[Development of barcode and proteome profiling of glioblastoma].

High grade glioma (glioblastoma) is the most common brain tumor. Its malignancy makes it the fourth biggest cause of cancer death. In our experiments we used several glioblastoma cell lines generated in our laboratory to obtain proteomics information specific for this disease. This study starts our developing the complete 2DE map of glioblastoma proteins. 2DE separation with following imaging, immunochemistry, spot picking, and mass-spectrometry allowed us detecting and identifying more than 100 proteins. Several of them have prominent differences in their level between norm and cancer. Among them are alpha-enolase (ENOA_HUMAN), pyruvate kinase isozymes M1/M2 (KPYM_HUMAN), cofilin 1 (COF1_HUMAN), translationally-controlled tumor protein TCTP_HUMAN, annexin 1 (ANXA1_HUMAN), PCNA (PCNA_HUMAN), p53 (TP53_HUMAN) and others. Most interesting results were obtained with protein p53. In all glioblastoma cell lines, its level was dramatically up regulated and enriched by multiple additional isoforms. This distribution is well correlated with presence of these proteins inside of cells themselves. At this initial step we suggest the panel of specific brain tumor markers (signature) to help creating noninvasive techniques to diagnose disease. These preliminary data point to these proteins as promising markers of glioblastoma.

[Isolation of protein phosphokinase from a complex form of DNA polymerase alpha from rat liver]. / Vydelenie proteinfosfokinazy iz kompleksnoi formy DNK-polimerazy alpha pecheni krysy.

Protein kinase activity was revealed in complex forms of rat liver DNA polymerase alpha containing 3'-5'-exonuclease, primase, helicase, DNA ligase. Protein kinase (mol. mass about 200 kDa) has been partially purified from a specimen of high molecular mass DNA polymerase alpha of nuclear membrane of regenerating liver. The protein kinase activity of the complex form of DNA polymerase alpha was maximal in the cytosol in normal rat liver cells and in the nuclear membrane in dividing cells (40 h after partial hepatectomy). The main phosphokinase properties of this enzyme were determined.

[Contribution of 3'---5'-exonuclease from rat liver nuclei in precision of DNA synthesis, catalyzed by mammalian DNA polymerase alpha]. / Vklad 3'---5'-ekzonukleaz iz iader pecheni krysy v tochnost' sinteza DNK, kataliziruemogo DNK-polimerazami alpha mlekopitaiushchikh.

Mammalian nuclear DNA polymerases alpha and beta are known to be devoid of editing 3'-->5'exonucleolytic activity. Presumably this activity could be effected by the exonucleases non-associated covalently with DNA polymerases. Two 3'-->5'exonucleases with molecular masses of 40 and 50 kDa have been isolated from rat liver nuclei and purified to near homogeneity. They are shown to excise mismatched nucleotides from a poly [d(A-T)] template respectively 10- and 2-fold faster than the matched ones. Upon addition of any of these exonucleases to DNA polymerase alpha from rat liver or calf thymus, the fidelity of in vitro reproduction of primed DNA from bacteriophage phi X174 amber 3 is increased 5-10-fold, the levels of exonuclease and polymerase activities being approximately the same. The extrapolation of replication fidelity to cellular activities of the exonucleases and alpha-polymerase suggests that exonuclease proofreading augments the accuracy of DNA synthesis at least by three orders of magnitude.

[3'-->5'-exonucleases of the rat liver and the correction of replication errors]. / 3'-->5'-ékzonukleazy pecheny krysy i korrektsiia oshibok replikatsii.

Mammalian nuclear DNA polymerases alpha and beta are lack of the proofreading 3'-->5' exonucleolytic activity. 40 and 50 kDa 3'-->5' exonucleases were isolated from rat liver. The exonucleases were shown to excise mismatched nucleotides from poly[d(A--T)] template 10 and 2 fold faster than matched ones. The addition of either exonuclease to DNA polymerase alpha from rat liver or calf thymus 5-10 times increased the accuracy of reproduction of primed DNA from bacteriophage phi X174 amber 3, values of exonuclease and DNA polymerase activities being approximately equal. The exonuclease activity surpasses the DNA polymerase one by an order of magnitude in chromatin and nuclear membrane. These data, taken together, are indicative of potent proofreading into hepatocytes.

