Preparation of Escherichia coli 70S ribosomes labeled with 35S

[35S]--70S ribosomes (150 Ci/mmol) were isolated from E. coli MRE-600 cells grown on glucose-mineral media in the presence of [35S] ammonium sulfate. The labeled 30S and 50S subunits were obtained from [35S] ribosomes by centrifugation in a sucrose density gradient of 10--30% under dissociating conditions (0.5 mM Mg2+). The activity of [35S]--70S ribosomes obtained by reassociation of the labeled subunits during poly(U)-dependent diphenylalanine synthesis was not less than 70%. The activity of [35S]--70S ribosomes during poly(U)-directed polyphenylalanine synthesis was nearly the same as that of the standard preparation of unlabeled ribosomes. The 23S, 16S and 5S RNAs isolated from labeled ribosomes as total rRNA contained no detectable amounts of their fragments as revealed by polyacrylamide gel electrophoresis. The [35S] ribosomal proteins isolated from labeled ribosomes were analyzed by two-dimensional gel electrophoresis. The [35S] label was found in all proteins, with the exception of L20, L24 and L33 which did not contain methionine or cysteine residues.     

G-specific RNA-cleaving conjugates of short peptides and oligodeoxyribonucleotides

Artificial ribonucleases, conjugates of short oligodeoxyribonucleotides and peptides built of arginine, leucine, proline, and serine, were synthesized and assessed in terms of ribonuclease activity and specificity of RNA cleavage. A specific group of the conjugates was identified that display T1-ribonuclease-like activity and cleave RNA predominantly at G-X sequences. Circular dichroism study of the structures of the most active conjugates, free peptide (LR)4G, and oligonucleotides revealed that conjugation of oligonucleotide to the peptide results in a specific peptide folding that possibly provides ribonuclease activity to the conjugate.     

Echinomycin production by Actinomadura sp. INA 654]

An actinomycete strain designated as Actinomadura sp. INA 654 was isolated from a chernozem soil sample in the Voronezh Region by the soil sample treatment with millimetric waves (EHF band). The strain produced an antibiotic complex of 2 components, named A-654-I and A-654-II. Investigation of their physico-chemical properties showed that A-654-I was identical to echinomycin, a heteropeptide lactone of the quinoxaline group with antitumor activity, while A-654-II proved to be likely a new natural compound. Production of echinomycin by a representative of the Actinomadura genus was detected for the first time. Up to now, only representatives of the Streptomyces genus were known to produce echinomycin.     

Mechanism and Specificity of RNA Cleavage by Chemical Ribonucleases

Abstract

Cleaving of model RNA substrates by chemical ribonucleases constructed by conjugation of 1,4 diazabicyclo[2,2,2]octane with histamine and histidine was investigated. Similarly to RNase A, the chemical RNases produce fragments with 5′ hydroxy-group and 3′-cyclophosphate. The cleavage occurs as the catalytic reaction: more than 150 phosphodiester bonds in RNA can be cleaved by one molecule of RNase mimic.     

Search for oligonucleotides selectively binding oncogenic miR-21

In the pathogenesis of malignancies, an active regulatory role belongs to small noncoding RNAs, miRNA (miR). miRNA expression profiles are often associated with the prognosis and therapeutic outcome of different oncological diseases. It is well known that in comparison with normal tissues cancer cells are characterized by hyperexpression of oncogenic miRNAs which leads to oncogenic transformation, carcinogenesis and metastasis progression. From this point of view, selective down-regulation of miRNA expression by specific agents, such as antisense oligonucleotides that recognize particular sequences, therefore, can be an effective tool to regulate the amount of miRNA in cancer cells and decrease tumor malignancy. In this paper, we have designed a series of antisense oligonucleotides addressed to the oncogenic miR-21 with a view to its selective binding and studied patterns of interaction of miR-21 with these oligonucleotides in vitro. The series included linear and hairpin oligonucleotides with the length of antisense fragment of 10–16 nucleotides (nt) complementary to the 5'- or the 3'-end of miRNA target. Hairpin oligonucleotides consist of a sequence complementary to miR-21 and a hairpin containing a four-nucleotide loop and stem of 6–9 bp necessary for stabilizing the complex with miR-21. It has been shown that inclusion of the hairpin with the stem of 6 bp to the oligonucleotide structure leads to a 1.6-fold increase in binding efficiency with miR-21 in comparison with a linear oligonucleotide and elongation of the stem from six to nine bp does not increase binding efficiency. Hairpin oligonucleotides with an antisense sequence of 14 nt effectively hybridize with miR-21 and are not inferior to 16-mer linear and hairpin oligonucleotides in the efficiency of complex formation. Thus, we have shown that hairpin oligonucleotides with antisense fragment of 14 nt and a hairpin, including the stem of 6 bp, are optimal for selective and effective sequestering of mature miR-21.     

