[Computer modeling of cytochrome P450 2E1 three-dimensional structure].

A computer model of human cytochrome P450 2E1 (CYP2E1) three-dimensional structure and active site was constructed based on homology with crystallographic coordinates of CYP2C5 and CYP2C9. A high degree of secondary structure homology for human, mouse, rat and rabbit CYP2E1 was demonstrated. The location of heme and the supporting alpha-helices was established. CYP2E1, CYP2C5 and CYP2C9 active sites are distinguished by pocket size and their amino acid residues composition. Key amino acid residues forming the active site channel and substrate-binding cavity are presented. Active site surface area and volume for CYP2E1, CYP2C5 and CYP2C9 were calculated.

Association of CYP2E1 gene polymorphism with predisposition to cancer development.

We summarize research results on association of cytochrome P450 2E1 (CYP2E1) genetic polymorphisms with the development of ecologically determined cancers. Cytochrome P450 monooxygenase enzyme system is involved in metabolic activation of procarcinogens, which is an important stage during normal cell transformation into a malignant one. Studies of association between CYP2E1 gene polymorphisms and the risk of cancer development are of particular interest, since the enzyme participates in bio-transformation of numerous procarcinogens. We place special emphasis on characterization of known CYP2E1 polymorphisms and analysis of their role in pathogenesis of various cancer types. Results obtained provide evidence on association of CYP2E1 genetic polymorphisms both with increased and decreased risk of development of malignant tumors. Ethnic and geographic peculiarities were also identified for frequencies of CYP2E1 variants distribution.

[Effect of factors from the Chernobyl exclusion zone on the level of cytochrome P-450 in mouse liver microsomes]. / Vliianie faktorov chernobyl'skoi zony otchuzhdeniia na soderzhanie tsitokhroma P-450 v mikrosomakh pecheni myshei.

The data concerning the influence of Chernobyl exclusive zone factors on cytochrome P-450 relative contents in liver microsome fractions of Balb/c, CC57W/Mv and C57 Black mice are submitted in this work Reliable diminishing of liver P-450 relative concentration in experimental animals, compared with respective isogenic control groups is shown to be varying depending on the mouse line and sex.

[The effect of monovalent cations on the structure of chromatin from Ehrlich ascites carcinoma cells]. / Vliiznie monovalentnykh kationov na strukturu khromatina kletok astsitnoi kartsinomy Erlikha.

Changes in the intracellular ratio of monovalent cations resulting in the alteration of transcriptional activity in some cell-specific genes have been investigated. It was found that at varying intracellular Na+/K+ concentration ratios the accessibility of the beta-actin gene to the effect of exogenous DNAaseI changes drastically. The use of hybridization of restricted DNA of the nuclear matrix with appropriate probes revealed that the number of copies for oncogenes c-fos and c-myc within this DNA changes with alteration of the intracellular ratio of monovalent cation concentrations.

Ascitic tumor cell cycle metabolism and energetics.

Data are presented on Ehrlich ascitic tumor cells energetic metabolism, activities of the glycolytic enzymes and the pentose phosphate pathway enzymes, contents and synthesis rates of the macromolecules at various cell cycle stages. An attempt was made to correct the direct measurement by taking into consideration a systematic error introduced in the experiment by incomplete cells synchronization. Cell metabolism activation sharply increased at two mitotic cycle stages. At the first stage (end of G1-period, beginning of S-period) the processes associated with the preparation to reduplication and DNA synthesis were activated. The second activation wave (end of S-period, G2-period) was connected with cell preparation of mitosis. The coordination of cell metabolism variations during the cell cycle is shown.

Intracellular ratio between monovalent cations (Na+/K+), and transcriptional activity of genes in tumour cells.