[Detection of DNA-binding proteins in polyacrylamide gel after denaturing electrophoresis]. / Obnaruzhenie DNK-sviazyvaiushchikh belkov v poliakrilamidnom gele posle denaturiruiushchego élektroforeza.

A simple method for detection of DNA-binding proteins is offered. These proteins can be revealed, following their electrophoretic separation in sodium dodecyl sulfate (SDS)-polyacrylamide gel containing labeled DNA, by washing the gel in buffer to remove SDS and to allow protein renaturation. Protein-free DNA is washed out, remaining in the DNA-binding proteins that restored their original characteristic. After autoradiography these proteins are seen as black bands (by one-dimensional gel electrophoresis) or spots (by two-dimensional gel electrophoresis) on a grey background. High sensitivity of the method is shown by using protein fractions of rat liver and a standard method.

[Homogeneous 3'----5'-exonucleases and their multienzyme complexes from the rat liver]. / Gomogennye 3'----5'-ékzonukleazy i ikh mul'tifermentnye kompleksy iz pecheni krysy.

Isolation and general properties of 3'-5' exonucleases I and II (EC 3.1.4.26), which are specific to single-stranded DNA, are described. Such enzymes, being components of replication complexes, could correct replication errors. Homogeneous exonucleases I and II consist of a single subunit with molecular mass of 50 and 40 kDa, respectively. These enzymes are located preferentially in the nuclear membrane and chromatin. They form complexes with nuclear DNA polymerases and some other proteins and are not observed practically in a free state. Molecular masses of the complexes amount from 70 to 1.500 kDa. The complexes dissociate as a result of solution hydrophobization and can be reconstituted after the decrease of hydrophobization. The heavy membrane complex form of 3'----5' exonuclease I manifests enzymatic activities of DNA polymerase alpha (EC 2.7.7.7), non-specific nucleoside triphosphatase (EC 3.1.3.2), nucleotidase (EC 3.1.3.31) and faint activity of endonuclease (EC 3.1.4.5). Complexes under study do not display activity of thymidine kinase (EC 2.7.1.21), marker protein of replitase, neither in G0 nor in S-period.

[Analysis of proteins in normal and regenerating rat liver using two-dimensional electrophoresis]. / Analiz belkov normal'noi i regeneriruiushchei pecheni krysy metodom dvumernogo élektroforeza.

The major proteins of homogenate, cytosol, nuclei and nuclear membrane extract from normal and regenerating rat liver were studied by two-dimensional electrophoresis with a view of detecting proteins involved in DNA replication regulation. Essential quantitative differences in three out of 200 polypeptides separated as spots and dyed with Coomassie R-250 on two-dimensional maps were revealed. The content of the p38 nuclear protein (Mr congruent to 38 kD, pI congruent to 4) increases 6-8-fold in the S-phase. The level of another nuclear protein, p50 (Mr congruent to 50 kD, pI congruent to 6.5) decreases 2-3-fold. The cytoplasmic protein p35 (Mr congruent to 35 kD, pI congruent to 8) also decreases 2-3-fold. Moreover, the p40 protein (Mr congruent to 40 kD, pI congruent to 6) whose content in the nuclei sharply rises up to 20 times after sham operation was revealed.

[Synthesis of heat-shock proteins in Saccharomyces cerevisiae protoplasts]. / Sintez belkov teplovogo shoka protoplastami drozhzhei Saccharomyces cerevisiae.

The effect of cellular capsule elimination in Saccharomyces cerevisiae yeasts (protoplast formation) on the heat-shock protein synthesis and the synthesis of the proteins in protoplasts were studied. The methods of mono- and dimeric electrophoresis have demonstrated that (1) about 18 heat-shock proteins with the molecular masses 26-98 Kd are synthesized in cells at 41 degrees C; (2) protoplast formation per se does not induce the synthesis of heat-shock proteins, but the induction of these proteins in protoplasts at 41 degrees C is similar to the one in intact cells. The protoplast formation induces the synthesis of specific proteins different from heat-shock proteins and the synthesis is inhibited by the heat-shock. The heat-shock induces modification of 88 and 86 Kd heat-shock proteins. It inhibits the synthesis of a number of peptides (15-50 Kd) in cells and protoplasts.

[Complexes of nuclear DNA-polymerases with 3'----5'-exonucleases from the rat liver]. / Kompleksy iadernykh DNK-polimeraz c 3'----5'-ékzonukleazami iz pecheni krysy.

