Comparison of polysomal and nuclear poly(A)-containing RNA populations from normal rat liver and Novikoff hepatoma.

Polysomal and nuclear poly(A)-containing RNA of normal rat liver and Novikoff hepatoma cells have been compared by cDNA.RNA hybridization kinetics. Homologous hybridization reactions revealed at total kinetic complexity of about 1.6 X 10(10) and 1.38 X 10(10) daltons for liver and Novikoff mRNA respectively. The high abundance component present in liver cannot be detected in Novikoff. It was found from heterologous reactions that about 30% by weight of mRNA sequences are specific to liver. Determination of the nuclear poly(A)-containing RNA complexities revealed that about 5.5% and 4% of the haploid genome is expressed in the liver and Novikoff respectively. In a heterologous reaction, up to 30% of the liver cDNA failed to form hybrids with Novikoff nuclear RNA. Cross hybridizations have further revealed abundance shifts in both nuclear and polysomal RNA populations. Some sequences abundant in liver are less abundant in Novikoff and some rare liver sequences are relatively abundant in Novikoff.     

Hepatocytes in heterokaryons can suppress entry into the S-period of fibroblast nuclei from murine NIH 3T3 cells

Mouse liver regeneration after partial hepatectomy can be considered as a spectacular example of controlled tissue increase. In this study serum-deprived (0.2%) resting and serum-stimulated (10%) proliferating NIH 3T3 mouse fibroblasts were fused with primary hepatocytes isolated from normal (intact) and regenerating adult mouse liver at different times after partial hepatectomy (1-15 days) to elucidate mechanisms of liver cell proliferation cessation at the regeneration end. DNA synthesis was investigated in the nuclei of heterokaryons and non-fused cells using radioautography. Hepatocytes isolated from regenerating liver within 1-12 days following operation did not retard the entry of stimulated fibroblast nuclei into the S-period. In contrast, hepatocytes isolated within 15 days after hepatectomy were found to have inhibitory effect on the entry of stimulated fibroblast nuclei into the S-period in heterokaryons. Preincubation of these hepatocytes with cyclocheximide for 2-4 h abolished their ability to suppress DNA synthesis in stimulated fibroblast nuclei in heterokaryons. Possible reasons of inhibitory effect of differentiated cells in heterokaryos are discussed. The data obtained enable us to conclude that the mechanism of proliferative process control in regenerating hepatocytes seems to be stopped being affected by the intracellular growth inhibitors, whose formation depends on protein synthesis.     

Immunocytochemical determination of ploidy class-dependent bromodeoxyuridine incorporation in rat liver parenchymal cells after partial hepatectomy

Immunocytochemistry of bromodeoxyuridine (BrdU) incorporated in DNA was performed on cryostat sections of rat liver and on isolated hepatocytes after partial hepatectomy using a two-step labeling technique. The method enabled the detection of S-phase nuclei in both tissue preparations. Quantification of the number of labeled nuclei in sections showed that the number of nuclei in S-phase increased from 0.3% in control liver to about 36% at 24 h after partial hepatectomy. The detection of BrdU in isolated hepatocytes showed the same labeling index of binuclear diploid, mononuclear tetraploid and binuclear tetraploid cells. A special role for mononuclear diploid cells in proliferation did not seem to occur.     

RNA metabolism and poly(A) distribution in mouse liver following administration of dimethylnitrosamine

Dimethylnitrosamine (DMNA) strongly inhibited RNA synthesis in mouse liver under conditions when the nucleotide pattern, rate of nucleotide synthesis and phosphorylation ratio were unaffected. (An unidentified, probably non-nucleotide, component in the acid-soluble liver fraction was selectively reduced.) The inhibition of RNA synthesis was associated with a decrease in the RNA polymerase activity of isolated liver nuclei, well established already 45 min after DMNA administration. The reduced activity included both Mg2+- and Mn2+/(NH4)2SO4-stimulated polymerase functions. The inhibition in vivo involved the whole complement of RNA, including poly (A)-containing RNA and isolated poly(A) sequences. The transfer of labelled RNA from the nucleus to the cytoplasm was not impaired. There was no detachment of poly(A)-containing RNA from the microsomes, and the proportion of tightly membrane-bound microsomal RNA and poly(A) sequences was not reduced as determined by use of a flotation technique. No breakage or shortening of the poly(A) chains was indicated by sedimentation analysis.     

