Studies of nuclei separated by zonal centrifugation from liver of rats treated with thioacetamide

González-Mujica, Freddy; Mathias, A. P.

doi:10.1042/bj1320163

Cited by 20 publications

(16 citation statements)

References 25 publications

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“…The value for the number of form B molecules measured in this study is in agreement with that published by Weaver et al [33] who estimated, from the specific activity of purified rat liver enzyme and the a-amanitin-sensitive activity in crude extracts, that 10000 molecules were present per nucleus. This value reduces to about 6500 molecules per diploid genome when corrected for the presence of tetraploid nuclei [22]. However this figure and our value are only 10 -20 % of that observed by Cochet-Meilhac et al [34] from experiments in which rat liver homogenates were titrated with O-[3H]methyl-demethyI-y-amanitin.…”

Section: Number Of Transcribing R N a Polymerase Moleculescontrasting

confidence: 51%

“…Elongation rates were calculated from the [3H]UMP/[3H]uridine ratios. The numbers of RNA chains being synthesised per diploid genome was calculated from the incorporation of label into uridine per unit DNA as described by Cox [21] assuming 5 pg DNA per diploid genome for rat liver [22]. DNA was estimated as described previously [23] on separate samples of the same batch of nuclei.…”

Section: Estimation Of the Elongation Rate For R N A Synthesis Andnummentioning

confidence: 99%

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The Mechanism by which Heparin Stimulates Transcription in Isolated Rat Liver Nuclei

Coupar

Chesterton

1977

European Journal of Biochemistry

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The polyanion heparin, whilst inhibiting free RNA polymerase, stimulates the rate of RNA synthesis in eucaryotic nuclei ; it has been suggested that this process involves either the inactivation of RNase or the activation of blocked transcription complexes. These hypotheses have been investigated in isolated rat liver nuclei at 22 "C. We confirm that heparin inhibits free form A and B RNA polymerases but has little effect on the transcribing enzymes. The sensitivity of these enzymes to x-amanitin is unchanged by the agent and the stimulation is dependent on the presence of (NH4)2S04 and Mn2+ ions. The size of the RNA product was analysed on denaturing formamide/ sucrose gradients. It shows only a small increase when heparin is included in the incubation. In addition, no significant size changes are observed if the incubation is continued in the presence of actinomycin D and a-amanitin which together inhibit 97 % of all RNA synthesis. These results show that inactivation of RNase cannot be an explanation of the stimulatory effect in this system.Polynucleotide elongation rates and numbers of transcribing RNA polymerase molecules were determined by alkaline degradation of RNA labelled from [3H]UTP during 1 -min incubations. The [3H]UMP and [3H]uridine, derived from internal nucleotides and 3'-terminal nucleosides respectively, were separated by thin-layer chromatography and their tritium content measured. From this data the above parameters can be calculated. We show that the activity of RNase and phosphatase do not significantly affect the data and that levels of endogenous UTP are negligible. Data from eight separate experiments show that isolated rat liver nuclei contain 25-35 x lo3 transcribing RNA polymerase molecules per diploid genome comprising 18 -20 x lo3 form A and 5-15 x lo3 form B. Form C was not detected. Addition of heparin has no effect on these values. Polyribonucleotide elongation rates in nuclei are approximately 0.15 and 0.4 nucleotide per second for form A and B respectively. In the presence of heparin the latter rate is increased by 2-4-fold but that of the form A enzyme remains essentially unchanged. Similar results were obtained using [3H]GTP in place of [3H]UTP indicating that polyribonucleotide termination was random. Our findings show that heparin exerts its stimulatory effect by increasing the form-B-mediated polyribonucleotide elongation rate. It does not activate previously inactive RNA polymerase molecules.

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Section: Number Of Transcribing R N a Polymerase Moleculescontrasting

confidence: 51%

Section: Estimation Of the Elongation Rate For R N A Synthesis Andnummentioning

confidence: 99%

The Mechanism by which Heparin Stimulates Transcription in Isolated Rat Liver Nuclei

Coupar

Chesterton

1977

European Journal of Biochemistry

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“…Reports on changes in nuclear ploidy during hepatocarcinogenesis in rat are numerous. Quite often, however, no distinction was made between nodular and surrounding tissue, due to the limitations of the chosen technique (10,11,12); moreover distinction between parenchymal cells and contaminating diploid non-parenchymal cells was not always possible when flow cytometry was applied. Often also studies were carried out on immature rat liver (7,11), which makes them difficult to compare with the data obtained from mature polyploid liver.…”

