[Monoclonal antibodies to 8-oxo-2'-deoxyguanosine (8-hydroxyguanosine). Characteristics and use for determining DNA damage by active forms of oxygen]. / Monoklonal'nye antitela k 8-okso-2'-dezoksiguanozinu (8-gidroksiguanozinu). Kharakteristika i ispol'zovanie dlia opredeleniia povrezhdeniia DNK aktivnymi formami kisloroda.

It has presently been established that the guanine base is one of the most sensitive and biologically significant target for the damaging action of active oxygen species on DNA, 7.8-dihydroxy-8-oxoguanine (8-oxoguanine, 8-hydroxyguanine) being the major degradation product and the most essential biomarker for DNA damage by active oxygen species. Murine monoclonal antibodies (MAbs) specifically recognizing 8-oxoguanine have been raised. Using competitive solid phase immunoenzymatic assay. (IEA) with peroxidase-antiperoxidase (PAP-method), a quantitative assay of this degradation product and characterization of affinity and specificity of MAbs have been carried out. The affinity constant (Kaff) Mabs for 8-oxo-2'-deoxyguanosine is equal to 1.3-10(6), that for 8-oxo-guanosine-to 1.10(6), exceeding by more than three orders of magnitude the Ka values for natural guanyl nucleosides and other possible cross-structure analogs. IEA was used to determine the DNA degradation product in gamma-irradiated DNA. The radiation-chemical yield of 8-oxoguanine (G = 0.57 molecules per 100 eV) is consistent with those obtained by other methods.

[Use of monoclonal antibodies for detecting Aujeszky's disease virus in culture media by an immunoenzyme method]. / Ispol'zovanie monoklonal'nykh antitel dlia opredeleniia immunofermentnym metodom virusa bolezni aueski v kul'tural'noi srede.

Three modifications of quantitative enzyme immunoassay (EIA) for assessment of Aujeszky's disease virus (ADV) in culture medium are compared, in which monoclonal antibodies (MAB) to ADV GH glycoprotein were used: nonconcurrent two-site "sandwich" EIA on the basis of two different MABs, indirect concurrent EIA, and direct concurrent EIA. MABs fit best of all for use in every of EIA modifications were selected. Conditions of the assays were optimized. Concurrent EIAs were 10-20 times less sensitive than sandwich EIA. The developed sandwich EIA permitted ADV assay in preparations of both live and formaldehyde-inactivated virus.

[The isolation and characteristics of monoclonal antibodies to the glycoprotein GII of Aujeszky's disease virus and their use for the epitopic mapping of GII]. / Poluchenie i kharakteristika monoklonal'nykh antitel k glikoproteinu GII virusa bolezni Aueski i ikh ispol'zovanie dlia épitopnogo kartirovaniia GII.

A panel of 24 different monoclonal antibodies (MAB) to Aujeszky's disease virus (ADV) proteins was prepared. Seven MABs were directed to ADV glycoprotein GII (6 MABs to GIIc chain and 1 MAB to GIIb chain). GII epitope mapping with the resultant MABs was carried out. Four ADV GII epitopes were detected: epitopes A, B, and C located on GIIc chain and epitope D located on GIIb chain. Epitope B overlaps epitopes A and C which do not overlap each other, epitope D does not overlap other epitopes. Epitopes A, B, and C are located in a restricted area of GIIc chain which seems to be one of GII immunodominant regions. The detected epitopes are mainly conformational, for they are sensitive to denaturation. Of all MABs obtained more than 30% were directed to ADV GII. This indicates that GII is one of the most important ADV antigens. All the seven MABs to GII interacted with all tested ADV strains (K. Arsky, VGNKI, BUK, and NIA-4), this being in line with the data on a highly conservative nature of ADV GII.

[The detection of lipofuscin in a culture of hybridoma cells]. / Obnaruzhenie lipofustsina v kul'ture gibridomnykh kletok.