Early nuclear cytochemical changes in regenerating mammalian liver

Various cytochemical parameters were studied cytophotometrically in parenchymal cell nuclei isolated from rat liver at different times following partial hepatectomy. The study was confined to the early period following operation, before DNA synthesis and before mitotic activity ensued. Binding of acridine orange was found to increase approx. 70% over normal controls at 3 h post-hepatectomy followed by a sharp decrease to 60% below controls by 12 h and a return to near control levels at 24 h. These changes were found in both diploid and tetraploid cell nuclei. The initial two-fold difference in AO binding observed between diploid and tetraploid nuclei in normal liver persisted at 3 h but decreased markedly from 6 to 24 h after operation. Chromatin thermal stability, determined by acridine orange microfluorimetry, showed a rapid decrease at 3 h in both diploid and tetraploid cell nuclei followed by an increase above control levels at 6 h which persisted up to 24 h after partial hepatectomy. Parallel measurements of Feulgen dye binding showed no change in the relative DNA content of diploid and tetraploid cells throughout the experimental period. Ratios of specific picric acid and alkaline bromphenol blue dye binding by histone arginine and lysine side chains were found to rise significantly over normal controls at 3 h post-hepatectomy and return to normal levels by 6 h.     

Immunocytochemical determination of ploidy class-dependent bromodeoxyuridine incorporation in rat liver parenchymal cells after partial hepatectomy

Summary Immunocytochemistry of bromodeoxyuridine (BrdU) incorporated in DNA was performed on cryostat sections of rat liver and on isolated hepatocytes after partial hepatectomy using a two-step labeling technique. The method enabled the detection of S-phase nuclei in both tissue preparations. Quantification of the number of labeled nuclei in sections showed that the number of nuclei in S-phase increased from 0.3% in control liver to about 36% at 24 h after partial hepatectomy. The detection of BrdU in isolated hepatocytes showed the same labeling index of binuclear diploid, mononuclear tetraploid and binuclear tetraploid cells. A special role for mononuclear diploid cells in proliferation did not seem to occur.     

Enhancement of neutral phosphatase activity in the cytosol and nuclei of regenerating rat liver: role of endogenous regucalcin.

The role of endogenous regucalcin (RC) in the regulation of neutral phosphatase activity in regenerating rat liver was investigated. The liver weight reduced by a partial hepatectomy (about 70%) was completely restored at 72 h after surgery. Phosphotyrosine, phosphoserine, and phosphothreonine were used as the substrate for the assay of phosphatase activity. Phosphatase activity toward phosphotyrosine in the hepatic cytosol and nuclei was significantly increased at 24-72 h after hepatectomy. Such an increase was not seen in the case of phosphoserine and phosphothreonine. However, the presence of anti-RC monoclonal antibody (200 ng/ml) in the enzyme reaction mixture caused a remarkable elevation of phosphatase activity toward three phosphoaminoacids in the hepatic cytosol at 24 and 48 h after hepatectomy. In the liver nuclei after sham operation or hepatectomy, phosphatase activity toward three phosphoaminoacids was significantly raised by the addition of anti-RC antibody (150 ng/ml). The nuclear phosphatase activity toward phosphothreonine in regenerating liver was significantly enhanced in the presence of anti-RC antibody (100 and 150 ng/ml). The effect of anti-RC antibody to increase phosphatase activity toward three phosphoaminoacids in the cytosol and nuclei of regenerating liver was completely blocked by the addition of exogenous RC (1.0 microM). The present study demonstrates that protein phosphatase activity in the cytoplasm and nuclei is enhanced in regenerating rat liver. This enhancement may be suppressed by endogenous RC.     

