[Effect of actinomycin D, cycloheximide, puromycin and mitomycin C on the biosynthesis of heat shock proteins in the nuclear matrix of Chinese hamster fibroblasts]. / Vliianie aktinomitsina D, tsiklogeksimida, puromitsina i mitomitsina C na novoobrazovanie belkov teplovogo shoka iadernogo matriksa fibroblastov kitaiskogo khomiachka.

The thermal shock proteins with molecular mass of 86 kD and pI 5.5 as well as of 70 kD and pI 5.2-5.4 were isolated by means of two-dimensional electrophoresis in polyacrylamide gel from nuclear matrix of chinese hamster fibroblasts after heating of the animals. Incorporation of 35S-methionine into the thermal shock proteins was completely inhibited by actinomycin D (2 micrograms/ml), if the antibiotic was added into the cell incubation medium before heating, while the incorporation of methionine was decreased only slightly when the antibiotic was added after heating of the cells. Thus, biosynthesis of mRNA of the thermal shock proteins of 86 and 70 kD in nuclear matrix was induced by means of an increase in temperature during the incubation of the cells. Biosynthesis of the thermal shock proteins in nuclear matrix was distinctly inhibited by cycloheximide (1 microgram/ml) and was not practically inhibited by puromycin (60 micrograms/ml).

[Heat-shock proteins in the nuclear matrix of Chinese hamster fibroblasts]. / Belki teplovogo shoka iadernogo matriksa fibroblastov kitaiskogo khomiachka.

Heating of Chinese hamster fibroblasts (46 degrees C, 10 min) results in sharp inhibition of protein biosynthesis in the homogenate, nuclei and, in a lesser degree, in the nuclear matrix. The ratio of specific radioactivity of nuclear matrix 35S-proteins to the homogenate radioactivity taken for 100% increases after the heat shock 2,5-fold. Thus, protein biosynthesis in the nuclear matrix is more stable to the damaging action of heat shock than that in the homogenate and nuclei. The nuclear matrix was shown to contain heat shock proteins with Mr of 82-84, 70 and 26 kD, the 70 kD polypeptide being predominant. This polypeptide revealed in 4 hours is intensively accumulated at the 6th hour and remains unchanged by the 17-24th hours after cell heating. The 70 kD heat shock polypeptide can be separated by two-dimensional electrophoresis into two subfractions differing in terms of pI from other proteins revealed within this time interval in the unheated cells.

[Effect of hyperthermia on the polypeptide composition of the nuclear matrix of the rat liver]. / Vliianie gipertermii na polipeptidnyi sostav iadernogo matriksa pecheni krysy.

The increase in rat body temperature by 2-3 degrees as a result of overheating (45 degrees C, 22% humidity) over 90 and 120 min is accompanied by changes in the rate of labeled precursors incorporation into rat liver protein fractions. The incorporation of labeled amino acids into liver nuclear matrix proteins within the first 90 min of overheating is somewhat decreased, whereas 120 min thereafter it exceeds by 30% the corresponding values in control animals kept at room temperature. The polypeptide pattern of the nuclear matrix in hyperthermia is characterized by an increased relative content of polypeptide components around Mr 100, 55, 40 and 30 kDa against a decreased level of several polypeptides as compared to the control.

[Inhibition by antibiotics of the incorporation of labelled amino acids into the nuclear matrix proteins of the Zajdela hepatoma]. / Podavlenie nekotorymi antibiotikami vkliucheniia mechenykh amminokislot v belki iadernogo matriksa gepatomy Zaidelia.

The incorporation of radioactivity into nuclear matrix proteins during incubation of Zajdela hepatoma cells with labelled amino acids was strongly inhibited by chloramphenicol and cycloheximide and slightly inhibited by actinomycin D and mitomycin C. The antibiotics studied inhibited the incorporation of the radioactive label preferentially into proteins with Mr 150 000-220 000, approximately 55 000 and less than 26 000. During incubation of ascites tumour cells with antibiotics, predominantly with chloramphenicol, a decrease in the content of some protein components was observed as well. As in the low molecular weight protein fraction, the intense inhibition of the radioactive label and a decrease of its content was observed, a conclusion is drawn that this protein fraction is characterized by a high turnover rate.