Lipofuscin granules (LG) are found in the cultured hybridoma cells producing monoclonal antibodies to phage. LG have been studied using light and electron microscopy. Luminescent spectra of LG clusters in hybridoma cells are presented. The increase of own luminescence intensity of LG in the course of excitation by ultraviolet (365/nm) is shown. The advantages of hybridoma cells culture for investigation of LG on the cell level are discussed.

[The demonstration of proliferating cells by using monoclonal antibodies to 5-bromo-2'-deoxyuridine in the peroxidase-antiperoxidase method]. / Vyiavlenie proliferiruiushchikh kletok s pomoshch'iu monoklonal'nykh antital k 5-brom-2'-dezoksiuridinu metodom peroksidaza--antiperoksidaza.

Monoclonal antibodies (MAb) to the thymidine analog 5-bromo-2'-deoxyuridine (BrdUrd) and to horseradish peroxidase (HRPO) have been produced and characterized. On the basis of Mab to HRPO, complexes of the antibodies and HRPO (PAP-complexes) for immunochemical investigations are prepared. The possibility to identify proliferating cells in cultures of mouse myeloma Sp2/0 and mouse fibroblasts NIH 3T3 using Mab to BrdURd by the PAP-method is shown. The conditions of performing the analysis were optimized. The effect of various techniques ot cell fixation and cell DNA denaturation on cell morphology and on specific staining of nuclei of BrdUrd-containing cells in investigated.

[Ultrastructural analysis of the action of centrophenoxine on retrovirus-transformed cells]. / Ul'trastrukturnyi analiz deistviia tsentrofenoksina na retrovirustransformirovannye kletki.

The ultrastructure of hybridoma cells cultured with 5.10(-4) centrophenoxine (CP) has been studied. It is shown that CP effects hybridomas and prevents retrovirus exocytosis. The effect of CP on Ca-calmodulin system associated with cytoskeleton is suggested.

[Antibody interaction with the amphotericin channel]. / Vzaimodeistvie antitel s amfoteritsinovym kanalom.

The monoclonal antibodies against asymmetric channel formed in the lipid bilayer of polyene antibiotic amphotericin B and cholesterol after addition of the antibiotic to the compartment from the cis side of the membrane were obtained. The effect of the antibodies on ion conductance of the channel depends on the distribution of cholesterol in the membrane. When cholesterol was present on both sides of the lipid bilayer, three antibody molecules bound to the channel from the trans side of the membrane, thus markedly increasing the lifetime of the open state of the channel. When cholesterol was present in the cis monolayer only, the antibodies, added to the trans compartment of the cell, reduced the membrane conduction.

[Regulation of the synthesis of total cellular proteins and monoclonal antibodies in a hybridoma cell culture]. / Reguliatsiia sinteza summarnykh kletochnykh belkov i monoklonal'nykh antitel v kul'ture gibridomnykh kletok.

A study was made of the regulation of total protein synthesis in cells of the mouse hybridoma producing monoclonal antibodies (McAb) against lambda phage, and in the course of hybridoma growth and at the change of fetal bovine serum (FBS) concentration. FBS strictly affected proliferation of hybridoma cells, the specific production of McAb per cell being unchanged. The rate of total cellular protein synthesis does depend on FBS concentration in the medium, whereas the rates of protein degradation and secretion do not. Evidence is presented that the reduction in the protein-synthesis rate, after the removal of FBS from the medium, is caused by a coordinated decrease in both the rate of protein synthesis initiation and the rates of polypeptide chain elongation and translation termination. The decrease in the protein synthesis rate at the stationary phase of cell growth was shown to be related to the three main factors: 1) a 15-25% decrease in ribosome content per cell; 2) a two-fold decrease of the ribosome portion involved in mRNA translation; 3) a 5 to 15% decrease in the rate of mRNA translation. Evidence is presented that the decrease in the portion of mRNA translating ribosomes is due to the decrease in the rate of protein synthesis initiation.

[Purification of restriction endonuclease EcoRII using monoclonal antibodies]. / Ochistka restriltsionnoi éndonukleazy EcoRII s ispol'zovaniem monoklonal'nykh antitel.