Intranucleolar localization of the RNA polymerase A activity in isolated nuclei of regenerating rat liver

Ultrastructural autoradiography was used to visualize RNA polymerase A activity in parenchymal cell nuclei isolated from normal and regenerating (3, 24, 36 and 48 h after partial hepatectomy) rat liver. High resolution autoradiography showed that the activity of RNA polymerase A which was not inhibited by α-amanitin in a concentration of 0.8 μg/ml, was restricted to the nucleolus. Both the distribution pattern and number of grains were similar in control liver and regenerating liver 3 h after hepatectomy. Twentyfour, 36, and 48 h after hepatectomy nucleoli were enlarged and labeling was distinctly increased. In all experimental groups the activity of RNA polymerase A was located within fibrillar components of the nucleolus. The association of enzyme activity with this component was especially distinct in later stages (36 and 48 h) of liver regeneration. These results suggest that the fibrillar component of the nucleolus contains the active template for ribosomal RNA (rRNA) synthesis in rat liver cell nuclei.     

Coordinate regulation of ribosomal protein mRNA level in regenerating rat liver. Study with the corresponding mouse cloned cDNAs.

Cloned mouse ribosomal protein (rp) cDNAs exhibit extensive homology with the corresponding rat sequences. The size of the rp-mRNAs and complexity of the rp-genes are very similar in the two species. Using the mouse rp-recombinant DNAs we find that the relative abundance of rat L7, L13, L18, L30, L32/33 and S16 mRNAs increases after partial hepatectomy. Their maximal level is about twice that of normal rat liver, and is achieved 12-18 h after the operation, while the relative abundance of albumin mRNA decreases to half the normal values 12 h after partial hepatectomy. This concomitant increase in the relative content of these rp-mRNAs indicates coordinate regulation of their level in the rat. The dissimilar behavior of L10 and L19 rp-mRNA suggests additional control mechanisms of rp-mRNA levels in the regenerating rat liver.     

Increased synthesis of low-molecular-weight nuclear ribonucleic acids of rat liver after gamma irradiation and hepatectomy.

Incorporation of [3H]orotic acid into low-molecular-weight nRNA of rat liver, fractionated on polyacrylamide gels, increased 6-12h after partial hepatectomy and 6h after gamma-irridation at 2000 R. The incorporation of orotic acid was particularly increased into the 4.5S, 5S and approx. 10S nRNA fractions. If the irradiation was given after 6h of regeneration and RNA was isolated from the nucleus 12h after hepatectomy then the incorporation of orotic acid into these low-molecular-weight nRNA components was greater than after hepatectomy or irradiation alone.     

The action of cordycepin on nascent nuclear RNA and poly(A) synthesis in regenerating liver.

Following a 5 min pulse of [5- 3H]orotic acid via the protal vein, the specific radioactivity of non-poly(A)heterogeneous nuclear RNA (HnRNA) reaches a peak at 12 h after partial hepatectomy. In contrast, poly(A)-HnRNA was maximally elevated only at 2 h after operation. After intraportal injection of cordycepin (3'-deoxyadenosine) 1 min before [5-3H]orotic acid, a dose-dependent inhibition of nuclear HnRNA and rRNA occurred. Fractionation of HnRNA on poly(U)-Sepharose following 20 mg/kg of cordycepin revealed that a 65% reduction occurred in the labeling of poly(A)-HnRNA while non-polyactivity of UTP in control and cordycepin-treated animals indicated no significant alterations in these parameters. Assessment of poly(A) size using poly(A)-HnRNA annealed with oligo(dT)10 as template primer for Escherichia coli DNA polymerase I, showed that 20 mg/kg of cordycepin inhibited nuclear polyadenylylation by 43%; no alteration in the binding of poly(A)-HnRNA to Millipore filters occurred at this dose of cordycepin. These results indicate that cordycepin is a non-selective inhibitor of nuclear RNA and poly(A)synthesis in regenerating rat liver.     

