Purification and properties of GTP-cyclohydrolase from Bacillus subtilis]

Highly purified GTP-cyclohydrolase was obtained by fractionation of cell extracts with ammonium sulfate, ion-exchange and hydrophobic chromatography. The N-terminal amino acid sequence and amino acid composition of the protein were determined. According to SDS-PAGE data, the molecular weight of the enzyme is 45 kDa. The active enzyme has several isoforms separable by native electrophoresis. The maximal enzyme activity is determined at 1.5 mM Mn2+; 70% of enzymatic activity is detected with Mg2+. The enzyme is inhibited by heavy metal ions and chelators and is inactive in the absence of thiol-reducing agents. The enzyme activity is detected in a broad range of pH with a maximum at pH 8.2. The pyrimidine product of the GTP-cyclohydrolase reaction. 2.5-diamino-6-hydroxy-4-ribosylaminopyrimidine-5'-phosphate was purified and identified. Another product of this reaction is pyrophosphate.     

Enzymatic activity of thymidine kinases of a herpes simplex virus strains resistant to acyclovir H-phosphonates

Some cloned laboratory mutant strains of herpes simplex virus type I resistant to acyclovir H-phosphonate were studied. As was shown for all the clones, the mutations augmented the susceptibility to cidîfovir. Thymidine kinase of the mutant viruses partially retained the capacity to phosphorylate thymidine, but lost the capacity to phosphorylate BVDU.     

Nonviral delivery systems for small interfering RNAs

RNA interference is now considered to be the most powerful and promising tool for gene-targeted therapy. Several problems are still to be solved for its successful use in medicine. One of the main issues is efficient siRNA delivery. The review considers various types of nonviral siRNA delivery systems.     

Prospects of antisense therapy technologies

Three variants of antisense technologies are presently known: antisense oligonucleotides, RNA interference, and ribozymes. In spite of the difference in the mechanisms of action, all of them are based on a common principle: an antisense preparation works after binding with an RNA target to form a duplex. All of the three variants are intensely used in experiments in vivo. The review considers the current situation in the field of using antisense technologies to treat various diseases. Key words: antisense therapy, antisense oligonucleotides, RNA interference, ribozymes     

Biodegradation of 125I-Labeled monoclonal antibodies after intravenous administration to rats with experimental C6 glioma

Employment of radiolabeled antibodies in biological studies, allows their specific accumulation in organs and tissues to be accurately detected. However, stability of such radiolabeled antibodies depends on both the method of labeling and particular experimental conditions. Therefore, stability of labeled antibodies should be determined in every particular experiment. In this study we have investigated stability of the ¹²⁵I-labeled monoclonal antibodies to gliofibrillary acidic protein (GFAP), endothelial antigen AMVB1, and non-specific mouse IgG at the stage of their synthesis and after their intravenous administration to rats with experimental C6 glioma. Stability of labeled antibodies was determined in blood samples and homogenates of organs by the method of their precipitation with trichloroacetic acid. The ¹²⁵I-radiolabeled antibodies were characterized by high radiochemical putiry, immunochemical activity and stability of the resultant preparations in blood and tissues, and the brain after administration in vivo. Electrophoretic analysis, thin-layer chromatography, and immunohistochemical tests have demonstrated the radiochemical purity, immunochemical competence, and stability of the labeled antibodies in vivo.     

The Effect of Pyrophosphate Analogues on the Activity of the Inorganic Pyrophosphatase from Escherichia coli

The effect of inorganic pyrophosphate analogues on the enzymic activity of inorganic pyrophosphatase from E. coli was studied. Hypophosphoric and diphosphonic acids were shown to inhibit inorganic pyrophosphatase, whereas pyrophosphorous acid exerts almost no effect on the hydrolysis of inorganic pyrophosphate.     

Studies of the functional topography of Escherichia coli RNA polymerase. A method for localization of the sites of affinity labelling

A method is proposed for localization of the sites of affinity labelling of the beta subunit of Escherichia coli RNA polymerase. The principle of this method is similar to that of the methods of rapid sequencing of nucleic acids. The polypeptide bearing a radioactive affinity label at one of the amino acid residues is subjected to short-term treatment with cyanogen bromide. The conditions of this reaction are selected in such a way that less than one cleavage occurs on average per polypeptide chain. Two series of radioactive peptides are formed, one involving all the possible N-terminal peptides and the other the C-terminal peptides. The distribution of the lengths of these peptides is studied by means of gel electrophoresis and compared with the theoretical ones based on the known amino acid sequence of the beta subunit. Obviously, the affinity label resides between the C-terminus of the shortest N-terminal radioactive peptide and the N-terminus of the shortest C-terminal radioactive peptide. In order to increase reliability and resolution of the method, partial trypsinolysis may be employed. The evidence obtained suggests that lysine residues over the regions 1036-1066, 1234-1242, and histidine-1237 are situated in the nearest neighbourhood to, or directly involved in the formation of the active center of initiating substrate binding of the beta subunit of E. coli RNA polymerase.