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2005 Vol III #2


This article is sponsored by Otava, Ltd.

S
tudies of mutagenic activity of fluorescent DNA-sensitive monomethinecyanine and carbocyanine dyes in Ames test


B.P. Matselyukh, D.Ya. Matselyukh, V.B. Kovalska
1,
K.D. Volkova
1, D.V. Kryvorotenko1, and S.M. Yarmoluk1

Danylo Zabolotny Institute of Microbiology and Virology, NAS of Ukraine
154 Zabolotny Str., Kyiv, 03143, Ukraine
1Institute of Molecular Biology and Genetics,
NAS of Ukraine
150 Zabolotny Str., Kyiv, 03143, Ukraine

Abstract: Cyanine dyes are widely used as fluorescent probes for visualization of nucleic acids in electrophoretic gels. Unfortunately, some of these dyes are known to have mutagenic and carcinogenic activity. Here, four cyanine dyes D-6, D-9, CPent V and CCyan 2-O, proposed before as fluorescent dyes for DNA visualization in gels [1], were evaluated for mutagenicity in the Salmonella typhimurium assay (Ames assay) and cytotoxicity. The most sensitive of the studied dyes, mesosubstituted carbocyanine CCyan 2-O, was selected for further examination in acute toxicity assay on laboratory animals. Results indicated that according to the ‘2-fold rule' of assessing mutagenicity in the Ames assay monomethinecyanines D-6 and D-9 are not mutagenic, and carbocyanines CCyan2-O and CPent-V appeared to be weak mutagens. Mutagenic activity of carbocyanines was up to two times higher as compared to monomethine dyes. Carbocyanines CCyan2-O and CPent-V demonstrated higher cytotoxicity, being tested against Salmonella cells strain TA98, than did monomethinecyanines D-6 and D-9. Acute toxicity test indicates that the oral LD50 of CCyan 2-O for albino mice and rats are equal to 53.0 and 56.5 mg/kg, respectively. The lowest dose causing lethality LDLO [2] for both species of rodents amounted to 20 mg/kg.

Key words: cyanine dyes, mutagenicity, acute toxicity, LD50, Salmonella mutation assay.

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P
reparation of highly purified human IgG, IgM, and IgA for immunization and immunoanalysis


I.V. Nikolayenko, O.Yu. Galkin, N.I. Grabchenko,
M.Ya. Spivak
1

Joint-Stock Company «DiaProphMed», 35a Svitlytski Str., Kyiv, 04123, Ukraine
1Danylo Zabolotny Institute of Microbiology and Virology, NAS of Ukraine
154 Zabolotny Str., Kyiv, 03143, Ukraine

Abstract: The authors have elaborated several protocols for isolation and purification of human IgG, IgA, and IgI using affinity chromatography and gel chromatography approaches. To control the process of globulin isolation and to evaluate globulin purity degree, the immunodiffusion reaction according to Ouchterlony and the SDS-PAGE were carried out. The immunoglobulin preparations obtained may be successfully used for animal immunization as well as for different immunoenzyme method modifications.

Key words: human IgG, IgM, and IgA, immunoglobulin isolation and purification, affinity chromatography, gel chromatography, immunodiffusion, electrophoresis.

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This article is sponsored by Otava, Ltd.

T
oxicity evaluation of the series of cyanine dyes used for nucleic acids and proteins detection


N.N. Nizamov, Z.F. Ismailov, E.N. Kurtaliev,
Sh.N. Nizamov, F.U. Khaydarova, G. Khodjayev
1,
O.P. Kukharenko
2, A.O. Balanda2, S.M. Yarmoluk2

Samarkand State University named A. Navoi
15 University blvd., Samarkand, 703004, Uzbekistan
1Samarkand Agricultural Institute
77 M. Ulugbek Str., Samarkand, 703003, Uzbekistan
2Institute of Molecular Biology and Genetics,
NAS of Ukraine
150 Zabolotny Str., Kyiv, 03143, Ukraine


Abstract: Potential toxicity of some commercial and novel dyes specifically interacting with biopolymer molecules was comparatively studied on bioobjects such as protozoa Paramecium caudatum, fungus Rhizoctonia solani and E. coli. The toxicity and critical concentrations were determined for compounds Rhodamine C, Cyan-40, SIL, SBT, D-174. Paramecium caudatum revealed the highest sensitivity to studied dyes and can be used as sensitive indicator organism in the toxicity tests.