[Selective inhibition by chloramphenicol of protein biosynthesis in ascites tumor cells]. / Izbiratel'noe podavlenie khloramfenikolom biosinteza belkov v kletkakh astsitnykh opukholei.

Chloramphenicol in a dose of 50-200 micrograms/ml sharply inhibits the incorporation of 14C-labelled amino acids into proteins of ascites Zajdela hepatoma cells while it has no effect on protein biosynthesis in rat liver cells. In vivo chloramphenicol selectively inhibits this process in ascites tumour cells of rat Zajdela hepatoma and mouse Ehrlich carcinoma and hepatoma 22a, without inhibiting the process in various organs of tumour-bearing animals. The inhibition of labelled amino acid incorporation into nuclear and especially nuclear matrix proteins is more pronounced than into the whole tissue. A certain degree of inhibition was revealed in liver cells as well.

[Peculiarities of protein composition of nuclear matrix in some tumors and cultures of embryonic fibroblasts of the Chinese hamster]. / Osobennosti belkovogo sostava iadernogo matriksa v nekotorykh opukholiakh i kul'turakh émbrional'nykh fibroblastov kitaiskogo khomiachka.

The polypeptide pattern of the nuclear matrix of hepatomas differs significantly from that of intact liver. On the contrary, the polypeptide pattern of the nuclear matrix of Chinese hamster fibroblast culture is similar to that of tumour cells. In our studies the nuclear matrix was characterized by an increased content of polypeptides with Mr = 120,000-135,000 and 150,000-200,000 and a protein group with Mr of about 13,000 and lower. In metabolically active hepatomas and fibroblasts of Chinese hamster the content of high and low molecular weight components of the protein band triplet (Mr = = 65,000-70,000) in the logarithmic phase is decreased, while in hepatomas the content of the middle component of the triplet is increased. It is assumed that these peculiarities are due to the inhibition of differentiation and to an increased metabolic activity of the cells.

[Localization of high-molecular alkali-insoluble polypeptides of rat liver cell nuclear matrix revealed by immunological methods]. / Lokalizatsiia vysokomolekuliarnykh shchelochenerastvorimykh polipeptidov iadernogo matriksa kletok pecheni krysy, vyiavliaemaia s pomoshch'iu immunologicheskikh metodov.

By means of chicken immunization, antibodies were obtained to two high molecular weight polypeptides of an alkali-insoluble highly dispersed fraction of rat liver cell nuclear matrix. Using methods of indirect immunofluorescence these antibodies were seen fixed selectively on the periphery of the cell nuclei of rat liver, cultured human fibroblasts and of cultured human fibrosarcoma 8387 cells. The same pattern of antibody fixation, with non-specific net-like staining in the cytoplasm, was observed in the light microscope after the immunoperoxidase staining. Using the similar peroxidase staining with electron microscopy, antibody fixation was recorded with nuclear pore complexes, ribosomes, fibrous lamina and intranuclear granular structures in isolated rat liver cell nuclei and in the nuclear matrix. In mitotic cells the cytoplasm displayed a bright fluorescence, whereas the condensed chromosomes showed a fainter fluorescence. Thus, the examined high molecular weight antigens revealed no organ or species specificity. They appear to be constituents of nonmembraneous structures of the nuclear envelope, more likely of the pore complexes.

[Inhibition by cycloheximide of the incorporation of radioactive amino acids into nuclear matrix proteins of Zajdela ascites hepatoma]. / Ingibirovanie tsiklogeksimidom vkliucheniia radioaktivnykh aninokislot v belki iadernogo matriksa astsitnoi gepatomy Zaidela.