Section: Discussionmentioning

confidence: 99%

“…This protocol can be supplemented with a promotion phase, that shortens the latency period before carcinoma arise and increases the yield and/or incidence of cancers (6). Several reports on changes in ploidy level during carcinogenesis in the normally polyploid mature rat liver or in newbom diploid liver have been published so far (7)(8)(9)(10)(11)(12)(13). However, only a very crude distinction between cells in and surrounding preneoplastic lésions can be made by techniques used for quantitative DNA estimation, e.g.…”

Section: Introductionmentioning

confidence: 99%

The influence of phenobarbital and butylated hydroxytoluene on the ploidy rate in rat hepatocarcinogenesis

et al. 1988

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The effect of the 'promoters' phenobarbital (PB) and butylated hydroxytoluene (BHT) on the ploidy changes during hepatocarcinogenesis in rats was compared in a densitometric analysis of Feulgen-stained nuclei on paraffin-embedded tissue slices. The triphasic Gerlans protocol for liver-cancer induction was applied. Initiation with a single dose of diethylnitrosamine (DEN), and selection with 2-acetylamino-fluorene (2-AAF) combined with a proliferative stimulus (CCl4 administration), was followed by a treatment with PB or BHT for periods up to 22 weeks. Control animals received no treatment after the initiation and selection procedure. Despite intra- and inter-individual variations, an increase in the amount of 2N nuclei is found in the putative preneoplastic lesions of animals that received initiation and selection (I-S) and 3 weeks basal diet (BD). When the diet is supplemented with PB (after I-S), the increase of diploid nuclei starts earlier. At the time carcinoma arise (22 weeks PB treatment) a decrease in the frequency of 2N nuclei is found. BHT-treated animals which develop no carcinoma within the considered timespan, show a clear increased amount of 2N nuclei in the precancerous lesions only after 14 weeks treatment. It seems that there is a positive correlation between the outgrowth of putative preneoplastic foci and nodules in rat liver and an increase of diploid nuclei in these lesions. PB, as promoter used after initiation and selection, speeds up the development of carcinoma in rat liver, and therefore also the shift to diploidization in these rats starts earlier in comparison with I-S-treated rats. Although BHT does not promote liver carcinogenesis, an increase of diploid nuclei is also observed here during lesion formation. It may, therefore, be concluded that the phenomenon of diploidization is closely linked to and probably necessary for preneoplastic development, but that it is not an absolute indicator for neoplastic transformation.

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“…In recent years a great deal of attention has been given to studies of the nuclear proteins because of their possible role as gene regulators [for review see Hnilica (1967) and Stellwagen & Cole (1969)]. The experiments reported in the present paper were undertaken because of the important changes observed in the liver nuclear proteins during the treatment of rats with thioacetamide, as described by Gonzalez-Mujica & Mathias (1973).…”

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confidence: 99%

Proteins from different classes of liver nuclei in normal and thioacetamide-treated rats

González-Mujica

Mathias

1973

Biochemical Journal

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1. In normal rats the amounts of each of the main types of nuclear protein, i.e. soluble proteins, histones, non-histone chromosomal proteins and residual proteins, vary within the different classes of rat liver nuclei fractionated by zonal centrifugation. 2. Heterogeneity is observed in the non-histone chromosomal proteins prepared from different classes of liver nuclei. These differences were observed by analysis of the proteins both by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis and electrofocusing electrophoresis. They are most evident between the non-histone chromosomal proteins obtained from stromal and parenchymal nuclei. However, some differences are also found for the parenchymal nuclei, between the diploid parenchymal and the tetraploid parenchymal, and between them and the nuclei involved in the synthesis of DNA respectively. 3. Drastic alterations in the nuclear proteins are found after the administration of thioacetamide. The changes observed are complex and not uniform. They vary with the age of the animal and the type of nucleus. In general an increase in the soluble proteins and non-histone chromosomal proteins and a decrease in the residual proteins is observed. There is a decrease in the specific radioactivity of soluble and residual proteins. 4. Electrophoretic analysis of the non-histone chromosomal proteins showed that specific changes occurred after administration of thioacetamide, which are different in adolescent and young adult rats.

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Studies of nuclei separated by zonal centrifugation from liver of rats treated with thioacetamide

Cited by 20 publications

References 25 publications

The Mechanism by which Heparin Stimulates Transcription in Isolated Rat Liver Nuclei

The Mechanism by which Heparin Stimulates Transcription in Isolated Rat Liver Nuclei

The influence of phenobarbital and butylated hydroxytoluene on the ploidy rate in rat hepatocarcinogenesis

Proteins from different classes of liver nuclei in normal and thioacetamide-treated rats

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