The effects of the intracellular Na+/K+ ratio on transcriptional activity of ribosomal, c-fos, beta-actin and histone genes of Ehrlich ascites tumour cells and P-388 leukaemia cells have been investigated. Optimum values of the Na+/K+ ratio for selective transcription of genes are shown, they are in accordance with the Na+/K+ ratio and mRNA content of respective genes during the cell cycle. Differential activation and repression of these genes do not correlate with the total synthesis rate of RNA during the cell cycle. The data indicate a selective nature of the action of monovalent cations on gene expression during the cell growth.

[Effects of intracellular Na+/K+ ratio on expression of c-fos oncogene and beta-actin gene in ascitic cells of leukemia P-388 and Ehrlich tumor]. / Vliianie vnutrikletochnogo sootnosheniia Na+/K+ na ékspressiiu onkogena c-fos i gena beta-aktina v astsitnykh kletkakh leikoza P-388 i raka Erlikha.

The intracellular Na+/K+ ratio was studied for its effect on expression gene beta-actin and oncogene c-fos and activity of these genes during the mitotic cycle in ascites cells of leukemia P-388 and Ehrlich tumour. It was established that gene beta-actin was activated at high and low ratios of Na+/K+, while c-fos only at high ones. The expression of these genes during the mitotic cell cycle was due to changes in the intracellular Na+/K+ ratio. The obtained data showed an important role of intracellular homeostasis of monovalent cations in regulation of gene expression during the mitotic cell cycle.

[Effects of DL-difluoromethylornithine on the cell surface charge of Ehrlich ascitic cancer during different phases of the mitotic cycle]. / Vliianie DL-diftormetilornitina na zariad poverkhnosti kletok astsitnogo raka Erlikha v razlichnye fazy mitoticheskogo tsikla.

Difluoromethylornithine (DFMO)-induced polyamide deficiency has been studied for its effect on the electrophoretic mobility of the Ehrlich ascitic cells in G1, S and G2 phases of the mitotic cycle. The DFMO treatment alters the pattern of the electrophoretic mobility distribution of Ehrlich ascite cells. The most profound changes of the electrophoretic mobility pattern are observed in G1-phase and at the S phase beginning. Exogenic polyamines decrease the electrophoretic mobility of cancer cells, which is accompanied by restoration of the initial pattern of the electrophoretic mobility distribution.

Energy supply of the mitotic cell cycle and the Na+/H+-antiport in ascites tumors.

The activation of Na+ transport is due to the exchange of protons formed via glucose conversion into lactate for Na+, i.e., to the stimulation of the Na+/H+-antiport. Experimental results and theoretical calculations suggest that in glucose-containing medium the Na+ transport increases from 0.75 to 1.78 pmol/hour per cell. The permeability of plasma membranes for K+ increases 2.75 fold, while the passive flux of Na+ diminishes. The intensity of O2 adsorption by ascites tumor cells does not practically depend on the monovalent cation concentration gradient between the cells and the culture medium, whereas the rate of glycolysis decreases simultaneously with the diminution of the concentration gradient. In synchronized cultures at the beginning of the mitotic cycle, the bulk of ATP resynthesized via glycolysis is utilized for the synthesis of biopolymers, whereas that at the end of the S-phase and in the G2-phase is utilized for cation transport across plasma membranes. From 35 to 100% of the whole amount of ATP resynthesized via glycolysis is utilized for transport purposes. It is concluded that the observed increase in the Na+/K+ ratio in ascites tumor cells is connected with their enhanced ability to synthesize lactic acid. Presumably, glycolysis is one of the regulatory mechanisms of intracellular ratios of monovalent cations.

[Morphometric characteristics and functional activity of the cells of Ehrlich ascites cancer found in different phases of the cell cycle]. / Morfometricheskaia kharakteristika i funktsional'naia aktivnost' kletok astsitnogo raka Erlikha, nakhodiashchikhsia v razlichnykh fazakh kletochnogo tsikla.

The Ehrlich ascites carcinoma cells at different interphase periods have been studied for dynamics of ultrastructural and morphometric characteristics and biochemical analysis of metabolism of the given cells has been made. A distinctive structure-function correlation detectable by stage coincidence in the re-structure with the cell metabolism alterations is shown.