Key words: fluorescent dyes, toxicity, Paramecium caudatum.

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S
econdary structure analysis of cytokine-like C-terminal module of mammalian (Bos taurus) tyrosyl-tRNA synthetase using methods of circular dichroism spectra and computer modeling


K.A. Odynets, A.I. Kornelyuk

Institute of Molecular Biology and Genetics,
NAS of Ukraine
150 Zabolotny Str., Kyiv, 03143, Ukraine


Abstract: COOH-Terminal module of mammalian cytoplasmic tyrosyl-tRNA synthetase (TyrRS) is an additional RNA-binding domain, which also has cytokine-like activities in isolated state after its proteolytic release from a catalytic core of the enzyme. In the article a comparative secondary structure analysis of this bifunctional C-module of bovine TyrRS (Bos taurus) is fulfilled using experimental and theoretical methods. The experimental determination of secondary structure was carried out by the method of circular dichroism (CD) spectra analysis for recombinant C-module. The theoretical prediction was carried out using: 1) the programs of protein secondary structure prediction from its amino acid sequence, and 2) the deduction algorithms of secondary structure elements from atomic coordinates (DSSP, STRIDE and P-SEA) of a model of C-module spatial structure, obtained by homology modeling technique using human cytokine EMAP II. Experimental CD data for C-module of TyrRS confirms the native state of protein YCD3k in obtained preparations.

Key words: tyrosyl-tRNA synthetase, C-terminal module, secondary structure prediction, circular dichroism spectra.

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I
nteraction of 4'-methoxyisoflavon-7-O-β-D-glucopyranoside (Ononin) with nitrogen containing binucleophiles

V.G. Pivovarenko, T.M. Tkachuk

Department of Chemistry, Taras Shevchenko National University
64 Volodymyrska Str., Kyiv, 01033, Ukraine

Abstract: The isoxazole, pyrazole and pyrimidine derivatives were synthesized by the recyclization of 4'-methoxyisoflavon-7-O-β-D-glucopyranoside under interaction of nitrogen containing binucleophilic reagents (hydrazine, hydroxylamine, amidines). The structure of the obtained compounds was confirmed by 1H-NMR-spectroscopy and mass-spectrometry data.

Key words: binucleophiles, isoxazole, pyrazole, pyrimidine, glucoside, ononine.

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S
ynthesis and structural characteristics of 4-(2-phenylethyl)-1,7-dithia-4-azaspiro[4.4]nonan-3-one 7,7-dioxide


P.V. Shaitanov, Yu.V. Bezugly, V.M. Charchenko,
S.M. Lukashov, S.M. Yarmoluk

Institute of Molecular Biology and Genetics,
NAS of Ukraine
150 Zabolotny Str., Kyiv, 03143, Ukraine

Abstract: 4-(2-phenylethyl)-1,7-dithia-4-azaspiro[4.4]nonan-3-one 7,7-dioxide, the derivative of new heterocyclic system 1,7-dithia-4-azaspiro[4.4]nonane, was obtained by interaction of 3-bromo-2,3-dihydrothiophene 1,1-dioxide with 2-mercapto-N-(2-phenethyl)-acetamide in the presence of the KOH in the alcohol solution. Its structure was proven with X-ray spectroscopy.

Key words: 1,7-dithia-4-azaspiro[4.4]nonane, 4-(2-phenylethyl)-1,7-dithia-4-azaspiro[4.4]nonan-3-one 7,7-dioxide, 3-bromo-2,3-dihydrothiophene 1,1-dioxide, 2-mercapto-N-(2-phenethyl)-acetamide, X-ray spectroscopy.

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