[ATP activity of myocardial actomyosin complex in patients with dilated cardiomyopathy]. / ATF-aznaia aktivnost' aktomiozinovogo kompleksa miokarda u bol'nykh s dilatatsionnoi kardiomiopatiei.

ATP activity of actomyosin in dilated cardiomyopathy (DCMP) was studied together with impact on the above activity of tropomyosin-troponin regulatory complexes recovered from the normal myocardium and cardiac muscle of a DCMP patient. Recordable in DCMP was a striking decline (1.5-fold) in ATP activity of actomyosin. But no significant difference was to be seen in sensitivity to Ca2+ ions of actomyosins obtained from the normal myocardium and cardiac muscle of the DCMP patient. The cardial tropomyosin-troponin regulatory complex from the DCMP patient's myocardium was shown to be endowed with somewhat more manifest activity compared to the analogous complex recovered from the normal myocardium.

Children of chernobyl cleanup workers do not show elevated rates of mutations in minisatellite alleles.

The disaster at the Chernobyl Nuclear Power Plant in April 1986 was accompanied by the release of large amounts of radioisotopes, resulting in the contamination of extensive regions of the Ukraine, Byelorus and the Russian Federation. Cleanup workers (liquidators) and people living on land contaminated with radioactive materials were most exposed. To assess the genetic effects of exposure to ionizing radiation after the Chernobyl accident, we have measured the frequency of inherited mutant alleles at seven hypermutable minisatellite loci in 183 children born to Chernobyl cleanup workers (liquidators) and 163 children born to control families living in nonirradiated areas of the Ukraine. There was no significant difference in the frequency of inherited mutant alleles between the exposed and control groups. The exposed group was then divided into two subgroups according to the time at which the children were conceived with respect to the fathers' work at the power plant. Eighty-eight children were conceived either while their fathers were working at the facility or up to 2 months later (Subgroup 1). The other 95 children were conceived at least 4 months after their fathers had stopped working at the Chernobyl site (Subgroup 2). The frequencies of mutant alleles were higher for the majority of loci (i.e. 1.44 times higher for CEB1) in Subgroup 1 than in Subgroup 2. This result, if confirmed, would reconcile the apparently conflicting results obtained in the chronically exposed Byelorus population and the Hiroshima-Nagasaki A-bomb survivors.

Negative regulation of PI 3-kinase by Ruk, a novel adaptor protein.

Class I(A) phosphatidylinositol 3-kinase (PI 3-kinase) is a key component of important intracellular signalling cascades. We have identified an adaptor protein, Ruk(l), which forms complexes with the PI 3-kinase holoenzyme in vitro and in vivo. This interaction involves the proline-rich region of Ruk and the SH3 domain of the p85 alpha regulatory subunit of the class I(A) PI 3-kinase. In contrast to many other adaptor proteins that activate PI 3-kinase, interaction with Ruk(l) substantially inhibits the lipid kinase activity of the enzyme. Overexpression of Ruk(l) in cultured primary neurons induces apoptosis, an effect that could be reversed by co-expression of constitutively activated forms of the p110 alpha catalytic subunit of PI 3-kinase or its downstream effector PKB/Akt. Our data provide evidence for the existence of a negative regulator of the PI 3-kinase signalling pathway that is essential for maintaining cellular homeostasis. Structural similarities between Ruk, CIN85 and CD2AP/CMS suggest that these proteins form a novel family of adaptor molecules that are involved in various intracellular signalling pathways.

Antiviral effect of trimeric 2',5'-oligoadenylic acid and some of its analogues.

The antiviral effect of 2',5'-trioligoadenylate (2',5'-A3) and some of its analogues was studied using several model cell culture systems and viruses: mice L929 fibroblast cells inoculated with vaccine virus, testicular piglet cells inoculated with Aueszki disease virus (strain BUK-628), and the same culture inoculated with a reference strain of transmissible gastroenteritis virus, strain Purdue-115. Our results suggest that both 2',5'-trioligoadenylate and its analogues are promising antiviral substances against DNA- and RNA-containing viruses.

The effect of trimeric 2',5'-oligoadenylic acid and its epoxy-derivative on human immunodeficiency virus (HIV-1) reproduction and retroviruses reverse transcriptase activity.

