Hemolysin from Vibrio cholerae eltor: cloning and expression of genes in Escherichia coli]

A 6.56-kb V. cholerae eltor DNA fragment encoding hemolysin synthesis was cloned in pUC18. The resultant recombinant plasmid pES4H (9.25 kb) was mapped by restriction analysis and shown to express in different E. coli strains as well as in nonhemolytic V. cholerae strains. Application of the cloned fragment as a molecular probe revealed homologous sequences in all V. cholerae strains tested independently on their biotypes, hemolytic activity and presence of vct-genes in their genomes while none of other Vibrio species and related microorganisms contained such sequences. A recombinant E. coli strain, a V. cholerae eltor hemolysin producer, was constructed. The simultaneous expression of hemolytic and toxinogenic properties by the same V. cholerae strains is discussed.     

Immunologic determination of an epidermal G2-chalone-like factor as a marker of squamous cell structures in the urinary bladder and urine in rats

The epidermal G2 chalone-like substance of the rat skin may be determined by immunochemical methods only in squamous epithelia and it is revealed neither in mucosa of the urinary bladder, nor in urine of control rats. The antigenic activity of the chalone was revealed in 37 extracts of 56 tumors of the rat bladder. All antigen-containing tumors were found to be squamous cell carcinomas, transitional cell carcinomas with squamous cell metaplasia or possessed ultramicroscopic signs of squamous cell metaplasia appearing as "pure" transitional cell carcinomas under light microscope. The epidermal G2 chalone-like substance was present in urine of 7 out of 10 rats bearing the antigen-positive tumors of the urinary bladder.     

Lysine Dendrimers and Their Starburst Polymer Derivatives: Possible Application for DNA Compaction and in vitro Delivery of Genetic Constructs

We attempted to find some compounds for the effective delivery of gene constructs into cells and obtained two trispherical dendrimers on the basis of lysine, (Lys)₈-(,-Lys)₄-(,-Lys)₂-(,-Lys)-Ala-NH₂ (D1) and ((Lys)₈-(,-Lys)₄-(,-Lys)₂-,-Lys)-Ala-[Lys(Plm)]₂-Ala-NH₂ (D2), as well as the starburst polymeric derivatives of D1, (pVIm) ₈ -D1 and (pLys) _n -D1, containing poly(N-vinylimidazole) and polylysine chains single-point bound to the dendrimer amino groups. The conditions of dendrimer–plasmid DNA complex formation were studied. The intracellular localization of these complexes and the expression of gene constructs delivered with their help were analyzed in transfection experiments on the HeLa cell cultures of human epithelial carcinoma and on mouse C2C12 myoblasts. It was found that the chemical structure of dendrimer D1 and its derivatives significantly affected the structure and properties of complex.     

Inhibitory action of polycationic peptides on hormonal regulation of adenylyl cyclase of the ciliate Dileptus anser

To analyse molecular mechanisms of regulatory action of different hormones on the activity of the adenylyl cyclase signaling system (ACS) of the ciliate Dileptus anser, we studied the influence on this process of six synthetic polycationic peptides and peptides, corresponding to C-terminal regions of mammalian G-protein 385-394 alphas- and 346-355 alphai2-subunits. As we reported earlier, these peptides block hormonal signal transduction in tissues of the higher eukaryotes. Now it has been found that both polycationic peptides, containing hydrophobic C to-radicals, and branched peptides decrease regulatory effects of peptide hormones (insulin, relaxin) and biogenic amines (serotonin, adrenaline) on adenylyl cyclase (AC) activity and GTP-binding. In regard to the following peptides Cys-epsilonAhx-Trp-Lys-Lys(C10)-Lys2-Lys(C10)-Lys3-Lys(C10)-Tyr-Lys-Lys(C10)-Lys-Lys-amide and [(Gly-Arg-Gly-Asp-Ser-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro- Pro-Gly)2-Lys-EAhx-Cys]2 (epsilonAhx - E-aminocaproyl, C10 - caprinoyl group) their dose-dependent inhibitory action is shown. In cell culture of D. anser with a lower basal AC activity, both hydrophobic and branched peptides stimulated AC and GTP-binding without hormones. The data give evidence that these peptides can activate ACS of ciliates in a receptor-independent manner. No influence of peptides 385-394 alphas and 346-355 alphai2 on hormonal signal transduction in D. anser was observed, due, presumably, to some structural differences of G-proteins of the lower and higher eukaryotes. A conclusion was made about an important role of polycationic regions for functional coupling of hormone-activated receptor and G-proteins in the ciliate D. anser.     

