Stimulating effect of histones on DNA synthesis in the regenerating rat liver]

Effect of exogenous histones, nuclear globulins and acid proteins on DNA synthesis is studied in regenerating liver of rats in which the synthesis of their own proteins and thus DNA replication are inhibited by cycloheximide. In these conditions histones from regenerating rat liver are found to stimulate 3H-thymidine incorporation into DNA of hepatectomyzed rat liver. Nuclear globulins and acid proteins from regenerating liver, and histones from intact liver produced no stimulating effect on DNA sythesis.     

Soluble and membrane-bound neuraminidase in regenerating rat liver]

Two forms of neuraminidase (soluble and membrane-bound) are found in regenerating rat liver. Specific activity of the soluble form was found to be maximal in 18 hours after partial hepatectomy, and that of the membrane-bound form-in 24 hours after the operation. Maximal specific activities of both neurominidase forms from regenerating rat liver considerably exceeded that from intact rat liver, shem-operated liver and also from embryonic and lactating rat liver.     

Lipoperoxides, vitamin E, and activities of superoxide dismutase, glutathione peroxidase, and catalase in regenerating rat liver

Lipoperoxides in homogenates of regenerating rat liver increased from 6 hours after the operation and reached a peak (about 7 times the control level) 18-24 hours after the operation. The concentration of blood lipoperoxides rapidly decreased after the operation. The enzymatic activities of superoxide dismutase, catalase, and glutathione peroxidase, and vitamin E content in regenerating livers were also determined. Among these antioxidant factors, the catalase level changed markedly.     

Lack of evidence for a pyruvate kinase isozyme shift in hepatocytes of the regenerating rat liver

1. Pyruvate kinase isozyme shift in a regenerating rat liver was studied. Rats were subjected to a 70% hepatectomy and the liver homogenate or hepatocyte preparations were obtained from the regenerating liver. 2. Using thin layer polyacrylamide gel electrophoresis, liver homogenates from an intact normal rat appeared to contain the L-type isozyme in the greatest number and M2-type to a lesser extent. 3. The ratio of the M2- to L-type increased in the preparations obtained from the regenerating liver. 4. In the hepatocyte preparations prepared from a regenerating rat liver by the conventional method, a small amount of M2-type isozyme was detected. 5. However, the M2-type isozyme was hardly detected in the highly purified hepatocyte preparations prepared using Percoll. 6. Similar results were obtained by separation of the enzyme by DEAE cellulose column chromatography. 7. These results suggest that there is no pyruvate kinase isozyme shift from L- to M2-type in hepatocytes in the course of regeneration. 8. The increased M2-type isozyme in the regenerating rat liver is considered to originate from nonparenchymal cells.     

Genome mutation yield in the cells of intact and regenerating liver of normal rats and following gamma irradiation

A comparative study was made of spontaneous and induced polyploidy in cells of resting and regenerating rat liver. Polyploidy was shown to play a major role in the ontogenesis and during regeneration after partial hepatectomy. An essential difference was revealed in the radiation response of cells of intact and regenerating liver with respect to the yield of polyploid cells. This distinction was referred to different effectiveness of the processes of repair and fixation of radiation damages in the actively proliferating and resting cells.     

Proliferation and cell death of hepatocytes in regenerating rat fetal liver

Proliferation and death of hepatocytes in regenerating liver of 17-day white rat fetuses were investigated. During 2 days after liver resection (20%), animals were sacrificed every 3 h. In experimental groups, the index of Ki67-positive hepatocytes increased sharply in 15 h after liver resection. In all experimental and control groups, the ratio of the metaphase, the longest phase of mitosis, and index to mitotic index remained unchanged, indicating identical duration of hepatocytes mitoses in regenerating liver. In the regenerating and intact liver hepatocytes labeled with antibodies to caspase 3 were not detected. Thus, resection of 20% rat fetal liver did not contribute to increased apoptosis of hepatocytes.     

Effect of ethanol on the DNA-polymerase activity in the liver subcellular fractions of adult and old rats

The effect of 5% ethanol on DNA polymerase activity in nuclei, mitochondria, microsomes and cytosol of intact and regenerating liver of adult and old rats has been studied. No changes in DNA polymerase activity were detected in subcellular fractions of adult rat liver. On the contrary, the increased activity of intact liver nuclei and decreased activity of regenerating liver microsomes was observed with ageing. These age-dependent peculiarities of DNA polymerase activity in response to 5% ethanol may be related to changes in the enzyme molecules or microenvironment associated with ageing.     