The effect of trimeric 2',5'-oligoadenylic acid (2',5' A3) and its epoxy-derivative (2',5'-A2 (RAA) on human immunodeficiency virus (HIV-1) reproduction was studied. An HIV-1 infectivity titer decrease was shown on the model of limphoblastoid cells when the substances under study were used. The interferonogenic effect of both substances was discovered. 2',5'-A2 (RAA) inhibited the activity of retrovirus reverse transcriptase (a C-type).

[Production of polyclonal antibodies to tyrosyl-tRNA-synthetase from the bovine liver and characterization of two forms of the enzyme]. / Poluchenie poliklonal'nykh antitel k tirozil-tRNK-sintetaze iz pecheni byka i kharakteristika dvukh form fermenta.

The polyclonal antibodies purified by affine chromatography against tyrosyl-tRNA synthetase (TyrRS) immobilized on the column with affigel-sepharose have been obtained from the bovine liver. The immunospecificity of these antibodies and their influence on enzymatic activity of TyrRS from the bovine liver have been investigated. We have stated that the polyclonal antibodies inhibited TyrRS enzymatic activity in aminoacylation of homologous tRNA(Tyr) by 47%. As it has been shown by immunoblotting the antibodies reacted both with the native enzyme (M(r) 2.59 kDa) and with the proteolytic cleaved functionally active form of the enzyme (M 2.39 kDa).

[Tyrosyl-tRNA-synthetase from bovine liver. Functional role of histidine residues]. / Tirozil-tRNK-sintetaza iz pecheni byka. Izuchenie funktsional'noi roli ostatkov gistidina.

A specific chemical modification of histidyl residues in tyrosyl-tRNA synthetase by diethyl pyrocarbonate was performed. It is shown that five of sixteen histidyl residues can react with diethyl pyrocarbonate in the native conditions. Modification of two histidyl residues per dimer results in the inactivation of tyrosyl-tRNA synthetase in both steps of the tRNATyr aminoacylation. All substrates protect tyrosyl-tRNA synthetase against inactivation with diethyl pyrocarbonate, the most effective protector being combination of ATP and tyrosine. Histidyl residues of tyrosyl-tRNA synthetase are suggested to be involved in the catalytic mechanism of aminoacylation of tRNATyr.

[Isolation and characteristics of functionally active proteolytically modified forms of tyrosyl-tRNA synthetase from bovine liver]. / Vydelenie i kharkteristkia funktsional'no aktivnoi proteoliticheski modifitsirovannoi formy tirozil-tRNK-sintetazy iz pecheni byka.

Functionally active proteolytic modified form of tyrosyl-tRNA-synthetase has been isolated in a homogeneous form from the bovine liver under incomplete blocking of endogenous proteolysis. The isolation scheme is described. From the data of gel electrophoresis under denaturing conditions the molecular weight of this form is 39 +/- 1.5 kDa and from the data of gel filtration under native conditions -84 kDa. Thus, this form as well as the native enzyme is a dimer of the alpha 2-type. As compared to the native enzyme (Mm 2 x 59 kDa) a proteolytically modified form has a fragment of the polypeptide chain about 20 kDa long split out (this fragment is not essential for catalytic activity). The values of catalytic characteristics of the modified form in tRNA(Tyr) aminoacylation reaction (Km = 1.19 microM and kcat = 2.99 min-1) are close to those obtained for the main form of the enzyme (0.69 microM and 2.97 min-1, respectively). Amino acid composition of the low-molecular form of tyrosyl-tRNA-synthetase has been determined. It was found that the fragment split out in limited proteolysis was characterized by very high content of positively charged lysine residues (46 residues). A proteolytically modified form of tyrosyl-tRNA-synthetase possesses, like the main form, the affinity to high-molecular rRNA but it is eluted from the column filled with rRNA-sepharose at lower salt concentration (50 mM KCl) as compared to the main form of the enzyme (100 mM KCl).

[Interaction of eukaryotic tyrosyl-tRNA-synthetase with high molecular weight RNA]. / Vzaimodeistvie éukarioticheskoi tirozil-tRNK-sintetazy s vysokomolekuliarnymi RNK.

