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1.
Nuclei were prepared from monkey hepatocytes by centrifugation of the homogenate on a cushion of 2.3 M sucrose, during 45 min at 100000 X g. The yield was 2.2 x 10(7) nuclei per g of liver, and 70% of te homogenate DNA was recovered in these nuclei. An electron microscopic study as well as a biochemical analysis of marker enzymes showed that the nuclei are not contaminated by other subcellular fractions, especially endoplasmic reticulum. A mannosyltransferase and an N-acetylglucosaminyltransferase, working on endogenous glycoproteic acceptors, are present in the nuclei for 1.4 and 6.5% of the homogenate activities, respectively. The nuclei are hydrolysed by DNAse I. The suspension, adjusted in 1.9 M sucrose, was centrifuged for 2 h at 100000 X g, under buffer layer. Purified nuclear membranes were collected at the interface. These membranes did not contain any more endoplasmic reticulum enzyme activities, but the mannosyl and N-acetylglucosaminyltransferase activities were still present. They essentially work on an exogenous chromatin acceptor, prepared by lysis of the nuclei. The eventual role of these glycosyltransferases in the glycosylation of non-histone proteins is discussed. 相似文献
2.
Quantitative changes of DNA synthesis in the hepatocytes of aging mice were studied by electron microscopic radioautography. Ultrastructural changes of labelled and unlabelled hepatocytes were estimated quantitatively by the image analysis. The results revealed that at various ages, the area of the cytoplasm and nuclei of labelled hepatocytes were more than those of the unlabelled cells. No significant changes were observed in the nucleoli. The area of the endoplasmic reticulum and mitochondria were less in the labelled hepatocytes than in the unlabelled hepatocytes. The number of the mitochondria was less in the labelled hepatocytes than in the unlabelled hepatocytes. The cell organelles of the hepatocytes that were synthesizing DNA were not well developed, as compared to those not synthesizing DNA during the postnatal development. 相似文献
3.
Hepatocyte proliferation in the liver regenerating after partial hepatectomy ceases when the organ is restored, and the mechanism of this phenomenon is still unclear. In the experiments on fusing hepatocytes from the regenerated mouse liver (15 days after partial hepatectomy) with NIH 3T3 mouse fibroblasts, we revealed no DNA synthesis in the nuclei of stimulated fibroblasts in heterokaryons (in the presence of hepatocyte nuclei), whereas DNA synthesis in nonfused cells was undisturbed. In this work, our purpose was to find out whether the suppression of DNA synthesis in heterokaryons could be due to the appearance in hepatocytes of some endogenous factors having an inhibitory effect on proliferation. To this end, hepatocytes from the mouse liver regenerated after partial hepatectomy were treated with cycloheximide for 1–4 h and were then fused with stimulated fibroblasts. Such a short-term treatment of hepatocytes with cycloheximide proved to result in the loss of their ability to inhibit DNA synthesis in the nuclei of stimulated or quiescent fibroblasts in heterokaryons, but hepatocytes proper actively proliferated in the medium with a low serum content (0.2%). When the mice with the liver regenerated after partial hepatectomy were treated with a single sublethal dose of cycloheximide (3 mg/kg), their hepatocytes taken two days after this treatment had no inhibitory effect. Puromycin, another inhibitor of protein synthesis, had the same effect on hepatocytes. These results may be interpreted as evidence that the final stage of liver regeneration after damage is controlled by the factors having a negative effect on cell proliferation. 相似文献
4.