[Effect of monovalent cations on the ratio of RNA transcripts from DNA with different repetitive sequences in leukemia P-388 cells]. / Vliianie monovalentnykh kationov na sootnoshenie RNK-transkriptov s raznykh po povtorennosti posledovatel'nostei DNK v kletkakh leikemii P-388.

Using leukemic P-388 cells, it was demonstrated that alterations of the intracellular Na+/K+ ratio results in qualitative changes of newly synthesized mRNA, which manifests itself as changes in the kinetics of hybridization of heterogeneous nuclear RNA (hnRNA) with DNA. The decrease of the Na+/K+ value from 3.8:1 to 1:1 leads to inhibition of mRNA synthesis in mRNA cells hybridized at 10(3) greater than Dot greater than 10(4), but weakly affects the transcription of mRNA sequences hybridized with DNA within the Dot interval of 10(3.4)-10(3.8). A similar phenomenon is observed during hybridization of hnRNA with homogeneous fractions of unique and averagely repeating sequences of DNA. The hybridization rate constants of hnRNA synthesized by cells at different values of Na+/K+ and the limit values of hybridization (H infinity) were calculated. It was shown that the rate constants for RNA hybridization with DNA decrease by more than two orders of magnitude during the transition of the averagely repeating DNA fraction to the unique one; however, in both cases these constants give equal values for the RNA synthesized by cells at different cationic balance. The H infinity values for the RNA synthesized by cells at higher Na+ ratio appeared to be 1.5-2.0 times as high as compared with those for the RNA in the cells, in which the Na+/K+ ratio was 1:1. Thus, the monovalent cation ratio seems to exert a strong influence on the expression of sequences of different repeatedness in the genome and to play a role in the regulation of proliferative activity of the cell.

[Energy characteristics of the transport of monovalent cations in ascites tumor cells]. / Energeticheskie osobennosti transporta odnovalentnykh kationov v kletkakh astsitnykh opukholei.

The intensity of O2 adsorption by ascite tumour cells does not practically depend on the monovalent cation concentration gradient between the cells and the incubation medium, whereas the rate of glycolysis decreases simultaneously with the diminution of the concentration gradient. In synchronized cultures at the beginning of the mitotic cycle, the bulk of ATP resynthesized via glycolysis is utilized for the synthesis of biopolymers, whereas that at the end of the S-phase and in the G2-phase--for cation transport across plasma membranes. From 35 to 100% of the whole amount of ATP resynthesized via glycolysis is utilized for transport purposes. The experimental results and theoretical calculations suggest that in glucose-containing media Na+ transport increases from 0.75 to 1.78 pmol/hour on a per cell basis. The activation of Na+ transport is due to the exchange of protons formed via glucose conversion into lactate for Na+, i.e., to the stimulation of Na+/H+ antiport. The permeability of plasma membranes for K+ increases 2.75-fold, while the passive flux of Na+ diminishes. It is concluded that the observed increase in the Na+/K+ ratio in ascite tumour cells is connected with their enhanced ability to synthesize lactic acid. Presumably, glycolysis is one of regulatory mechanisms of intracellular ratios of monovalent cations.

[Biosynthesis of protein and RNA during changes in the Na and K ratios in Ehrlich carcinoma cells]. / Biosintez belka i RNK pri izmenenii sootnosheniia Na+ i K+ v kletakh raka Erlikha.

The changes in the intracellular ratios of Na+ and K+ by equilibration of Ehrlich carcinoma cells in salt media at 0-4 degrees C leads to the changes in the rate of rRNA and protein synthesis. Using competitive hydridization, it was shown that with a decrease of the Na+/K+ ratio from 1:1 to 1:2, the rate of pre-rRNA synthesis is markedly diminished. The incorporation of labelled amino acids into histones, nonhistone proteins, and cytoplasmic proteins also depends on the ratio of monovalent cations in cancer cells. At the translation level, Na+ and K+ are responsible for the selectivity of protein synthesis, whereas upon transcription these cations determine the rate of synthesis of corresponding mRNAs. It is concluded that the intracellular ratio of monovalent cations is one of the mechanisms, by means of which the cells operate their genetic control.