The affinity of eukaryotic tyrosyl-tRNA synthetases from bovine liver and from yeast for E. coli ribosomal RNA and synthetic polyribonucleotides has been studied by protein binding on the rRNA-Sepharose column and enzyme inhibition by high molecular weight RNAs. Tyrosyl-tRNA synthetase from bovine liver (Mr 2.59 kDa) was fully retained on the rRNA-Sepharose and eluted by buffer with 100 mM KCl. The functionally active modified form of bovine liver tyrosyl-tRNA synthetase obtained by endogenous limited proteolysis (Mr 2.38 kDa) partially maintains the affinity for rRNA and is eluted by 50 mM KCl. The highest rRNA-binding ability was revealed for yeast tyrosyl-tRNA synthetase eluted by 200 mM KCl. The E. coli tyrosyl-tRNA synthetase was not retained on rRNA-Sepharose. The aminoacylation activities of both bovine liver and yeast tyrosyl-tRNA synthetases were efficiently inhibited by rRNA and the inhibition was partially competitive in respect to tRNA(Tyr). At the same time the activities of proteolytically modified bovine tyrosyl-tRNA synthetase and E. coli tyrosyl-tRNA synthetase were not influenced by the addition of rRNA. Synthetic single- and double-stranded polyribonucleotides specifically inhibited the activity of bovine tyrosyl-tRNA synthetase to different extent. The inhibition degree of bovine liver tyrosyl-tRNA synthetase decreased in the order: poly (G) greater than poly (I) greater than poly (I).poly (C) greater than poly (G).poly (C) greater than poly (C) greater than poly (A). Poly (U) did not inhibit the activity of bovine liver tyrosyl-tRNA synthetase.

[Determination of interacting segments of tRNA(Leu) from cow mammary glands with homologous aminoacyl-tRNA-synthetase by a chemical modification method]. / Opredelenie uchastkov vzaimodeistviia tRNK(Leu) iz molochnoi zhelezy korov s gomologichnoi aminoatsil-tRNK-sintetazoi metodom khimicheskoi modifikatsii.

The interaction of the cow mammary gland tRNA(IAGLeu), having a long variable loop, with the cognate aminoacyl-tRNA synthetase has been studied by the alkylation with ethylnitrosourea. It was shown that leucyl-tRNA synthetase protects from alkylation 3'-phosphates of the nucleotides 12-13 in D-loop, 23-24 in D-stem and 37-43 in the anticodon arm of tRNA(IAGLeu). All regions of interaction with the aminoacyl-tRNA synthetase are located in the same plane of tRNA whereas the long variable loop is in another plane.

Evidence for the ability of L10 ribosomal proteins of Salmonella typhimurium and Klebsiella pneumoniae to regulate rplJL gene expression in Escherichia coli.

Genes rplJ, coding for ribosomal protein L10 of Salmonella typhimurium and Klebsiella pneumoniae, have been cloned on pUC plasmid. The resultant multicopy recombinant plasmids were detrimental for the growth of normal JM101 E. coli host cells and harmless for the mutant JF3029 host. This negative effect is the evidence for the ability of heterologous L10 proteins to regulate expression of rplJL genes in E. coli. Nucleotide sequence was determined completely for S. typhimurium rplJL' DNA portion and partially for rplJL' genes of K. pneumoniae. According to the nucleotide sequence data obtained three amino acid substitutions differ L10 proteins of S. typhimurium and E. coli and the long range, providing for the coupled translations of L10 and L7/L12 cistrons in E. coli mRNA is also valid for S. typhimurium and K. pneumoniae.

[Isolation of tyrosyl-tRNA-synthetase from Thermus thermophilus HB-27]. / Vydelenie tirozil-tRNK-sintetazy iz Thermus thermophilus HB-27.

A method for isolating tyrosyl-tRNA synthetase from Thermus thermophilus is described, including ammonium sulfate fractionation, chromatography on DEAE-sepharose, hydroxyapatite, heparin-sepharose and hydrophobic chromatography on Toyopearl HW-65. The yield of the purified enzyme was 1.6 mg per 1 kg of T. thermophilus cells. The enzyme is a dimer protein of the alpha 2 type with molecular weight of 100 kDa.

[Primary structure of tRNA1ser from bovine liver]. / Pervinna struktura tRNK1ser pechinki bika.

The primary structure of tRNA(1Ser) from the bovine liver has been studied. pG- A-C-G-A-G-G-U-G-G-C-ac4C-G-A-G-D-Gm-G-D-D-A-A-G-G- C-m2(2)-G-A-psi-G-G-A-m3C-U-G-C-U-A*-A-psi-C-C-A-U-Um-G-psi- G-C-U-m3C-U-G-C-A-C-G-m5C-G-U-G-G-G-T-psi-C-G-m1A-A- U-C-C-C-A-U-C-C-U-C-G-U-C-G-C-C-AOH. A comparison of the nucleotide sequence of tRNA(1Ser) from the bovine liver with already known sequences of serine tRNA revealed a number of common nucleotides, some of them, probably, participated in specific interaction with seryl-tRNA synthetase.