Effects of partial hepatectomy on RNA polymerase activities in mouse liver

Chromatin-bound and poly[d(A-T)]dependent RNA polymerase I plus III and II activities of mouse liver were analysed 24 and 48 hr after partial hepatectomy. Chromatin-bound RNA polymerase I plus III activity showed an increase of 57% at 24 hr and 51% at 48 hr after partial hepatectomy. There was a decrease in chromatin-bound RNA polymerase II activity of 15% at 24 hr and 34% at 48 hr after partial hepatectomy. There was no significant changes in poly[d(A-T)]dependent RNA polymerase activities. Heparin caused an approximately 10-fold increase in chromatin-bound RNA polymerase II activity. The stimulation by heparin was significantly increased 48 h after partial hepatectomy. Anaesthesia and/or surgery had great influence on RNA polymerase activities. At 24 hr after operation, chromatin-bound RNA polymerase I plus III and II activities were depressed, and the liver cell chromatin was more susceptible to stimulation by heparin.     

Cytophotometric study of nuclear proteins during gametogenesis in Ascaris lumbricoides.

Reduction in the number of nucleoli/nucleus and increase in their size were usually observed in rat liver after partial hepatectomy. These changes of nucleoli were greatest 16–18 h after the operation, when RNA biosynthesis in the nucleoli is reported to be highest. Approx. 50% of the nuclei had one enlarged nucleolus at this time but after the increase in nuclear DNA synthesis less than 15% of the nuclei had one nucleolus, as in normal liver. Before the next peak of nuclear DNA synthesis, nucleolar changes appeared again, though less conspicuously.The enlarged nucleoli of regenerating liver were separated from smaller ones by discontinuous sucrose gradient centrifugation and the contents of nucleic acid and ribosomal cistrons in different-sized nucleoli were measured. The large nucleoli in regenerating liver were found to have increased DNA content, whereas smaller ones had the normal content. The total number of ribosomal cistrons in the enlarged nucleoli from regenerating liver was also increased roughly in proportion to the DNA content. No significant difference was found between the percentages of ribosomal cistrons in whole nuclear DNAs from regenerating and normal liver. Small but reproducible [³H]TdR incorporation into nucleolar DNA was observed and this was similar in normal liver and regenerating liver 12 h after partial hepatectomy. Therefore, the nucleolar changes in regenerating liver were not accompanied by any particular DNA synthesis in the nucleolus itself. These results suggest that in the nuclei of regenerating liver nucleolar chromatins may be redistributed and assembled into large nucleoli, rather than that any amplification of ribosomal cistrons occurs.     

Effect of the parenteral administration of energy substrates in different postoperation phases on the initiation and development of liver regeneration in rats subjected to partial hepatectomy

DNA specific activity in the liver, the total DNA content of the liver and the mitotic index of the hepatocytes were studied after the infusion of glucose or lipid emulsions in female laboratory rats with a mean pre-operation weight of 250 +/- 30 g after partial (65-70%) hepatectomy (PH). The infusions were administered in the early prereplication phase (the 1st to 6th hour after the operation), in the late prereplication phase (the 7th to 12th hour after the operation), or continuously from the 1st to the 12th, or the 1st to the 24th, hour after partial hepatectomy. The effect of these parenterally administered energy substrates on the initiation of liver regeneration was evaluated 18 and 24 hours after partial hepatectomy. The results indicate that the infusion of glucose, in any interval after the operation, inhibited the initial phases of liver DNA synthesis (18 h after PH), but not its further development (24 h after PH). Neither the mitotic index of the hepatocytes, nor the total DNA content of the liver differed from the control groups in the case of rats given a glucose infusion. In the experimental groups given lipid emulsions, inhibition of liver DNA synthesis was recorded 18 h after PH only when the infusions were given from the 1st to the 12th or the 1st to the 18th hour after PH. The total DNA content of the liver 18 h after PH was raised in all the experimental groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Galactose metabolism in regenerating rat liver.