Isolation of low molecular protein P-9 from nuclei of NK/Ly lymphoma and Guerin carcinoma.

Protein with molecular weight 9000 (protein-9) was found in nuclei of resting cells of NK/Ly lymphoma and Guerin carcinoma by electrophoresis in polyacrylamide gel. Method of preparative isolation P-9 concluding in fractionation of nuclei from phenol extract by acetone at different pH and following electrophoresis in polyacrylamide gel was made. Electrophoretically homogeneous preparation of protein P-9 from nuclei of NK/Ly lymphoma and Guerin carcinoma was obtained.

[Effects of monovalent cations on synthesis and degradation kinetics of RNA in cells of Ehrlich carcinoma]. / Vliianie monovalentnykh kationov na sintez i kinetiku raspada RNK v kletkakh raka Erlikha.

The changes in intracellular ratio of Na+/K+ by equilibrating the cells in saline media at 0-4 degrees C cause changes in the rate of RNA synthesis in the cells. The optimum of [3H]uridine incorporation lies within the range of the Na+/K+-ratio of 7 : 1 to 3 : 1. Within the Na+/K+ ratio of 3 : 1 to 1 : 1 the rate of RNA synthesis is decreased about 2.5-fold, showing a further fall at the Na+/K+ ratio of about 1 : 3. Using competitive hybridization of labelled and non-labelled heterogenous nuclear RNA with DNA, it was shown that the total rate of synthesis and the nature of newly formed transcripts are changed. When the cells equilibrated with a certain saline medium were labelled with [3H]uridine for 15 min and their cationic equilibrium was changed by another equilibration in a medium having a different Na+/K+ ratio, the processing of labelled heterogenous nuclear RNA was drastically changed, i.e. part of the labelled RNA was rapidly depolymerized, while the other part was deposited in the nuclei or cell cytoplasm. Hence the intracellular cationic homeostasis can control the transcription and its changes cause recoding of gene expression. Besides, the cells contain an active system which controls the transcription program and depends on intracellular ionic homeostasis. This system prevents the release of non-essential mRNAs into the cytoplasm by depolymerizing or depositing them in the nuclei.

[Isolation and physico-chemical properties of P-9 protein, marker for the resting cells of lymphoma NK/Ly and Gerene carcinoma]. / Vydelenie i fiziko-khimicheskie svoistva belka P-9, markiruiushchego pokoiashchiesia kletki limfomy NK/Ly i kartsinomy Gerena.

A fraction of tumour cells in the state of proliferative rest was prepared by sedimentation from aged Ehrlich tumor and lymphoma NK/Ly. These cells do not incorporate [3H]-thymidine and contain fewer proteins and RNAs than the proliferating cells. The incorporation of [3H]-uridine by these cells makes up to 3.5-10% of the maximal incorporation by proliferating cells at the S-phase. The resting cells have a low rate of glycolysis and a high rate of respiration. The activity of lactate dehydrogenase, glucose-6-phosphate dehydrogenase and malate dehydrogenase in these cells is considerably reduced. The resting cell nuclei were found to contain a low molecular weight protein with molecular weight of 9000 +/- 500 (protein P-9) absent in proliferating cell nuclei. Using acetone fractionation and a subsequent preparative electrophoresis in a phenol solution, protein P-9 was isolated from the nuclei of lymphoma NK/Ly and Gerene carcinoma in a pure state. The physico-chemical properties of the protein from various sources were found to be similar. Thus, protein P-9 can be attributed to low molecular weight non-histone proteins with pI at the weakly alkaline region of pH.