass-spectrometry confirm that the preparation is an N-terminal pegylated IFN alpha-2b that contains no more than 10% of dipegylated IFN alpha-2b. The comparison of PEG-IFN alpha-2b with its foreign analogue has revealed the similarity of their biological activity and pharmacokinetic parameters.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Interferón-alfa/química , Interferón-alfa/farmacología , Tecnología Farmacéutica/métodos , Animales , Antivirales/administración & dosificación , Antivirales/sangre , Antivirales/aislamiento & purificación , Bovinos , Línea Celular , Efecto Citopatogénico Viral/efectos de los fármacos , Preparaciones de Acción Retardada , Portadores de Fármacos/química , Humanos , Interferón alfa-2 , Interferón-alfa/administración & dosificación , Interferón-alfa/sangre , Interferón-alfa/aislamiento & purificación , Modelos Moleculares , Polietilenglicoles , Ratas , Ratas Wistar , Proteínas Recombinantes , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Protein splicing is a post-translational autocatalytic process that results in excision of internal peptide (intein) from a precursor protein and the ligation of the flanking protein sequences (exteins). High specificity of the intein-mediated excision of protein precursors allows the use of protein splicing in biotechnology. This work was aimed at the obtaining of human growth hormone with a native N-terminus in E. coli. Chimerical protein consisting of a short N-terminal peptide, Mxe GyrA intein and human growth hormone was created. During the translation formyl-methionine modified N-terminal peptide should have been removed by splicing. Intein was shown to mediate the cleavage of exteins, but their subsequent ligation was not observed. That allowed the preparation of human growth hormone with a native N-terminus. The effect of various factors on cleavage efficiency was studied. The most efficient cleavage of chimeric protein (60-80%) was achieved in the presence of inductor (100 mM beta-mercaptoethanol) upon the incubation for 4-6 days.
Asunto(s)
Escherichia coli/genética , Hormona del Crecimiento/biosíntesis , Inteínas/genética , Ingeniería de Proteínas , Empalme de Proteína/genética , Proteínas Recombinantes de Fusión/biosíntesis , Catálisis , Hormona del Crecimiento/genética , Humanos , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/genética , Proteínas Recombinantes de Fusión/genéticaRESUMEN
The delivery of peptide drugs via the buccal mucosa is more convenient and safe approach than most other delivery methods. However, the efficiency of the buccal system of protein delivery is not yet able to compete with injection method. To improve its efficacy a new generation of absorption promoters that would be sufficiently enchance penetration and at the same time cause no irritation or unpleasant taste should be developed. Lysalbinic acid, a product of the alkaline hydrolysis of egg albumin, meets those requirements. The paper describes production and general physico-chemical properties of the lysalbinic acid. Lysalbinic acid has been shown to increase permeability of the oral mucosa for some peptide compounds. It is recommended to use lysalbinic acid as an absorption enhancer for the development of novel buccal peptide drugs.
Asunto(s)
Albúminas/farmacología , Portadores de Fármacos/farmacología , Insulina/administración & dosificación , Mucosa Bucal/metabolismo , Péptidos/administración & dosificación , Absorción , Administración Bucal , Albúminas/química , Animales , Transporte Biológico , Cricetinae , Portadores de Fármacos/química , Técnicas In Vitro , Insulina/farmacocinética , Peso Molecular , Mucosa Bucal/efectos de los fármacos , Péptidos/farmacocinéticaRESUMEN
Inhibition of mollicutes by synthetic oligonucleotides and their analogs complementary to specific "signature" regions of 16S rRNA and corresponding sequences of ribosomal operon DNA was studied. It was shown that antisignature oligonucleotides inhibited transcription in vitro for above 79% interacting specifically with ribosomal operon and non-specific with DNA-dependent RNA-polymerase. The inhibition efficiency depended on oligonucleotide sequence and type of modification. Translation in vitro was suppressed most efficiently (up to 60%) by oligonucleotides complementary to 3'-end region of 16S rRNA, also depending on their modification. Translation in vivo was inhibited most efficiently (up to 73%) by thiophosphate analogs of oligonucleotides complementary to sequences 499-507 and 523-532 of 16S rRNA responsible for binding of ribosomal "core" protein S4 starting the assembly of 30S ribosome subunit. With the simultaneous use of the last two oligonucleotides, the growth of mollicutes in SM IMV-72 medium rich in exogenous sources of nucleosides was suppressed for over 90%. It is supposed that under conditions where mollicutes have no free access to starting materials for their own synthesis of nucleic acid these nucleotides could suppress microorganisms completely. Antisignature oligonucleotides are considered as superspecific agents not leading to the development of resistance of mollicutes and believed to be the main future remedy against diseased caused by microorganisms lacking the system of nucleoside synthesis.
Asunto(s)
Acholeplasma laidlawii/efectos de los fármacos , VIH-1 , Mycoplasma fermentans/efectos de los fármacos , Oligonucleótidos Antisentido/farmacología , Acholeplasma laidlawii/genética , Secuencia de Bases , ADN Bacteriano/efectos de los fármacos , ADN Bacteriano/genética , Depresión Química , Datos de Secuencia Molecular , Mycoplasma fermentans/genética , Biosíntesis de Proteínas/efectos de los fármacos , ARN Bacteriano/efectos de los fármacos , ARN Bacteriano/genética , ARN Ribosómico 16S/efectos de los fármacos , ARN Ribosómico 16S/genética , Transcripción Genética/efectos de los fármacos , Operón de ARNr/efectos de los fármacos , Operón de ARNr/genéticaRESUMEN
It is shown that the concentration of "antisignature" phosphorothioate analogs of oligodeoxynucleotides, complementary to the region of 165 rRNA Acholeplasma laidlawii PG-8 and Mycoplasma fermentans PG-18 responsible tor binding with ribosomal protein S4 being 0.5--1 microM synthesis of proteins in vivo decreases to 70%. A model of mechanisms is suggested to block oligonucleotides of the process of in vivo translation in mollicutes by "antisignature" phosphorothioate analogs. The advantages of the use of antisense oligonucleotides complementary to functionally significant plots of 16S rRNA to inhibit the in vivo translation are discussed in comparison with oligonucleotides, 5-nontranslated regions of mRNA serving a target for them.
Asunto(s)
Acholeplasma laidlawii/efectos de los fármacos , Mycoplasma fermentans/efectos de los fármacos , Oligodesoxirribonucleótidos/farmacología , Fosfatos/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Tionucleótidos/farmacología , Acholeplasma laidlawii/metabolismo , Radioisótopos de Carbono , Depresión Química , Mycoplasma fermentans/metabolismo , ARN Bacteriano/efectos de los fármacos , ARN Bacteriano/metabolismo , ARN Ribosómico 16S/efectos de los fármacos , ARN Ribosómico 16S/metabolismo , Factores de TiempoRESUMEN
The mechanism of the internucleotide condensation and side-reactions in H-phosphonate approach has been investigated with the help of NMR-spectroscopy. On the basis of the results obtained a modification minimizing side-reactions in the course of the nucleotide component preactivation has been developed. It includes the use of acetonitrile--quinoline (4:1) mixture as the solvent in coupling reactions. The efficiency of the method is illustrated by the synthesis of oligodeoxynucleotides.