Nicotinamide coenzyme content in the normal and regenerating liver of rats

The content of NADH and NADPH was measured in the intact and regenerating rat liver. In the intact rat liver, the content of NAD+, NADH, NADP+ and NADPH was 235 +/- 6.4, 66.6 +/- 4.3, 73.3 +/- 2.5 and 148.0 +/- 4.6 micrograms/g crude liver weight, respectively. Seasonal alterations in the rat liver content of coenzymes were established. No changes were found in the content of nicotinamide coenzymes in the regenerating liver 4 and 18 h after operation. Twenty-four hours after operation, a 25.6% increase in the content of NAD+ and a 57.8% reduction in the NADH content were recorded in the liver of hepatectomized animals. At the same time the total content of NAD+ plus NADH changed but insignificantly (14.7%). The total content of NADP+ plus NADPH dropped by 29.8% (within the above period). Thirty-two hours after operation the content of all the nicotinamide coenzymes returned to the initial level.     

Electron paramagnetic resonance spectra of catalase in mammalian tissues.

The relatively small number of paramagnetic species and the high concentration of catalase in mammalian liver and blood make it possible to directly study this enzyme in frozen whole tissue. The EPR spectra of catalase are dependent on the heme environment and in human blood only catalase A, gxy = 6.48, 5.36 is observed whereas in liver a second spectrum, catalase B, gxy = 6.80, 5.07 can also be seen. Using rapid freeze techniques it has been shown that in rat liver catalase A corresponds to the in vivo steady state and that after death this is largely converted into catalase B. Data from the perfusion of rat livers with oxygenated and deoxygenated blood and dextran solutions together with results from in vitro studies of catalase are interpreted as indicating that catalase B results from the interaction of catalase with an organic acid, most probably formic acid, that the acid is a peroxidative substrate for catalase in vivo and that peroxidation of the acid is not the major role for catalase in rat liver. Catalase binding with other small molecules in intact liver has been demonstrated by perfusion with nitrite-containing dextrans and by intraperitoneal injection of 3-amino-1,2,4-triazole.     

Stabilization of ornithine decarboxylase and N1-spermidine acetyltransferase in rat liver by methylglyoxal bis(guanylhydrazone)

The activities of ornithine decarboxylase and spermidine N1-acetyltransferase started to rise in normal rat liver 4 h after the intraperitoneal injection of methylglyoxal bis(guanylhydrazone) (MGBG; 80 mg/kg). Ornithine decarboxylase had its greatest activity 24 h after a single injection of MGBG and the acetyltransferase peaked 8 h after the injection. Measurement of the apparent half-life of ornithine decarboxylase after MGBG treatment revealed a clear decrease in the decay rate of the enzyme in both normal and regenerating rat liver. MGBG slowed the decay of the transferase also in normal rat liver, as well as inhibiting its activity in vitro. The stabilization by MGBG of these two short-lived proteins involved in metabolism of polyamines should lead to their accumulation in liver, thus explaining their increased activities. In the case of ornithine decarboxylase, studies with a specific antibody against mouse kidney ornithine decarboxylase showed that the rise in ornithine decarboxylase activity after MGBG application was not due to the appearance of an immunologically different isozyme.     

Three-dimensional reconstruction of a peroxisomal reticulum in regenerating rat liver: evidence of interconnections between heterogeneous segments

The three-dimensional (3-D) form and the interrelationship of peroxisomes (Po) in the model of regenerating rat liver after partial hepatectomy were studied by computer-assisted 3-D reconstruction of serial ultrathin sections. Po were labeled cytochemically for either catalase, which stains them all uniformly, or for D-amino acid oxidase (DAA-OX), which gives a heterogeneous reaction with lightly and darkly stained PO. In regenerating rat liver, Po exhibit marked pleomorphism with some budding forms and dumbbell-shaped ones. The 3-D reconstruction revealed many single spherical Po measuring 0.15-0.8 micron in diameter. In addition, two to five Po were found interconnected with each other via narrow 30-50-nm hourglass-shaped bridges forming a reticulum. Such aggregates of Po measured 1.5-2.5 microns across. Whereas all segments of this reticulum stained homogeneously for catalase, they exhibited a marked difference in the intensity of the DAA-OX reaction. These observations are consistent with the view of peroxisomal proliferation by budding or fragmentation from preexisting ones. Under such conditions of rapid growth as in regenerating liver, Po may be interconnected forming a reticulum. The interconnections between Po with differing DAA-OX activities suggest that they originate from the same parent organelle.     