Proteins of nuclear matrix readily incorporated labelled amino acids after incubation of ascites Zajdela hepatoma cells with 14C-hydrolyzate of chlorella proteins. Cycloheximide at the concentrations, inhibiting the cytoplasmic protein synthesizing system, significantly decreased the labelled amino acid incorporation both into nuclear proteins and into proteins of nuclear matrix but the antibiotic effect was less distinct in the matrix. The incorporation into proteins with molecular mass below 26 KD and above 150 KD was especially distinctly inhibited as shown in experiments on separation of the matrix proteins by means of SDS-polyacrylamide gel electrophoresis. Besides the inhibition of the incorporation of amino acids, a decrease in content was found in studies of the fraction of matrix low molecular proteins. The high rate of metabolism of nuclear matrix low molecular proteins is discussed.

[Electron microscope characteristics of the nuclear matrix and its fractions]. / Elektronno-mikroskopicheskaia kharakteristika iadernogo matriksa i ego fraktsii.

The rat liver nuclear matrix retains the shape of the nucleus and reveals a sponge-like structure in negative staining and scanning electron microscopy. A fibrous layer (dense lamina) with associated pore complexes are preserved on the surface of the nuclear matrix. The cytoplasmic face of the nuclear matrix is perceived as a network consisting of cells (or units) of 10-30 nm in diameter in negative staining as well as in high resolution scanning electron microscopy. In sections, a fibrous layer, 15-30 nm in width with granules of 7-10 nm in diameter, can be observed. In pore complexes associated with the fibrous layer granular and fibrillar components rather than central granules are observed. The pore complexes differ in arrangement of the annular granules. Structures similar to pore complexes are revealed in close proximity to the nucleoli. The biogenesis of the pore complexes is discussed. A few morphologically different structures could be derived be fractionation of the nuclear matrix. A fraction rich in pore complexes, and a fraction retaining the shape of the nucleus with spongy or alveolar structure were isolated. The latter fraction is regarded to form a protein framework or skeleton of the nucleus.

[Fractionation and biosynthesis of rat liver and Zajdela hepatoma nuclear matrix proteins]. / Belki iadernogo matriksa i ikh biosintez v kletkakh pecheni Krysb i gepatomy Zaidelia.

A comparative study of the nuclear matrix proteins of rat liver and Zajdela hepatoma cells was performed. The polyacrylamide SDS electrophoretic profile of the hepatoma nuclear matrix proteins differed from those of the liver by the presence of high molecular weight (over 135 KD) bands. Four nuclear matrix fractions were isolated by a subsequent treatment of the preparation with an aqueous solution of EDTA and 0,025 N sodium hydroxide. The bulk of the nuclear matrix proteins of both liver and hepatoma were alkali-soluble. The percentage of the alkali-insoluble residue and of the water-soluble fraction in the Zajdela hepatoma nuclear matrix was 3.5 and 1.7 times that of the liver, respectively. In the course of 60 min incubation of the liver mince or Zajdela hepatoma cells with 14C-Chlorella protein hydrolyzate in vitro the nuclear matrix proteins incorporated by 10-20% more label than did the total nuclear protein, the specific activity of the alkali-insoluble residue being twice higher that of the whole nuclear matrix protein. After 15 min of incubation the label was rather evenly spread along the gel, containing labelled protein bands separated according to their molecular weight. However, after 30 min and especially 60 min of incubation the label markedly prevailed in the high molecular weight proteins.

[Nuclear pore density in rat liver cells during regeneration and x-ray irradiation of the animals]. / Plotnost' iadernykh por v kletkakh pecheni krysy pri regeneratsii i rentgenovskom obluchenii zhivotnykh.

Cytoplasmic as well as nucleoplasmic surfaces of the pore complexes (PC) could be observed using freeze-etching method. The density of PCs per 1 micron2 of nuclear envelope (NE) surface in regenerating liver (9.9) is twice as that in resting liver (5.3). 1 hour after 1200 R X-ray irradiation the pore density in regenerating liver decreases 5.8-fold, consisting only of 1.7 PCs per 1 micron2 of the NE. The structure of the PC after irradiation undergoes degradation and normal PCs practically disappear; only their "ghosts" remain. Peripheral and possibly central granules of the PC appear to consist of some subunits with their diameter of 4--5 nm. The central granule forms a channel through which RNA containing material may be transported from the nucleus to the cytoplasm. The non-uniform state of the PC, observed on platinum-carbon replicas of cleaved nuclei, and the non-altered PC associate with the dense lamina of the NE, after detergent treatment of isolated nuclei indicate that the PC could be formed inside the nuclei and to be "inserted" into the NE membranes in the course of their processing.