Monoclonal antibodies against EcoRII endonuclease were obtained after immunization of two BALB/c mice with a homogeneous enzyme prepared by conventional methods. IgG from ascitic fluid was purified and coupled to CNBr-activated Sepharose 4B to give a specific column used to isolate EcoRII endonuclease. The isolated EcoRII endonuclease produced a single band during SDS gel electrophoresis.

[The use of monoclonal antibodies for purification of ribonuclease H from E. coli]. / Ispol'zovanie monoklonal'nykh antitel dlia ochistki ribonukleazy H E. coli.

Monoclonal antibodies to E. coli ribonuclease H were obtained from two hybrid clones. Using the monoclonal antibodies two immunosorbents were synthesized for RNase H which have a slight difference in the capacity and do not differ in the conditions of antigen elution. A homogeneous (according to electrophoresis in PAAG) preparation of the enzyme was obtained using the synthesized immunosorbents.

[Monoclonal antibody metabolism during the growth of hybridoma cells]. / Metabolizm monoklonal'nykh antitel v protsesse rosta gibridomnykh kletok.

Metabolism of monoclonal antibodies (MAb) during the growth of mouse hybridoma, producing MAb to phage lambda, has been studied. It was shown that the specific production of MAb decreased by 25-35% in the stationary phase of growth in comparison with the middle of the exponential growth phase, which was associated with the decrease in the rate of MAb synthesis. The secretion kinetics of MAb did not change during the growth of hybridoma cells. MAb did not degrade inside the cells and in the culture medium after being secreted. The ratio of the synthesis rate of MAb to that of cellular proteins increased from 7-10% in the exponential growth phase to 14-18% in the stationary phase, which points to a specific regulation of MAb synthesis in comparison with cellular proteins. Possible regulation mechanisms for synthesis of MAb and cellular proteins during the growth of hybridoma cells are discussed.

[Protein metabolism during the growth of hybridoma cells]. / Belkovyi metabolizm v protsesse rosta gibridomnykh kletok.

Protein metabolism during the growth of mouse hybridoma, producing monoclonal antibodies to phage lambda has been studied. The rate of protein accumulation Va at any time of growth was equal to that of protein synthesis Vs minus the degradative rate Vd and protein secretion rate Vsecr (Vn = Vs--Vd--Vsecr). Cellular protein metabolism was described in terms of the relationship 6.3% hr = 8.1%/hr--1%/hr for the middle of exponential growth phase, and in terms of the relationship 1.4%/hr = 2.9%/hr = 0.8%/hr--0.7%/hr for the early stationary phase. The results obtained enabled us to conclude that the synthesis of cellular proteins may be the main process regulating the protein metabolism during hybridoma cell growth.

[Effect of SH-reagents on Ca2+ level in lymphocyte cytoplasm]. / Vliianie SH-reagentov na kontsentratsiiu Ca2+ v tsitoplazme limfotsitov.

The effect of SH-reagents on cytoplasmic free Ca2+ concentration [( Ca2+]i) in rat thymocytes and B lymphoma Raji cells has been studied by means of fluorescent Ca2+ indicator quin-2. N-ethylmaleimide and ethylmercurythiosalicylate have been found to induce a dose-dependent increase of Ca2+ concentration from about 100 nM in the control cells up to 1000 nM. The effect is weakened with a decrease of the external Ca2+ concentration and is not observed already with Ca2+ concentration in the medium less than 0.2 mM. Reduction of the level of intracellular ATP does not suppress the Ca2+ response to SH-reagents. The effect of SH-reagents is weakened with a decrease of the temperature from 37 to 24 degrees C. Addition of 1 mM Mn2+ or Ca2+ into the standard medium containing 1 mM CaCl2 prevents Ca2+ concentration increase in the cytoplasm under the action of SH-reagents. The conclusion is made that in lymphocytes Ca2+ permeability is regulated by a protein(s) sensitive to the SH-reagent and that a high level of SH-group oxidation is necessary to maintain the low Ca2+ permeability of lymphocyte plasma membrane. Mechanisms of SH-reagents action on the Ca2+ level in the cell are discussed.