[A study of the conformation of tRNA(IAGLeu) from the cow mammary gland using chemical modification methods]. / Issledovanie élementov prostranstvennoi struktury tRNK(IAGLeu) molochnoi zhelezy korov metodami khimicheskoi modifikatsii.

The nucleosides of tRNA(IAGLeu) (with a long variable loop) from the cow mammary gland included in formation of the three-dimensional structure have been analysed by the chemical modification methods. Exposed guanosine and cytidine residues were detected by means of dimethylsulfate, whereas diethylpyrocarbonate was used to detect exposed adenosine residues. The low level of the modification was characteristic of guanosine residues in positions 10 (m2G), 13, 15, 23, 24, 29, 30, 47 H, 51, 52, 53, 57; of cytidine residues in positions 48 (m5C), 56 and those involved in Watson--Crick pairing; of adenosine residues in positions 14, 22, 31, 42, 59, 64. Most bases of tRNA(IAGLeu) thus detected are similarly located in the yeast tRNA(Phe) molecule, which suggests a common role of these bases in the formation of the spacial structure of both tRNAs.

[Tyrosyl-tRNA synthetase from the bovine liver. Isolation and physico-chemical properties]. / Tirozil-tRNK-sintetaza iz pecheni byka. Vydelenie i fiziko-khimicheskie svoistva.

Tyrosyl-tRNA synthetase of beef liver has been isolated and its properties have been studied. Tyrosyl-tRNA synthetase is a structural dimer of alpha 2 type. Mr of the enzyme subunit is about 59 kDa. Km values for substrates have been determined and compared with kinetic properties of tyrosyl-tRNA synthetases from different sources. The polymorphism of tyrosyl-tRNA synthetase was studied. The enzyme was separated into two different forms by chromatography on phosphocellulose P 11. P1-form is active only in the amino acid activation reaction. This form is not due to the phosphorylation of the enzyme. The low molecular weight form (38 kDa) was also isolated. This form appeared due to the limited endogenic proteolysis of the main form and retained full activity in the aminoacylation reaction. Tyrosyl-tRNA synthetase from beef liver has non-specific affinity to rRNA-sepharose.

[Determination of phosphate residues participating in the formation of the spatial structure of tRNA- Leu IAG from cow mammary glands]. / Opredelenie ostatkov fosfornoi kisloty, uchastvuiushchikh v obrazovanii prostranstvennoi struktury tRNK- Leu IAG molochnoi zhele korov.

The phosphates of the tRNA-Leu IAG from cow mammary gland (tRNA which has a long variable loop) participating in the formation of three-dimensional structure were studied by alkylation with ethylnitrosourea and methylnitrosourea. A low degree of modification was observed for the phosphates of the following nucleotides: 7, 8, 9, 10 (at the bend site between the acceptor and D-stem); 18, 19, 20A and 21 in the D-loop; 47H and 49 at the joint of variable and T-stem; 57, 58 and 59 in the T-loop.

[Chemical modification of tryptophan residues of leucyl tRNA synthetase by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide]. / Khimicheskaia modifikatsiia triptofanovykh ostatkov leitsil-tRNK-sintetazy N-bromsuktsinimidom i 2-oksi-5-nitrobenzilbromidom.

The structural accessibility of tryptophan residues in leucyl-tRNA synthetase from cow mammary gland has been studied using chemical modifications by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide. The modifications were monitored by UV absorbance and intrinsic fluorescence of the enzyme's tryptophan residues. Under native conditions, at pH 7,8, only two exposed tryptophan residues are modified in each subunit of the dimeric enzyme. Under denaturing conditions, in 6 M guanidine hydrochloride solution, internal tryptophan residues are also modified as a consequence of unfolding of the native tertiary structure of the enzyme. Modifications of tryptophan residues resulted in inactivation of leucyl-tRNA synthetase both in aminoacylation and ATP-PPi exchange reactions. In the specific complex of leucyl-tRNA synthetase with the cognate tRNALeu one of exposed tryptophan residues is protected by tRNALeu and is not modified by the above reagents.