The effect of the ODC-factor, which was partially purified from ascites fluid of mice bearing Ehrlich ascites hepatoma, on DNA synthesis in the normal mouse liver and spleen was studied, and target cells for the factor in the liver were examined. DNA synthesis in the liver increased about 4-fold over the basal level 39-42 h after the increase of ODC activity induced by injection of the factor into normal mice. This increase of DNA synthesis was inhibited by repeated injection of DAPol. The inhibition was completely reversed by the administration of an appropriate amount of putrescine at about the same time. TK activity also increased in parallel with DNA synthesis. Normal mice with and without treatment with the factor were used to examine which cell population in the liver is the real target for the factor. The livers were dispersed and three cell populations (heavy, medium, and light) were separated by centrifugation. The heavy and light cell populations were characterized as mature hepatocytes and a cell population consisting mainly of immature hepatocytes and non-hepatocytes by analysis of marker enzymes, pyruvate kinase isozymes, L and M2, respectively. The factor stimulated ODC induction, with concomitant increases in TK and DNA poly activities and DNA synthesis, most effectively in the light cell fraction followed in order by the medium and heavy fractions. A nutritional factor (a high protein diet), which is a potent inducer of liver ODC, appeared to act on liver in a different way from the ODC-factor, judging from the results of studies of both whole liver and the fractionated cell system described above. Autoradiography of [3H]thymidine incorporation into liver cells showed that DNA synthesis in mature hepatocytes as well as nonhepatocytes was enhanced by injection of the factor. Stimulation of non-hepatocytes seems to be suggestive evidence that an immunologic response of mice might be developed by the factor. In fact, ODC activity, DNA synthesis, and DNA poly activity (but not TK) in the spleen significantly increased in response to the factor and their increments were suppressed by DAPol, though less sensitively than those in the light cell fraction of the liver. 相似文献
5.
Nuclei were prepared from monkey hepatocytes by centrifugation of the homogenate on a cushion of 2.3 M sucrose, during 45 min at 100 000 × g. The yield was 2.2 · 107 nuclei per g of liver, and 70% of the homogenate DNA was recovered in these nuclei. An electron microscopic study as well as a biochemical analysis of marker enzymes showed that the nuclei are not contaminated by other subcellular fractions, especially endoplasmic reticulum. A mannosyltransferase and an N-acetylglucosaminyltransferase, working on endogenous glycoproteic acceptors, are present in the nuclei for 1.4 and 6.5% of the homogenate activities, respectively.The nuclei are hydrolysed by DNAase I. The suspension, adjusted in 1.9 M sucrose, was centrifuged for 2 h at 100 000 × g, under a buffer layer. Purified nuclear membranes were collected at the interface. These membranes did not contain any more endoplasmic reticulum enzyme activities, but the mannosyl and N-acetylglucosaminyltransferase activities were still present. They essentially work on an exogenous chromatin acceptor, prepared by lysis of the nuclei. The eventual role of these glycosyltransferases in the glycosylation of non-histone proteins is discussed. 相似文献
6.
L Haim-Muchnik S Iapalucci-Espinoza M T Franze-Fernández 《Biochemistry international》1986,12(6):821-829
Shortly after feeding protein-depleted mice on a meal containing proteins, the RNA polymerase I activity in isolated liver nuclei shows a two fold to threefold activation over the basal value in nuclei of either normal or protein-depleted mice. This activation can be accounted for by the increase in the number of growing rRNA chains. Moreover, the template-bound RNA polymerase I fraction in nuclei from re-fed mice is about three times that from protein-depleted animals. An excess of template- unbound enzyme was found in liver nuclei from animals under either nutritional condition. Shortly after inhibition of protein synthesis by pactamycin administration to re-fed mice, the number of transcribing RNA polymerase I molecules in liver nuclei decreases to the basal level found in nuclei from protein-depleted mice, while in the latter, protein synthesis inhibition has no effect. These results support the suggestion that short-lived proteins may enhance the initiation frequency by RNA polymerase I after re-feeding. 相似文献
7.
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. 相似文献
8.
The quantitative ratio between labelled pre-and postmitotic nuclei of hepatocytes was studied 8 hr after H-3-thymidine administration in the 12-15 g mice. The karyoautoradiographic analysis showed that DNA-synthetizing cells are concentrated at a certain stage of S-period. This indicates the complete synchronization of DNA synthesis in the liver parenchyma. The times during which all the hepatocytes enter the S-period did not exceed 4 hr. 相似文献
9.
H J Ma F L Gao M T Olea T Nagata 《Cellular and molecular biology, including cyto-enzymology》1991,37(6):607-615
The application of 3H-leucine results in labeling of the liver cells of mice in which protein is synthesized at various ages of the animals. Quantitative changes of protein synthesis in the hepatocytes of aging mice were studied by electron microscopic radioautography. The silver grains in the hepatocytes were mainly located over the rough surfaced endoplasmic reticulum, mitochondria, Golgi apparatus, cytoplasmic matrix, and a few over the nuclei. The number of silver grains in the cytoplasm and nuclei of the hepatocytes gradually increased after birth, reached the maximum at 1 month after birth, thereafter it continued to decrease with aging until the 24th month. The number of silver grains in the hepatocyte cytoplasm was more than that in nuclei at various ages. The number of silver grains in the rough surfaced endoplasmic reticulum and mitochondria gradually increased from embryo to 1 month after birth, thereafter it continued to decrease with aging until the 24th month. The number of silver grains in the Golgi apparatus showed almost no change from fetal stage to 6 months after birth, thereafter it continued to decrease with aging until the 24th month. The number of silver grains in the cytoplasmic matrix gradually increased from fetal stage to 2 months after birth, then decreased with aging until the 24th month. These changes reflect the quantity of protein synthesized in each cell organelle at various ages of animals. 相似文献
10.