Dendrimers based lysine and their "starburst" polymeric derivatives: prospects of use in compacting DNA and in vitro delivery of genetic constructs

We attempted to find some compounds for the effective delivery of gene constructs into cells and obtained two trispherical dendrimers on the basis of lysine, (Lys)8-(alpha, epsilon-Lys)4-(alpha, epsilon-Lys)2-(alpha, epsilon-Lys)-Ala-NH2 (D1) and (Lys)8-(alpha, epsilon-Lys)4-(alpha, epsilon-Lys)2-(alpha, epsilon-Lys)-Ala-[Lys(Plm)]2-Ala-NH2 (D2), as well as the starburst polymeric derivatives of D1, (pVIm)8-D1 and (pLys)n-D1, containing poly(N-vinylimidazole) and polylysine chains bound at a single point to the dendrimer amino groups. The conditions of dendrimer-plasmid DNA complex formation were studied. The intracellular localization of these complexes and the expression of gene constructs delivered with their help were analyzed in transfection experiments on the HeLa cell cultures of human epithelial carcinoma and on C2C12 mouse myoblasts. It was found that the chemical structure of dendrimer D1 and its derivatives significantly affected the structure and properties of complex. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 1; see also http://www.maik.ru.     

Regularities of changes in microcirculatory flow through different parts of the heart in patients with acquired valvular disease before and after surgery

Microcirculation in the subepicardium was studied in 30 patients with aortic acquired valvular disease (AVD) and 20 with mitral AVD with the use of an ALF-21 laser Doppler blood flowmeter (Transonic Systems). In total, 845 measurements were made. Subepimyocardial flow (EMF) was measured on the anterior surface of the right and left atria and on the anterior, posterior, and diaphragmatic surfaces of the left and right ventricles of the heart before and after surgery. A decrease in the hyperfunctioning of the chambers of the heart led to a redistribution of myocardial flow. Differences (EMF) between EMF values observed before and after treatment were analyzed, and the coefficients for the linear equation EMF = a + kEMF_bs were computed by the least-squares method. It was found that blood flow decreased when it was enhanced before treatment and increased when it was weak initially. Thus, blood flow was balanced, approaching a value that did not change after treatment, and heterogeneity of coronary flow in the microcirculatory link decreased. Control mechanisms were assumed to change blood flow so that it acquires stability, which is needed to preserve and maintain normal energy parameters of the functioning myocardium.Translated from Fiziologiya Cheloveka, Vol. 31, No. 1, 2005, pp. 40–48.Original Russian Text Copyright © 2005 by Okuneva, Vlasov, Karaskov, Nazarov, Bulatetskaya, Voronova, Dukhnov, Zhelezchikov, Zheleznev.     

Mechanism for suppression mRNA translation with antisense oligonucleotides

Experimental studies of the effects of antisense oligonucleotides on translation of mRNAs in cell-free systems are reviewed. Oligonucleotides complementary to the leader sequences or to the sequence overlapping the initiating codon region of mRNAs inhibit translation of the messengers. In the presence of ribonuclease H, oligodeoxyribonucleotides and their phosphorothioate analogs complementary either to the mentioned mRNA regions or to the mRNA coding sequence suppress the translation due to the RNAs cleavage. This inhibition-enhancing mechanism does not operate in the case of the oligonucleotide analogs--oligonucleoside methylphosphonates and oligonucleotides built of the alpha-nucleosides, since the complexes formed by RNA and these analogs are not substrates of the ribonuclease H. The translation inhibition efficiency is determined by the oligonucleotides lengths and by the availability of the complementary sequence in the mRNA structure. The oligonucleotides inhibitory power can be improved by the coupling to the oligonucleotides of the intercalating groups and the reactive groups.