[Preparation of biologically transformed raw material from woolly foxglove Digitalis lanata Ehrh and isolation of digoxin from it]. / Poluchenie biotransformirovannogo syr'ia naperstianki sherstistoi Digitalis lanata Ehrh i vydelenie iz nego digoksina.

A biotechnological approach is proposed for conservation of a terraneous part of woolly foxglove under anaerobic conditions with a subsequent air-sun drying of the biologically transformed raw material. During the conservation primary foxglove glycosides completely convert to secondary ones which do not transform further. A simple method is described for preparation from the transformed raw material of an enriched glycoside fraction with the yield of 3.6% and for isolation from this fraction of highly purified digoxin with the yield of 0.06% of the starting raw material, and the other secondary glycosides can be also isolated.

Formation and reactivity of 2,4-ditriazolyl pyrimidine C-nucleoside derived from pseudouridine.

Thymine and 2',3',5'-tri-O-acetyl-psi-uridine (1) was converted into the corresponding 2,4-ditriazolyl derivatives 5 and 2, respectively. Of these two substituents, the C4-triazolyl group was found to be quite susceptible to nucleophilic substitution while the other triazolyl is resistant.

Correction of chromosomal point mutations in human cells with bifunctional oligonucleotides.

A sequence-specific genomic delivery system for the correction of chromosomal mutations was designed by incorporating two different binding domains into a single-stranded oligonucleotide. A repair domain (RD) contained the native sequence of the target region. A third strand-forming domain (TFD) was designed to form a triplex by Hoogsteen interactions. The design was based upon the premise that the RD will rapidly form a heteroduplex that is anchored synergistically by the TFD. Deoxyoligonucleotides were designed to form triplexes in the human adenosine deaminase (ADA) and p53 genes adjacent to known point mutations. Transfection of ADA-deficient human lymphocytes corrected the mutant sequence in 1-2% of cells. Neither the RD or TFD individually corrected the mutation. Transfection of p53 mutant human glioblastoma cells corrected the mutation and induced apoptosis in 7.5% of cells.

Bicyclic monoterpene diols induce differentiation of S91 melanoma and PC12 pheochromocytoma cells by a cyclic guanosine-monophosphate-dependent pathway.

Previously, we showed that 5-norbornene-2,2-dimethanol (5-NBene-2,2-DM) is an effective inducer of melanogenesis in cultured cells and guinea-pig skin [Brown et al. (1998) J. Invest. Dermatol., 110:428-437]. This study shows that 2,3-cis/exo-pinanediol (2,3-cs/ex-PinD) is a more effective inducer of melanogenesis than 5-NBene-2,2-DM in S91 mouse melanoma cells. Furthermore, 2,3-cs/ex-PinD appears to penetrate guinea-pig skin better than 5-NBene-2,2-DM and to induce higher levels of pigmentation. Both 5-NBene-2,2-DM and 2,3-cs/ex-PinD induce synthesis of nitric oxide (NO) in S91 cells, and the melanogenic activity of both compounds is reduced by inhibitors of the NO/cyclic guanosine monophosphate (cGMP)/protein kinase(PK) G signaling pathway, but not by inhibitors of the PKC or PKA pathways. Thus, these bicyclic monoterpene diols appear to induce melanogenesis by the same pathway in S91 cells as that shown previously for ultraviolet radiation in melanocytes (Romero-Graillet et al. (1996) J. Biol. Chem., 271:28052-28056). These compounds also induce NO synthesis, neurite outgrowth, and tyrosine hydroxylase activity in PC12 pheochromocytoma cells. Neurite outgrowth in PC12 cells is blocked by the guanylate cyclase inhibitor, LY83583 (6-anilino-2,8-quinolinequinone), indicating that, similar to S91 cells, the induction of morphological differentiation of PC12 cells by bicyclic monoterpene diols is regulated by a cGMP-dependent pathway.

Targeted gene knockout mediated by triple helix forming oligonucleotides.

Triple helix forming oligonucleotides (TFOs) recognize and bind sequences in duplex DNA and have received considerable attention because of their potential for targeting specific genomic sites. TFOs can deliver DNA reactive reagents to specific sequences in purified chromosomal DNA (ref. 4) and nuclei. However, chromosome targeting in viable cells has not been demonstrated, and in vitro experiments indicate that chromatin structure is incompatible with triplex formation. We have prepared modified TFOs, linked to the DNA-crosslinking reagent psoralen, directed at a site in the Hprt gene. We show that stable Hprt-deficient clones can be recovered following introduction of the TFOs into viable cells and photoactivation of the psoralen. Analysis of 282 clones indicated that 85% contained mutations in the triplex target region. We observed mainly deletions and some insertions. These data indicate that appropriately constructed TFOs can find chromosomal targets, and suggest that the chromatin structure in the target region is more dynamic than predicted by the in vitro experiments.