M P Viola-Magni P B Gahan E Albi R Iapoce P F Gentilucci 《Basic and applied histochemistry》1987,31(3):355-364
The presence of a phospholipid fraction associated with chromatin has been demonstrated by biochemical technique in rat hepatocytes. The composition of this fraction determined by chromatography with respect to that of the nuclei is characterized by low content of phosphatidylserine and high content in phosphatidylethanolamine. Also the synthesis and turnover studied after injection of [32P]O4(2-) show a different behaviour: the peak of activity is after 6 hrs in nuclei and microsomes, whereas in chromatin it occurs after 9 hrs. A second peak is evident after 24 hrs in chromatin and microsome phospholipids. Differences have been also shown by analyzing the single phospholipid radioactivity in time. The behaviour of chromatin phospholipids has also been studied during DNA premitotic synthesis in regenerating liver. It has been shown that there is no difference in synthesis in relation to that of DNA in nuclear phospholipids, whereas the specific activity of chromatin phospholipids begins to increase twelve hours after hepatectomy and continues throughout the period of the first mitotic wave, thus bringing to a summation with the beginning of the second wave. The role of this phospholipid fraction in relation to DNA synthesis and gene expression is discussed. 相似文献
11.
Budker VG Subbotin VM Budker T Sebestyén MG Zhang G Wolff JA 《The journal of gene medicine》2006,8(7):874-888
BACKGROUND: The efficient delivery of plasmid DNA (pDNA) to hepatocytes by a hydrodynamic tail vein (HTV) procedure has greatly popularized the use of naked nucleic acids. The hydrodynamic process renders onto the tissue increased physical forces in terms of increased pressures and shear forces that could lead to transient or permanent membrane damage. It can also trigger a series of cellular events to seal or reorganize the stretched membrane. Our goal was to study the uptake mechanism by following the morphological changes in the liver and correlate these with the fate of the injected plasmid DNA. METHODS: We utilized both light microscopic (LM) and electron microscopic (EM) techniques to determine the effect of the HTV procedure on hepatocytes and non-parenchymal cells at various times after injection. The LM studies used paraffin-embedded livers with hematoxylin and eosin (H&E) staining. The immune-EM studies used antibodies labeled with sub-nanometer gold particles followed by silver enhancement to identify the location of injected pDNA at the subcellular level. The level of overall damage to liver cells was estimated based on alanine aminotransferase (ALT) release and clearance. RESULTS: Both the LM and EM results showed the appearance of large vesicles in hepatocytes as early as 5 min post-injection. The number of vesicles decreased by 20-60 min. Plasmid DNA molecules often appeared to be associated with or inside such vesicles. DNA could also be detected in the space of Disse, in the cytoplasm and in nuclei. Non-parenchymal cells also contained DNA, but HTV-induced vesicles could not be observed in them. CONCLUSIONS: Our studies suggest an alternative or additional pathway for naked DNA into hepatocytes besides direct entry via membrane pores. It may be difficult to prove which of these pathways lead to gene expression, but the membrane pore hypothesis alone appears insufficient to explain why expression happens preferentially in hepatocytes. Further study is needed to delineate the importance of each of these putative pathways and their interrelationship in enabling oligonucleotide (siRNA) activity and pDNA expression. 相似文献
12.
The comparative analysis of DNA-synthetase activity of hepatocytes, isolated nuclei and nuclear matrix from normal and regenerating rat liver was performed. The highest enrichment with newly-synthesized DNA was registered in the DNA fraction associated with the nuclear matrix both in vivo and in vitro. The functioning of DNA polymerases alpha and beta in the matrix was shown. Our results indicate that DNA polymerase beta is more firmly bound with the nuclear matrix in the cells of normal liver but this enzyme is eluted almost completely from the nuclei of regenerating liver cells. At the first moment after gamma-irradiation of rats the preferential initiation of unscheduled DNA synthesis in vivo has been observed on the nuclear skeletal structures. This may serve as an indication on the possibility that DNA repair process occurs on the nuclear matrix. 相似文献
13.
