Radioautographic studies with methionine-3H and cytidine-3H in protein-fed,protein-deprived and starved rats

Summary Male, growing rats were either fed on a protein-rich or a protein-free diet or starved. At various intervals before they were killed, they were given either cytidine-³H or methionine-³H subcutaneously. Radioautographs of several organs were prepared. Grain counts were performed on sections of liver, pancreas, kidney, adrenal, spleen, stomach, duodenum, heart, lung, striated muscle, testis, skin and cerebellum. They revealed inter alia an increased labelling, especially of ribose nucleic acid, in the liver during protein depletion and starvation. These changes were also found in the ribose nucleic acid labelling of nucleoli, nucleolus-associated chromatin and non-nucleolar nucleus in liver cell nuclei. Changes were also found in many other organs. The findings might be interpreted as signs of a shift of nucleic acid and protein synthesis during protein deprivation and starvation from less essential to more essential' organs. — Some aspects of the intracellular ribose nucleic acid and protein metabolism are discussed.The investigation was supported by a grant from the Swedish Medical Research Foundation.     

The effects of chlorphentermine and phentermine on certain metabolic parameters associated with development of rat kidney and liver.

Daily oral administration of the anorexigenic agents chlorphentermine or phentermine (60 mg/kg) to rats for either 1, 3, 5 or 7 days resulted in a significant fall in the incorporation of [¹⁴C]thymidine into renal and hepatic DNA throughout the course of the experiment. Although 24 h after treatment with either drug there was no dramatic change in the incorporation of [¹⁴C]orotic acid into liver RNA, a statistically significant reduction was noted after 3, 5 and 7 days. In rat kidney, the incorporation of [¹⁴C]orotic acid into RNA was only significantly depressed by chlorphentermine at 5 days and by phentermine at 3 days. In general, treatment with either anorexigenic agent tended to significantly lower or not affect the endogenous concentrations of renal and hepatic putrescine, spermidine and spermine. The chlorphentermine-induced decrease in liver and kidney nucleic acid synthesis was accompanied by depression in the levels of cyclic AMP in both tissues as well as a reduction in the activity of adenylate cyclase in renal tissue. In contrast, chlorphentermine produced a rise in hepatic adenylate cyclase at 5 days followed by a return to control values after 7 days. The phentermine-induced alterations in nucleic acid metabolism appeared generally to occur independent of any changes in the adenylate cyclase-cyclic AMP system of renal and hepatic tissues. In view of the fact that nucleic acids, polyamines and cyclic AMP constitute integral components of the growth process, our data suggest that chlorphentermine and phentermine interfere with certain biochemical parameters associated with the development of kidney and liver.     

Macromolecular synthesis in the livers of aging mice as revealed by electron microscopic radioautography

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.     

Stimulation by abscisic acid of RNA synthesis in discs from healthy and tobacco mosaic virus-infected tobacco leaves

Uptake of abscisic acid from the culture medium by discs of healthy and tobacco mosaic virus-infected tobacco leaves was measured. Small (two to five-fold) increases in abscisic acid concentration in discs caused increases in rates of [³H]uridine and [³H]adenine incorporation into total nucleic acid, virus RNA and host ribosomal RNA. Net accumulation of virus RNA was also enhanced by abscisic acid. This evidence for stimulation of RNA synthesis is compared with previous reports showing inhibition of RNA synthesis in other tissues. It is suggested that the increase in endogenous abscisic acid caused by tobacco mosaic virus infection may be at least partly responsible for observed increases in rates of RNA synthesis after infection.Abbreviations ABA abscisic acid - TMV tobacco mosaic virus     

Nucleic Acid Precursor Incorporation by Eimeria nieschulzi (Protozoa: Apicomplexa) and Jejunal Villus Epithelium*

Autoradiography was used to investigate incorporation of tritiated adenine, adenosine, guanosine and thymidine by Eimeria nieschulzi and rat jejunal villus epithelial cells. At 2 1/2 days postinoculation, parasitized and control tissues were incubated for 20 min in oxygenated Tyrode's solution (37 C, pH 7.5) containing 30 μCi/ml of each nucleic acid precursor. Treatment of tissues with ribonuclease revealed that E. nieschulzi incorporated label from [³H]adenine primarily into RNA while that from [³H]adenosine and [³H]guanosine was present mainly in DNA. Label from [³H]thymidine was not utilized by parasites. Host villus epithelial cells incorporated label from [³H]purines primarily into RNA. Labeled cytoplasmic RNA was significantly increased in parasitized cells after incubation in [³H]adenine. Tritiated nuclear RNA and cytoplasmic RNA were significantly decreased in parasitized cells after incubation in [³H]adenosine. Incorporation of label from [³H]guanosine was similar for parasitized and control cells. A small quantity of label from each [³H]precursor was incorporated into DNA of villus epithelial cell nuclei.     

Some properties of cortisol-receptor complex isolated from cytoplasm of rat liver and its effect on biosynthesis of nuclear RNA

The cortisol binding compound, from the cytoplasm of rat liver, was purified by gel filtration and precipitation with ammonium sulphate. The binding compound from rat liver cytoplasm, has been found to consist of two distinct protein fractions. The proteins eluted from DEAE-cellulose column chromatography at 0.04 M and 0.18 M concentrations of KCl, have two different isoelectric points, one at pH 4.85–5.00, and another at pH 5.85–6,10, but only the fraction eluted at 0.04 M KCl was found to be able to stimulate the incorporation of ³²P into RNA of incubated rat liver nuclei. The highest values of ³²P incorporation in the nucleic acids of incubated nuclei were obtained with partially purified hormone protein (s) complexes wich were precipitated with ammonium sulphate saturation between 20–25% and 25–30%.     