Proliferation and cell death of hepatocytes in regenerating fetal rat liver

Proliferation and death of hepatocytes in regenerating liver were studied in 17-day-old fetal white rats. Two days after liver resection (20%), animals were sacrificed every 3 h. In experimental groups, the index of Ki67-positive hepatocytes increased sharply 15 h after liver resection. In all experimental and control groups, the ratio of the index of the metaphase, the longest phase of mitosis, to the mitotic index remained unchanged, indicating the same duration of hepatocyte mitoses in regenerating liver. In regenerating and intact liver, hepatocytes labeled with antibodies to caspase 3 were not detected. Thus, resection of 20% fetal rat liver did not promote enhancement of apoptosis of hepatocytes.     

CFUs of the normal and regenerating liver in the sexually mature mouse

Functional properties of CFUs have been studied in intact and regenerating liver of mice. According to a number of properties (proliferative activity, character of colony growth) CFUs in the liver are similar to CFUs in the peripheral blood and, probably, make the same population. In the regenerating liver relative contents of CFUs 3-5 days after a partial resection is substantially increasing. Concentration of CFUs (endogenic) increases significantly also in a locally injured and regenerating lobe, comparing to the intact lobe that is in the same organ. The transplanted bone marrow CFUs prevail in number in the regenerating liver over the intact liver. It is concluded that increasing contents of CFUs in the regenerating liver depend mainly on its creased property to invade and/or to hold the extrahepatic CFUs.     

Effect of low doses of the hydra peptide morphogen on protein biosynthesis in the intact and regenerating rat liver

The dependence of ornithine decarboxylase (ODC) activity on hydra peptide morphogen doses has been established. The parameters of protein synthesis were determined in normal and regenerating rat liver, using the peptide in a dose of 20 micrograms/kg body weight, initiating maximum enzyme activity. It was established that intraperitoneal injection of the peptide in partially hepatectomized animals stimulated ODS activity in dose-dependent manner and was dome-shaped. The peptide injection in intact animals does not affect the intensity of 3H-leucine inclusion into the liver protein and the protein content in rat liver. However, the peptide injection in partially hepatectomized animals increased the level of 3H-leucine inclusion into the protein of regenerating liver and stimulated protein accumulation in this type of tissue.     

A comparative study of plasma membrane Mg2+-ATPase activities in normal,regenerating and malignant cells

Plasma membranes have been prepared from rat normal liver cells, regenerating liver cells and Yoshida ascites hepatoma 66 cells after intact cells were first bound to polylysine-coated polyacrylamide beads, and the membrane-associated Mg²⁺-ATPase activity was assayed directly on beads with membrane attached. With plasma membranes from normal liver cells, K_m for ATP and V were found to be higher than those in regenerating liver cells and hepatoma cells. Vanadate caused a different sensitivity of the activity, without an effect in normal liver cells and with an inhibition in regenerating liver cells and hepatoma cells. The activity in normal and regenerating liver cells decreased with increasing temperature above 24–30°C, while the activity in hepatoma cells continued to increase linearly to 37°C. Unlike the enzyme in normal and regenerating liver cells, the hepatoma enzyme was shown to have a higher phase transition temperature and lower activation energies. In all three kinds of cells the activity was increased by the dephosphorylation of plasma membranes and unaffected by the phosphorylation. By means of histochemical Mg²⁺-ATPase staining applied on polyacrylamide gels, at least three major bands which show the enzymic activity were visible in normal and regenerating liver and a single band was detected in hepatoma cells.     