S Papa S Capitani A Matteucci M Vitale P Santi A M Martelli N M Maraldi F A Manzoli 《Cytometry》1987,8(6):595-601
The development of hepatocyte polyploidy in rats aged up to 4 months was analyzed by flow cytometry using both scatter and fluorescent parameters to distinguish DNA diploid and DNA tetraploid populations and to discriminate between parenchymal and non-parenchymal compartments. The precise origin of each class of nuclei was assessed in whole liver homogenate using purified hepatocytes, obtained by liver perfusion followed by separation on Percoll gradient, and identifying the peaks corresponding to parenchymal nuclei. The results indicate that preparative procedures involving homogenization of the rat liver tissue caused loss of the DNA octaploid population. Data on the relative proportion of the different DNA ploidy elements during rat liver development, which are in good agreement with those observed by cell analysis by means of microspectrophotometry, indicate the usefulness of flow cytometry as a choice method for the analysis of ploidy distribution. 相似文献
14.
15.
Tetsuji Nagata 《Progress in histochemistry and cytochemistry》2010,45(1):1-291
For the purpose of studying the aging changes of macromolecular synthesis in animal cells, we studied many groups of aging mice during development and aging from fetal day 19 to postnatal newborn, juvenile, young adults, aged and senescent adults up to 12 and month 24 (2 years). They were injected with 3H-thymidine, 3H-uridine or 3H-leucine, precursors for DNA, RNA and proteins, as well as 3H-glucose, 3H-glucosamine, 35S-sulfuric acid, or 3H-glycerol for glucide and lipid precursors, respectively, then sacrificed and the liver tissues were taken out, fixed and processed for light and electron microscopic radioautography. On many radioautograms the localization of silver grains demonstrating DNA, RNA and proteins in hepatocytes in respective aging groups were analyzed qualitatively. The number of silver grains and the number of cell organelles in each cell of each animal in respective aging groups were analyzed quantitatively in relation to the aging of individual animals. The results revealed that the localization of respective precursors as well as the number of silver grains in cell nuclei, cell organelles, changed with the aging of animals. The numbers of labeled nuclei and cell organelles, as well as the numbers of silver grains in nuclei and cell organelles changed due to aging of individual animals. The number of mitochondria, the number of labeled mitochondria and the mitochondrial labeling index labeled with silver grains were counted in each hepatocyte. It was demonstrated that the numbers of mitochondria, the numbers of labeled mitochondria and the labeling indices showing DNA, RNA and protein synthesis at various ages from embryonic day 19 to postnatal newborn day 1, 3, 9, 14, adult month 1, 2 and 6, reaching the maxima, then decreased to senile year 1 to 2, indicating the aging changes. The results indicated that mitochondria in hepatocytes synthesized nucleic acids and proteins independently from the nuclei, but their synthetic activities were affected from the aging of the individual animals. 相似文献
16.
In order to clarify the underlying mechanism of malignant transformation from cirrhosis to hepatoma the cell kinetics of hepatocytes were studied in these two conditions. The content and synthesis of DNA in hepatocyte nuclei were investigated, by means of Feulgen-microspectrophotometry and tritiated thymidine radioautography, in cirrhotic and noncancerous parts of hepatoma with concomitant cirrhosis. The distribution of ploidy patterns was widely spread, from hypodiploid to hyperpolyploid, in the noncancerous parts of a cirrhotic liver containing hepatoma. In normal liver, each paired nuclear DNA content of a binucleate cell recorded almost the same amount, whereas in the noncancerous as well as in hepatoma cells much difference of DNA content was observed between the paired nuclei of the binucleate cells. The ploidy pattern of hepatocytes in patients with liver cirrhosis, who had developed hepatoma during follow-up periods of several months to several years, appeared to resemble that in noncancerous parts of hepatoma cases. On the other hand, the incorporation of tritiated thymidine into hepatocytes was found to be markedly increased in noncancerous parts as well as in cirrhotic liver developing hepatoma during follow-up periods. These results suggest the possibility that the hepatocytes in noncancerous parts of hepatoma have deranged cell-kinetics which might be a driving factor for the development of malignancy. 相似文献
17.