Aliphatic and alicyclic diols induce melanogenesis in cultured cells and guinea pig skin.

We have found that several aliphatic and alicyclic diols induce melanogenesis in cultured S91 mouse melanoma cells and normal human epidermal melanocytes (NHEM). In addition, these compounds induce melanogenesis when applied to guinea pig skin, with transfer of melanin to keratinocytes and formation of "supranuclear caps," as occurs in naturally pigmented skin. The relative order of potency of some of these diols in NHEM is 5-norbornene-2,2-dimethanol > 3,3-dimethyl-1,2-butanediol > cis-1,2-cyclopentanediol > 2,3-dimethyl-2,3-butanediol > 1,2-propanediol. Following treatment with these diols or 3-isobutyl-1-methylxanthine, melanin and tyrosinase activity are increased within S91 cells and NHEM; however, for cultured NHEM, the largest increases of melanin and tyrosinase occur in an extracellular particulate fraction, shown by electron microscopy to consist almost entirely of stage III and IV melanosomes. These results indicate that cultured NHEM treated with diols export melanosomes in a fashion that is commensurate with natural melanogenic processes. In contrast, S91 mouse melanoma cells exhibit aberrant melanosomal trafficking, in accordance with the known defect in myosin-V mediated melanosomal transport. Both S91 cells and NHEM exhibit morphologic changes and growth arrest indicative of differentiation following treatment with diols. The diols described in this report are candidates for use as cosmeceutical tanning agents.

Trivaline molecular complexes with trinucleotides form in solution the extended, about 1000 A in length structures.

The fluorescence, flow linear dichroism and electron microscopy (EM) have shown the trivaline ability to interact in solution with certain molecules of trinucleotides. This interaction results in formation of extended structures up to several thousand angstroms in length. Such structures were observed for trivaline complexes with homopurine, homopyrimidine or random sequences of deoxyribo- and ribonucleotides, independently of the presence or absence of the terminal 5'-phosphate residue. A model of such a structural organization is proposed. An elementary structural unit consists of a trivaline beta-dimer and adsorbed trinucleotide. So, "dimeric" complex is formed. Two such "dimeric" complexes combine with each other by means of peptide-peptide contacts (as with beta-sandwich). So, "tetrameric" complex is formed. It has a dyad axis. Two such structural units combine with each other by means of Hoogsteen's hydrogen bonds. So, "octameric" complex is formed. It has three mutually perpendicular dyad axes. The "octameric" complexes appear to be able to combine with each other by means of stacking interactions, and to form the regular organized aggregates consisting of many dozens of elementary units. So, "stacking" structure is formed. The "octameric" complex is the symmetry translational unit of such a structure. The spatial position of the bases in all these structures is additionally fixed by the nucleo-peptide interactions. These aggregates have the appearance of extended structures on electron micrographs.

Chemical synthesis of a biologically active natural tRNA with its minor bases.

The complete chemical synthesis of an E. coli tRNA(Ala) with its specific minor nucleosides, dihydrouridine, ribothymidine and pseudouridine, is reported. The method makes use of protected 2'-O-tertiobutyldimethylsilyl-ribonucleoside-3'-O-(2-cyanoethyl-N- ethyl-N- methyl)phosphoramidites. The exocyclic amino functions of the bases were protected by the phenoxyacetyl group for purines and acetyl for cytosine. The assembling has been performed on a silica support with coupling yield better than 98% within 2 min of condensation. Triethylamine tris-hydrofluoride allowed a clean and complete deprotection of the tBDMS groups. The synthetic tRNA(Ala) has been transcribed into cDNA by reverse transcriptase and sequenced. With E. coli alanyl-tRNA synthetase the alanyl acceptance activity and kcat/Km were 672 pmol/A260 and 6 x 10(4)M-1s-1, respectively.

Trivaline 'catalyzes' 5'-pdGTT oligomerization in solution.

We have found that the 5'-pdGTT molecules at a concentration of 10(-4) M are oligomerized in solution in the presence of 10(-4) M tripeptide-(L-Val)3-NH-NH-DNS.CF3COOH and the condensation reagents (carbodiimide and imidazole). Oligonucleotides not less than 12 bases long were formed in the yield which was over 15%. It is known that in the absence of peptide 10(-2) M mono- or dinucleotides are required. Thus trivaline can be considered as one of the simplest enzymes. This oligomerization seems to be an essential way for the synthesis of long enough oligonucleotides of the random GC-sequence, which could be used at the earliest steps of evolution.