"Editing" 3'----5' exonuclease activity of DNA polymerases corrects replication errors. This activity associated with procaryotic DNA polymerases is not intrinsic to purified mammalian DNA polymerases. By means of extraction and subsequent gel filtration, several subspecies of complexes of 3'----5' exonuclease (E.C. 3.1.4.26) with DNA polymerases alpha, beta (E.C. 2.7.7.7) and some other proteins were isolated from chromatin, nucleoplasm, nuclear membrane, and cytosol. Complexes containing 3'----5' exonuclease manifest from 40 to 70% of total DNA polymerase activity revealed in different compartments of a hepatocyte. Molecular masses of the complexes amount from 250 to 1500 kDa They dissociate as a result of solution hydrophobization. DNA polymerase alpha activity enhances 5--8 folds during cell transition from G0 to S-period. The value of the ratio of 3'----5' exonuclease activity of different complexes to their DNA polymerase activity varies from 0.5 to 12. Other cases of discovery of the complexes of DNA polymerases with 3'----5' exonucleases are discussed. It is suggested that the absence of 3'----5' exonuclease active site in the DNA polymerase polypeptide is compensated by the complex formation of the corresponding enzymes.

[Effect of the antitumor antibiotic bleomycin on DNA polymerase activity]. / Deistvie protivoopukholevogo antibiotika bleomitsina na DNK-polimeraznuiu aktivnost'.

Treatment with bleomycin activates considerably a repair synthesis of DNA in rat liver chromatin in vitro and can cause loosening of the nucleoprotein complex, which facilitates the accessibility or repair enzymes for lesions in chromatin DNA. The bleomycin action on DNA-template increases severalfold the rate of synthesis catalyzed by DNA polymerase beta inhibits the activity of DNA polymerase I from Escherichia coli and suppresses severalfold the activity of DNA polymerase alpha and DNA polymerase of bacteriophage T4. The effect of bleomycin consists in a prevailing increase of nicks and minimal gaps in DNA as compared to the rise of moderate gaps, thus suggesting that bleomycin is a gamma-mimetic.

[Non-specific acid nucleoside triphosphatase from cytosol and chromatin of rat liver: partial purification and general properties]. / Nespetsificheskaia kislaia nukleozidtrifosfataza tsitozolia i khromatina pecheni krysy: chastichnaia ochistka i osnovnye svoistva.

The nucleoside triphosphatase (EC 3.6.1.15) was isolated from rat liver cytosol and purified 600-fold. The enzyme hydrolyzes all NTPs and dNTPs, splits NDPs and dNDPs at a low rate and does not destroy NMPs and dNMPs. The phosphatase consists of a single subunit with molecular weight of 65 000. The chromatin fraction of the enzyme is fully bound to the membrane, whereas the cytosol fraction contains 15-30% of the membrane-bound enzyme. Both free and membrane-bound phosphatases possess identical functional properties. The enzymatic activity has a pH-optimum of 4.0--4.5, is increased in the presence of Me2+ and is unaffected by ouabain, Triton X-100, N-ethylmaleimide, NaF or DNA, but is inhibited by NaCl, Pi and PPi. The value of Km is equal to 20 microM for TTP splitting. Since the NTP pool is essentially changed throughout the cell cycle, it is suggested that the nucleoside triphosphatase can participate in the nucleotide pool regulation.

[Mechanism of activating action of ATP on repair synthesis of DNA in chromatin]. / Mekhanizm aktiviruiushchego deistviiv ATP na reparativnyi sintez RNK v khromatine.

The mechanism of activating action of ATP on the repair synthesis of DNA was studied in the chromatin isolated from rat liver (G0). It was shown that chromatin catalyzed the conversion dNTP leads to dNDP leads to dNMP leads to NdR. The principal mechanism of activating action of ATP is the maintenance of dNTP levels. The maintenance is carried out mainly by the inhibition of dNTP's phosphatases and in less extent by the means of reaction dNDP leads to dNTP. Besides that, ATP partially suppresses 3' leads to 5' exonuclease of chromatin which degrades the nascent DNA. The activating action of ATP is connected neither with phosphorylation of histones, nor with the activities of ATP-dependent endo- or exonucleases, DNA gyrase, polynucleotide ligase, or DNA unwinding protein.