1. Rats trained on a controlled lighting and feeding schedule were subjected to partial hepatectomy or sham operation. 2. After a large period of about 6h the activity of UDP-galactose 4-epimerase increased threefold, reaching a maximum 4 days after partial hepatectomy, and returned to normal values within a fortnight. 3. The enzyme pattern of the UDP-galactose-glycoprotein galactosyltransferase was biphasic, one peak appearing at 20 h, the second at 72 h after partial hepatectomy. 4. The rise in enzyme activities could be blocked by the injection of actinomycin D, and the Km values for UDP-glucose and UDP-galactose were nearly identical in regenerating and adult liver. It is therefore concluded that the increase in enzyme activity is due to synthesis de novo of enzyme protein.     

Distribution of mRNAS of fibrinogen polypeptides and albumin in free and membrane-bound polyribosomes and induction of alpha-fetoprotein mRNA synthesis during liver regeneration after partial hepatectomy

To study the effect of regenerative response of the liver following partial hepatectomy on the synthesis of major plasma proteins (secretory proteins), we have determined the sequence contents and the distribution of albumin and fibrinogen polypeptide mRNAs in rat liver at intervals after partial hepatectomy and sham operation. Using a quantitative technique for the isolation of polyribosomes, we demonstrated that the distribution of RNA between free and membrane-bound polyribosomal fraction was unchanged in these experiments. There was no shift in the polyribosomal population to favor free polyribosomes after partial hepatectomy. However, there was a dramatic increase (5-6-fold) of the fibrinogen polypeptide mRNA concentration during the first 24 h after resection. In contrast, the albumin mRNA concentration decreased (2-3-fold). There were no alpha-fetoprotein mRNA sequences detectable in any liver RNA fraction in these experimental animals. In sham-operated rats with intact livers, similar changes of fibrinogen polypeptide and albumin mRNA concentrations as described in regenerating liver after partial hepatectomy, were observed. These results suggest that albumin and fibrinogen synthesis after partial hepatectomy is reciprocally regulated at the mRNA level and represents a nonspecific acute phase response to surgical trauma.     

Chromatin structure through the cell cycle. Studies with regeneration rat liver.

Liver nuclei were prepared through the first cell cycle in partially hepatectomized young rats showing 30% parenchymal cell synchrony. To determine if nucleosome structure altered during this period, liver nuclei from sham-operated rats were compared with nuclei isolated at various times after partial hepatectomy. These nuclei were exposed to deoxyribonuclease I (EC 3.1.4.5), deoxyribonuclease II (EC 3.1.4.6) or micrococcal nuclease (EC 3.1.4.7) and the nucleosome-associated DNA length was ascertained. In no case was a difference in the DNA lengths associated with nucleosome structure observed. Differences were observed with regard to the histones and their relative association with nuclear material. When nuclei from normal rat livers were incubated in hypo-osmolar medium 9% of histone 1 and 4% of the other histones were released. These released histones, unlike those remaining bound to the nuclei, showed high [3H]adenosine and [3H]acetate uptakes in vivo. [32P]P1 uptake was also much greater into released than bound histones 1 and 3, but was not different for histone2A. At 3.5-4.5 h after partial hepatectomy, the release of histone 1 was trebled and that of histone 4 doubled. By 13.5 h, when phosphorylation of the bound forms of histones 2A and especially 1 was increased, no further changes in histone release in hypo-osmolar medium were found. The released histones from partially hepatectomized livers had indistinguishable [3H]adenosine uptakes from controls. The roles are discussed of phosphorylation and ADP-ribosylation in labilizing histone binding.     

Increased synthesis of ribonucleic acids in mouse liver after treatment with a nonionic detergent.

A single injection of Tween 40 (polyoxyethylene(20)sorbitan monohexanoate) in the dose range of 600--800 mg/kg body weight induced an increased short-term labeling especially of poly(A)-containing mRNAs in mouse liver (using either [3H]orotic acid or [32P]orthophosphate as RNA precursors), and apparently increased the turnover rates of both rRNAs and mRNAs in this organ over a period of 24 h. In the early period (4 h) after the injection of Tween 40, there was also a significant increase in the content of 32P-labeled adenylic acid in microsomal poly(A)-mRNA fraction. The activity of DNA-dependent RNA polymerases in the nuclei of treated animals was stimulated up to 60% over that in control nuclei in the same period after detergent injection.