[Interaction of trivaline with single-stranded polyribonucleotides]. / Vzaimodeistvie trivalina s odnonitevymi poliribonukleotidami.

Binding of tripeptide H-Val3-(NH)2-Dns (TVP) to polyribonucleotides was studied by fluorescence methods, circular and flow linear dichroism, equilibrium dialysis and electron microscopy. It was found that TVP binds to poly(U) in monomer, dimer and tetramer forms with binding constants of about 10(3), 40, 18.10(4) M, respectively. The cooperativity parameter for peptide dimer binding is 2000. The peptide forms tetramer complexes with poly(A), poly(C), poly(G) also. The formation of a complex between the peptide tetramer and nucleic acid is accompanied by a significant increase in the fluorescence intensity. The cooperative binding of TVP dimers to poly(U), poly(A), poly(C) is accompanied by a dramatic decrease in the flexibility of polynucleotide chains. However, it has a small effect (if any) on the flexibility of the poly(G) chain. The observed similarity of thermodynamic, optical and hydrodynamic++ properties of TVP complexes with single-stranded and double-stranded nucleic acids may reflect a similarity in the geometries of peptide complexes with nucleic acids. Electron microscopy studies show that peptide binding to poly(U) and dsDNA leads to compactization of the nucleic acids caused by interaction between the peptide tetramers bound to a nucleic acid. At the first stage of the compactization process the well-organized rod-like particles are formed, each consisting of one or more single-stranded polynucleotide fibers. Increasing the peptide concentration stimulates a side-by-side association and folding of the rods with the formation of macromolecular "leech-like" structures with the thickness of 20-50 nm.

[Hybridization of DNA with oligonucleotides immobilized in a gel: a convenient method for recording single base replacements]. / Gibridizatsiia DNK s oligonukleotidami, immobilizovannymi v gele: udobnyi metod registratsii odinochnykh zamen osnovanii.

In an attempt to develop a reliable system for DNA sequence analysis with multiple hybridization probes, oligonucleotides down to 8 bases long were covalently immobilized in a thin layer of polyacrylamide gel fixed on a glass plate. It was shown possible to detect single base changes in DNA by hybridization of the immobilized oligonucleotides with radioactively and fluorescently labeled DNA fragments. Moreover, it was found that dissociation temperatures of differently GC-rich duplexes could be equalized by appropriate choice of immobilized oligonucleotides concentrations. A model accounting for this phenomenon is presented. In order to make the system more compact, a rectangular matrix of 200 mm dots of immobilized oligonucleotides ("hybridization chip") was designed which offered the sensitivity of 20 attomoles per dot for fluorescent DNA fragment. The applications and perspectives of the approach are discussed.

A method for DNA sequencing by hybridization with oligonucleotide matrix.

A new technique of DNA sequencing by hybridization with oligonucleotide matrix (SHOM) which could also be applied for DNA mapping and fingerprinting, mutant diagnostics, etc., has been tested in model experiments. A dot matrix was prepared which contained 9 overlapping octanucleotides (8-mers) complementary to a common 17-mer. Each of the 8-mers was immobilized as individual dot in thin layer of polyacrylamide gel fixed on a glass plate. The matrix was hybridized with the 32P-labeled 17-mer and three other 17-mers differing from the first one by a single base change. The hybridization enabled us to distinguish perfect duplexes from those containing mismatches in 32 out of 35 cases. These results are discussed with respect to the applicability of the approach for sequencing. It was shown that hybridization of DNA with an immobilized 8-mer in the presence of a labeled 5-mer led to the formation of a stable duplex with the 5-mer only if the 5- and the 8-mers were in continuous stacking making a perfect nicked duplex 13 (5+8) base pairs long. These experiments and computer simulations suggest that continuous stacking hybridization may increase the efficiency of sequencing so that random or natural coding DNA fragments about 1000 bases long could be sequenced in more than 97% of cases. Miniaturized matrices or sequencing chips were designed, where oligonucleotides were immobilized within 100 x 100 micron dots disposed at 100 micron intervals. Hybridization of fluorescently labeled DNA fragments with microchips may simplify sequencing and ensure sensitivity of at least 10 attomoles per dot. The perspectives and limitations of SHOM are discussed.

[Compounds similar to acyclovir. VI. Synthesis of optically active 1',2'-seconucleosides]. / Soedineniia, podobnye atsikloviru. VI. Sintez opticheski aktivnykh 1',2'-sekonukleozidov.