Biogenesis of peroxisomes in regenerating rat liver. I. Sequential changes of catalase and urate oxidase detected by ultrastructural cytochemistry

The biogenesis of peroxisomes has been investigated in the model of regenerating rat liver after partial hepatectomy using ultrastructural cytochemical staining methods: catalase as a marker of the peroxisomal matrix and uricase for the cores. The peroxisomes in regenerating rat liver showed several distinctive features: a) marked variation in shape and size, e.g., peroxisomes with tail-like extensions and tortuously elongated rod-shaped ones, b) formation of peroxisomal clusters and, c) interconnections between adjacent peroxisomes suggesting cleavage or budding. Whereas the reaction product for catalase was present at all intervals after hepatectomy in the matrix of all peroxisomes, the pattern of localization of uricase case varied with the time. It was confined to the cores in controls and at 10 days after the operation, while at 24 and 48 h it showed, in addition, a diffuse reaction in the matrix of some peroxisomes. In interconnected apparently dividing peroxisomes, the core with positive uricase reaction was present only in one half, while the other half was devoid of the reaction product. Similarly, the diffuse uricase staining was confined to the half which contained the core with the other half remaining unstained. These observations are consistent with the concept that new peroxisomes are formed from preexisting ones by budding and segmentation. While catalase is transferred uniformly to all new segments, uricase is compartmentalized in certain portions, of the apparently growing "peroxisomal reticulum".     

Immunochemical demonstration of increased accumulation of ornithine decarboxylase in rat liver after partial hepatectomy and growth hormone induction

Antiserum against ornithine decarboxylase (EC 4.1.1.17) was prepared in rabbits using purified ornithine decarboxylase from rat liver as the antigen. Immunoglobulins from the immune sera were covalently coupled to agarose by cyanogen bromide activation. With the aid of this immunoadsorbent against the enzyme it has been shown that following partial hepatectomy and growth hormone administration, the ornithine decarboxylase activity is elevated concomitantly with the increase in the immunoreactive enzyme protein. In addition, the rapid decay in ornithine decarboxylase activity in regenerating rat liver after cycloheximide injection is accompanied by a decrease in the immunoreactive protein. These results suggest that the activity of ornithine decarboxylase in rat liver is regulated through rapid changes in de novo synthesis and degradation of the enzyme protein.     

Gylcine-14C incorporation into the plasma membrane proteins of normal and regenerating liver

Incorporation of 14C-glycine in plasmatic membrane proteins of the intact and regenerating liver was studied at the beginning of G1 period of the cell cycle. The electrophoretic study of 0.05 M Na2CO3 soluble plasmatic membrane proteins of the regenerating liver showed that maximal incorporation of 14C-glycine was associated with the proteins having molecular weight of about 60 000. The pattern of incorporation of 14C-glycine in different fractions of 0.05 M Na2CO3 insoluble proteins of the plasmatic membranes of the intact and regenrating liver did not differ essentially.     

Comparison of transcription stimulating, phenol-soluble non-histone chromosomal proteins in normal rat liver and transplantable hepatocellular carcinomas.

The non-histone chromosomal proteins (NHCP) of a rapidly and slowly proliferating transplantable hepatocellular carcinoma (THC) were compared to those of normal and regenerating rat liver. The total quantity of NHCP is approximately threefold higher in the THCs than in either normal rat liver at 4 h and 44 h regenerating rat liver. Only those NHCP that can be extracted from chromatin by 0.35 M NaCl were further examined and it was observed that the proteins of this highly complex fraction could be further fractionated by their differential phenol-solubility. The phenol-soluble 0.35 NHCP contained protein(s) capable of stimulating the level of DNA-directed RNA synthesis in vitro. The total amount of this stimulatory activity was 5 times higher in the rapidly growing THC and 1.6 times higher in the slowly growing THC than in normal rat liver. In order to assess the contribution of cell-cycle dependent alterations on the increase in the amount of stimulatory activity in the THCs, 44 h regenerating rat livers were examined. This tissue represents a mix of cells in various stages of the cell cycle which is similar to that found in the THCs. It was found that the total quantity of NHCP in the 44 h regenerating rat liver was the same as in normal rat liver. The total amount of the stimulatory activity also was similar in both the normal and 44 h regenerating rat liver. The amount of the stimulatory activity was found to double in 4 h regenerating rat liver, however. These data suggest that the alterations observed in the NHCP of the THCs are not due solely to cell cycle dependent changes, but may represent malignancy dependent alterations.