Y. Koike Y. Suzuki A. Nagata S. Furuta T. Nagata 《Histochemistry and cell biology》1982,73(4):549-562
Summary In order to clarify the underlying mechanism of malignant transformation from cirrhosis to hepatoma the cell kinetics of hepatocytes were studied in these two conditions. The content and synthesis of DNA in hepatocyte nuclei were investigated, by means of Feulgen-microspectrophotometry and tritiated thymidine radioautography, in cirrhotic and noncancerous parts of hepatoma with concomitant cirrhosis. The distribution of ploidy patterns was widely spread, from hypodiploid to hyperpolyploid, in the noncancerous parts of a cirrhotic liver containing hepatoma. In normal liver, each paired nuclear DNA content of a binucleate cell recorded almost the same amount, whereas in the noncancerous as well as in hepatoma cells much difference of DNA content was observed between the paired nuclei of the binucleate cells. The ploidy pattern of hepatocytes in patients with liver cirrhosis, who had developed hepatoma during follow-up periods of several months to several years, appeared to resemble that in noncancerous parts of hepatoma cases. On the other hand, the incorporation of tritiated thymidine into hepatocytes was found to be markedly increased in noncancerous parts as well as in cirrhotic liver developing hepatoma during follow-up periods. These results suggest the possibility that the hepatocytes in noncancerous parts of hepatoma have deranged cell-kinetics which might be a driving factor for the development of malignancy. 相似文献
18.
Effects of exogenous human heparin-binding epidermal growth factor-like growth factor on DNA synthesis of hepatocytes in normal mouse liver. 总被引:2,自引:0,他引:2
S Kiso S Kawata S Tamura S Umeki N Ito H Tsushima A Yamada J Miyagawa S Higashiyama N Taniguchi Y Matsuzawa 《Biochemical and biophysical research communications》1999,259(3):683-687
19.
DNA synthesis in the renal parenchyma was studied by electron microscopic autoradiography 48 and 72 hours after unilateral nephrectomy in mice and weanling rats. The proportion of labelled nuclei belonging to the epithelium, endothelium, interstitial areas, or circulating cells was evaluated. Most of cells showing DNA synthesis were epithelial but many belonged to stroma. All the nephron segments were found to participate in compensatory hyperplasia, with a greater contribution, however, of the proximal convoluted tubules. DNA synthesis by the capillary endothelial cells occurred later than the peak epithelial mitotic activity. The site of DNA synthesis in nuclei was the euchromatin, the silver grains being uniformly distributed throughout the nuclear areas with condensed chromatin, and more seldom in the nuclear envelope areas or that of the perinucleolar satellites. 相似文献
20.
R Bucki M Górska M Zendzian-Piotrowska J Górski 《Journal of physiology and pharmacology》2000,51(3):535-540
The aim of the present study was to examine the effect of triiodothyronine (T3) on the content of phospholipids and on the incorporation of blood-borne palmitic acid into the phospholipid moieties in the nuclei of the rat liver. T3 was administered daily for 7 days, 10 microg x 100 g(-1). The control rats were treated with saline. Each rat received 14C-palmitic acid, intravenously suspended in serum. 30 min after administration of the label, samples of the liver were taken. The nuclei were isolated in sucrose gradient. Phospholipids were extracted from the nuclei fraction and from the liver homogenate. They were separated into the following fractions: sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine and cardiolipin. The content and radioactivity of each fraction was measured. It was found that treatment with T3 reduced the content of phosphatidylinositol and increased the content of cardiolipin in the nuclear fraction. In the liver homogenate, the content of phosphatidylinositol decreased and the content of phosphatidylethanolamine and cardiolipin increased after treatment with T3. The total content of phospholipids after treatment with T3 remained unchanged, both in the nuclear fraction and in the liver homogenate. T3 reduced the specific activity of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine and cardiolipin and had no effect on the specific activity of sphingomyelin and phosphatidylinositol both in the fraction of the nuclei and the liver homogenate. It is concluded that excess of triiodothyronine affects the content of phospholipids in the nuclei. The changes in the content of phospholipids in the nuclei largely reflect changes in their content in the liver. 相似文献