A convenient method is suggested of synthesis of (3R, 4R)-, (3S, 4S)- and (3R/S, 4S/R)-dihydroxy-3-hydroxymethyl-2-oxapentyl derivatives of the, cytosine, uracyl, adenine and guanine ("full" acyclic analogues of nucleosides with C1'-C2' bond cleaved) by condensation of trimethylsilyl derivatives of nucleic bases (sodium salt in case of adenine) with (3$, 4R)-, (3S )-, (3S, 4S)- and (3R/S, 4S/R)-4,5-diacetoxy-3-acetoxymethyl-1-chloro-2-oxapentanes without catalyst followed by deacetylation.

[Compounds similar to acyclovir. VII. Attempts to construct an anti-herpetic agent (6-hydroxy-2-oxa-4-hexenyl derivatives of nucleic bases]. / Soedineniia, podobnye atsikloviru. VII. Popytka konstruirovaniia protivogerpeticheskogo preparata (6-gidroksi-2-oksa-4-geksenil'nye proizvodnye nukleinovykh osnovanii.

Based on the available data on the acyclovir's mechanism of action we attempted to predict the antiherpetic activity of 6-hydroxy-2-oxahexen-4-yl derivatives of nucleic bases. In terms of this model 9-(6-hydroxy-2-oxahexen-4-yl) guanine might be active. 6-Hydroxy-2-oxahexen-4-yl derivatives of adenine, guanine, cytosine, thymine, uracil, 1,2,4-triazole-3 and 1,2,4-triazole-5-carboxamide have been synthesized and their activity against herpes virus I investigated. The guanine derivative proved to possess rather high activity (chemotherapeutical index 8).

[Inhibition of HIV reproduction in cell culture by 5'-phosphonates of 3'-azido-2',3'-dideoxynucleosides]. / Ingibirovanie reproduktsii virusa immunodefitsita cheloveka v kul'ture kletok 5'-fosfonatami 3'-azido-2',3'-dideoksinukleozidov.

The antiviral activity of 3'-azido-2',3'-dideoxynucleoside 5'-phosphate analogues: 5'-phosphonomethylene-3'-azido-2',3'-dideoxythymidine, 5'-methylphosphonate and 5'-phosphite of 3'-azido-2',3'-dideoxythymidine, 5'-phosphites of 3'-azido-2',3'-dideoxyadenosine and guanosine was investigated in HIV-infected cell cultures (human lymphoblastoid cells). The effectivity of inhibition of HIV-reproduction in cells by these substances was close or even higher than that for the corresponding 3'-azido-2',3'-dideoxynucleosides, whereas their toxicity was lower than that of nucleosides. These substances are supposed to be transported into the cells and to be transformed into the corresponding 5'-triphosphate analogues under the action of cell kinases. It is possible that such agents are terminator substrates of virus reverse transcriptases and thus inhibit the biosynthesis of DNA chains.

[Glycosylation of glycoconjugates on thymocyte surfaces]. / Glikozilirovanie glikokon''iugatov poverkhnosti timotsitov.

The ectosialation and ectogalactosylation of mouse thymocyte surface were studied. The incorporation of labeled monosaccharides (N-acetylneuraminic acid and galactose) into cell surface glycoproteins and glycolipids were demonstrated. Identification of glycolipids was carried out. The effect of glycosylation on the immune properties of thymocytes was established.

[Compounds, similar to acyclovir V. Synthesis and antiviral activity of carbethoxyalkoxymethyl derivatives of nucleic bases]. / Soedineniia, podobnye atsikloviru V. Sintez i protivovirusnaia aktivnost' karbétoksialkoksimetil'nykh proizvodnykh nukleinovykh osnovanii.

Ethyl esters and amides of carboxymethoxy-, alpha- and beta-carboxyethoxy- and gamma-carboxypropoxymethyl derivatives of adenine, guanine, cytosine, uracil, thymine, 1,2,4-triazole-3- and -5-carboxamide were synthesized and their antiviral activity was studied.

[Compounds similar to acyclovir. IV. A convenient method of synthesizing adenallene]. / Soedineniia, podobnye atsikloviru. IV. Udobnyi metod sinteza adenallena.

A convenient method for the synthesis of adenallen (9-(4-hydroxy-1,2-butadienil)adenine) is proposed. Adenallen was prepared in 30% yield by alkaline treatment of 9-(4-acetoxy-2-butinyl)adenine synthesized, in turn, by condensation of sodium derivative of adenine with 4-acetoxy-1-chlorbutine-2.