[Hybridoma cultures using porcine serum not containing immunoglobulin and dialysis fractions]. / Kul'tivirovanie gibridom s ispol'zovaniem svinoi syvorotki, ne soderzhashchei immunoglobulinovoi i dializnoi fraktsii.

The whole swine serum was treated with ammonium sulphate to precipitate immunoglobulins. The remained IgG was removed with the use of protein A-sepharose. The hybridoma cells producing monoclonal antibodies to lambda phage (class IgG) were cultured in Dalbecco's modified Eagle medium with addition of a 5% whole swine serum or of a treated unwhole one (final concentration of the protein being 3 mg/ml). Upon these conditions, hybridoma cells had similar growth rate and population density (1-1.3 X 10(6) cells/ml). Maximal antibody concentration was almost similar (80-90 mcg/ml). Purity of a sample of monoclonal antibodies isolated by the method of chromatography with the use of protein A-sepharose from supernatant containing the unwhole serum was no less than 99%, whereas it was considerably lower (12-15%) in the case of the whole serum.

[DNA degradation during radiation death of cultured cells]. / Zakonomernosti degradatsii DNK pri radiatsionnoi gibeli kletok v kul'ture.

The method of flow cytofluorometry was used to study postirradiation changes in plasma membrane permeability and DNA content of the Burkitt's lymphoma cells (Raji line). At a dose of 10 Gy, the increase in membrane permeability preceded the appearance of cells with diminished DNA content. The synchronization of cells in phase G2 was associated with a virtually complete radiation arrest of mitoses. The postirradiation electrophoretic analysis of DNA of the Burkitt's lymphoma cells and of Syrian and Chinese hamster fibroblasts showed that the DNA degradation is unordered and results perhaps from activation of hydrolases in the dead cells.

[Swine serum increases hybridoma stability]. / Svinaia syvorotka povyshaet stabil'nost' gibridom.

Three hybridoma cell lines producing monoclonal antibodies to lambda phage were cultured for 9 months in Dulbecco's modified Eagl's medium supplemented with 10% fetal calf serum or with 5% swine serum. Under these conditions all the clones had the same proliferation rates and maximum population densities. The cultuvation in the medium with swine serum enhanced the stability of hybridomas: all clones retained the ability to produce antibodies throughout the experiment; one of the clones examined enhanced the activity by 10(3)-10(4) times. When the same three clones were cultured in the medium with the fetal calf serum, two of them lost their ability to produce antibodies 3-15 weeks after the beginning of the experiment.

[Action of secondary radiation from 70-Gev protons on the cellular DNA of mammals]. / Issledovanie deistviia vtorichnogo izlucheniia ot protonov 70 Gév na DNK kletok mlekopitaiushchikh.

A study was made of the effect of secondary radiation of 70 GeV protons on DNA of Chinese hamster cells. With a reference to fibroblast DNA, lymphoid cell DNA, and the lethal effect of radiation on the survival of Chinese hamster cells the RBE was 1.6-7.6, 1.1-3.8 and 1.14-1.7, respectively. DNA breaks were repaired to an equal level after exposure to secondary radiation from the accelerator and gamma-radiation from 60Co in equally effective doses.

[Advantages of using swine serum in culturing animal and human cell lines]. / O preimushchestvakh ispol'zovaniia svinoi syvorotki pri kul'tivirovanii nekotorykh linii kletok zhivotnykh i cheloveka.

Using the culture of lymphoid human cells, mice myeloma cells and hybridoma (the latter was obtained by fusion of myeloma cells with those of mice spleen immunized with phage lambda), the fetal serum was shown to be surpassed in growth activity by that from adult swines. This fact is especially pronounced at small serum concentrations. When hybridoma cells were cultured there were no differences in titre of monoclonal antibodies specific to phage lambda with the use of both sera. The possibility of substitution of swine serum for human one was demonstrated using the culture of lymphocytes from human peripheral blood.