Affinity modification of Escherichia coli ribosomes with 2',3'-O-[4-(N-2-chloroethyl)-N-methylamino]-benzylidene derivative of AUGU3 in the 70S initiation complexes

Affinity labelling of the Escherichia coli ribosomes with the 2',3'-O-[4-(N-(2-chloroethyl)-N-methylamino]benzylidene derivative of AUGU3(AUGU3[14C]CHRCl) has been studied within 70S initiation complexes ribosome.AUGU3[14C]CHRCl.fMet-tRNA(Metf) and binary complex ribosome.AUGU3[14C]CHRCl. Various ways of the 70S initiation complex formation resulted in differently labelled products. Proteins S5, S7, S9, L1, L16 were thus identified as cross-linked with AUGU3[14C]CHRCl within an initiation complex obtained in the presence of initiation factors IF-1, IF-2, IF-3, whereas only proteins S5 and S7 were cross-linked within the complex obtained with the sole factor IF-2. Proteins S1, S3, L1 and L33 were labelled within the initiation complex obtained nonenzymatically but only protein S1 within the binary complex. In all complexes formed with use of initiation factors labelling of IF-2 factor was invariably observed.     

Downregulation of PGY1/MDR1 mRNA level in human KB cells by antisense oligonucleotide conjugates. RNA accessibility in vitro and intracellular antisense activity

Inhibition of PGY1/MDR1 (multidrug resistance gene 1) mRNA expression in multidrug resistant KB-8-5 cells by 5'-bis-pyrenyl-3'-aminohexyl oligodeoxyribonucleotide conjugates targeted to four sites of this mRNA has been investigated. Three of the tested oligonucleotide conjugates specifically inhibited the expression of PGY1/MDR1 mRNA as monitored by the RT-PCR assay. The oligonucleotide conjugate targeted to the region (+178; +194) of the PGY1/MDR1 mRNA decreased level of this mRNA to 10% compared to the control. Nuclease-resistant analogs of oligonucleotide, complementary to this MDR1 mRNA region therefore, might be considered as a prototype compounds for development of gene-targeted therapeutic agents for overcoming the MDR phenotype caused by the overexpression of the PGY1/MDR1 gene.     

The hypolipidemic action of antibiotic 86/88 (enniatin B) in a hepatoblastoma G2 cell culture]

A cyclodepsipeptide antibiotic 86/88 (enniatin B) with strong hypolipidemic action was isolated from the culture liquid of the fungus INA F-86/88 identified as Fusarium lateritium Nees var. stilboides (Wr.) Bilai. In the Hep G2 cell culture the antibiotic suppressed 14C-acetate incorporation into cholesterol (IC50 1.75 microM), cholesterol ethers (IC50 1 microM), triglycerides (IC50 1.3 microM) and free fatty acids (IC50 2.2 microM). The most pronounced effect of the drugs, i.e. the suppression of the cholesterol ethers synthesis is likely due not only to the ACAT inhibition but also to the inhibition of the triglyceride synthesis and the diminishing of the free fatty acids pool in the cells.     

Complementary addressed alkylation of 16s rRNA from Escherichia coli by 2',3'-0-[4-N-(2-chloroethyl)-N-methylamino]benzylidene derivatives of oligodeoxyribonucleotides. III. Segments of 16s rRNA interacting with the benzylidene derivative of d(pACCTTGTT)rA

16S rRNA chain was cleaved by RNase H within covalent adduct with a 2',3'-0-[4-N-(2-chloroethyl)-N-methylamino]benzylidene derivative of d(pACCTTGTT)rA. It was shown that no less than 50% of cleavage occurs at the 1498-1506 site. The selectivity of alkylation and correspondingly the cleavage by RNase H, at this site practically does not increase when RNA was alkylated to a low extent, and also when a small excess of the reagent in respect to rRNA was used.