[Action of x-ray irradiation on the nuclear envelope of rat liver cells]. / Deitsvie rentgenovskogo oblucheniia na iadernuiu obolochku kletok pecheni krysy.

X-irradiation of isolated nuclear envelopes (NE) has revealed their high radiosensitivity, while irradiation of isolated intact nuclei in vitro, in the doses up to 5000 r 18--20 hours after partial hepatectomy, produced no morphological changes in NE. The damaging effect of irradiation on both nuclei and mitochondria (Mt) was revealed only with a decrease in cytochrome-c-oxidase (CO) activity in parallel with an increase in the radiation dose. One hour after the whole body irradiation of rats in the beginning of S-period, the damaging effect was recorded in both NE and Mt at the doses of 50 and 150 t, and was enhanced with the increase of irradiation dose. Morphological changes were observed mostly in the outer nuclear membrane, which lost its distinct outline and disappeared from some nuclear regions. Lethal radiation doses produced a decrease in the number of pore complexes (PC) with their evident segregation from the membranes. After irradiation in a dose of 1200 r, only the residue or "ghosts" of the PCs remained. After irradiation in doses up to 400 r, the CO-activity recovered during the first hour in Mt and during first two hours in the nuclei.

[Oxidation and phosphorylation in rat liver nuclei and nuclear membranes in postnatal development and regeneration after partial hepatectomy]. / Okislenie i fosforilirovanie v iadrakh i iadernykh obolochkakh kletok pecheni krysy v protsesse postnatal'nogo razvitiia i regeneratsii posle chastichnoi gepatektomii.

Specific oxygen consumption by isolated nuclei of liver cells of newborn rats is higher and phosphorylation is lower as compared to adult animals. This is correlated with a higher free cytochrome oxidase activity as determined in the absence of detergents or tetramethyl-p-phenylenediamine. Correspondingly, oxygen consumption by isolated nuclear membranes of rat liver 44 hrs after partial hepatectomy is also increased and the P/O ratio is decreased 2.2-fold as compared to the controls.

[Cytochrome oxidase of rat liver nuclear membranes and its interaction with mitochondrial cytochrome oxidase]. / Tsitokhromoksidaza iadernykh obolochek pecheni krysy i ee vzaimootnoshenie s mitokhondrial'noi tsitokhromoksidazoi

The percent of mitochondrial protein contamination in nuclei decreased 10-fold (from 18 to 1.8%) under purification of protein-labelled mitochondria before their introduction into nuclei-free homogenate, cytochromoxidase activity being unchanged. Thus, cytochromoxidase activity of nuclei does not correlate with the amount of nuclei-adsorbed mitochondrial protein, which demonstrates the presence of nuclear cytochromoxidase independent on mitochondrial protein. Radioactivity of protein-labelled mitochondria is proportially distributed between globuline, deoxyribonucleoprotein, acid and residual nuclear proteins, as it is shown under fractionation of nuclei isolated from protein-labeled mitochondria containing homogenate. The comparison of mitochondrial protein contamination of nuclear membranes and their possible contamination with cytochromoxidase and suecinate-cytochrome-c-reducatase activities revealed that cytochromoxidase activity of nuclear membranes is twice higher and succinate-cytochrome-c-reductase activity is considerably lower than it can be referred to mitochondrial protein contamination. The ratio of cytochrome-c-oxidase and succinate-cytochrome-c-reductase activities in isolated nuclear membranes is 4-7 times as high as that in mitochondrial membranes under the same isolation procedure. The data obtained make possible to consider the cytochromoxidase activity of nuclear membranes to be really nuclear enzyme, and not a contominant of nucleipreparation with mitochondrial membranes.