The regulation of liver glycogen synthetase D phosphatase by ATP and glucose

Cortisol at a concentration of 5×10^?6M induces profound changes in U.V. absorption spectra of isolated nuclei from rat liver and thymus. The changes occur within the first 10 min of cortisol action. In both types of nuclei, a blue shift of 5–10 nm from the normal absorption maximum at 260–270 nm is evident. In addition, liver nuclei exhibit an elevation of the spectrum at 230–270 nm (increased U.V. absorption), while the spectrum of thymus nuclei becomes flattened. No such changes occur in nuclei exposed to a physiologically inactive hormone (pregnenolone). The results are interpreted as evidence for cortisol-induced perturbations in deoxyribonucleoprotein structure with consequent changes in the degree of condensation of nuclear chromatin.     

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.     

A cytochemical study of pycnotic nuclear degeneration

Pycnotic degeneration of neoplastic and normal nuclei of mice has been studied cytologically and cytochemically, in pieces of tissues removed some time after subcutaneous transplantation. The cytological changes in pycnosis were found to be about as they have often been described earlier: the nuclei become spherical, shrink progressively, the nucleoli become lost, the chromatin becomes homogeneous. The relative changes in total desoxyribose nucleic acid were followed by the Feulgen reaction; the methylgreen stain was used as an index of nuclei acid polymerization; the Millon reaction was used for the detection of the protein changes. In order to measure the amounts of the Feulgen and methylgreen dye, respectively the color developed by the Millon reaction within individual nuclei, the photometric microscopic method after Pollister and Ris was used, which allows the estimation of relative amounts of colored precipitates within individual nuclei of fixed and stained sections. Using these methods the nucleoprotein composition of resting nuclei of a viable tumortissue was compared with that of three pycnotic stages (Stage I, II, III) in nuclei of necrotic areas of the same tumor tissue (Sarcoma 180). In Sarcoma 180 the change from a fresh tumor cell to Pycnosis I involves loss of nearly half the protein, no significant decrease in desoxyribose nucleic acid, and depolymerisation of over half of the desoxyribose nucleic acid. Later these processes continue, and there is added progressive loss of DNA. It is pointed out that at any pycnotic stage there is a total protein equivalent to about 20 times the highly polymerised (methylgreen positive) DNA, which is the ratio found in non-pycnotic nuclei of the type from which these were derived. It is suggested that this part of the pycnotic chromatin represents the unaltered nucleoprotein. In transplanted liver nuclei pycnosis is much more rapid than in neoplastic tissue, but otherwise the two processes are similar both cytologically and chemically. The discussion points out that: a) there is no real increase in chromatin stainability in pycnotic nuclei; b) the change of shape in pycnosis may be due to lower nuclear viscosity accompanying the DNA depolymerisation; c) the highly polymerised state of the DNA may depend upon the presence of some particular protein, possibly histone; and d) that pyenosis can be interpreted as showing high proteolytic activity in the nucleus and a delayed nuclease activity.     

Effect of undernutrition on DNA and RNA synthesis in subcellular fractions from different regions of the developing rat brain

The effect of undernutrition on the incorporation of [methyl-³H]thymidine into DNA and of 5-[³H]uridine into RNA of cerebral hemispheres, cerebellum, and brain stem was studied in vivo and in vitro in rats. The labeling of DNA from nuclei and mitochondria and of RNA from nuclei, mitochondria, microsomes, and soluble fractions, was also measured in vitro. The results demonstrate that nucleic acid synthesis is impaired and delayed during undernutrition. Specific effects were observed for the different brain regions and subcellular fractions: at 10 days nuclear and mitochondrial DNA and RNA synthesis was impaired, whereas at 30 days only the mitochondrial nucleic acid synthesis was affected.The delay of DNA and RNA labeling, caused by undernutrition, was most evident in the cerebellum, probably due to its intense cell proliferation during postnatal development. The specific sensitivity of mitochondria as compared to other subcellular fractions, may be due to the intense biogenesis and/or turnover of nucleic acids in brain mitochondria not only during postnatal development, but also in the adult animal.     

THE RELATIONS BETWEEN DNA, RNA, AND PROTEIN IN NORMAL EMBRYONIC CELL NUCLEI AND SPONTANEOUS TUMOUR CELL NUCLEI

Interferometric and photometric measurements have been made successively on individual cell nuclei derived from normal embryonic tissues and spontaneous tumour tissues of the mouse grown in vivo. From the measurements, the relations between nucleic acid and dry mass content have been studied in the two types of nuclei and the results shown to be consistent with differences in cell metabolism previously reported to exist in vitro. In the nuclei of normal embryonic cells, the syntheses of DNA, nuclear RNA, and protein appear to be closely associated, whereas in the tumour cell nuclei an appreciable fraction of the chromatin RNA and protein synthesis is dissociated from the replication of DNA.     

Nucleic acid synthesis in proliferating peroxisomes of rat liver as revealed by electron microscopical radioautography

Summary Thirty albino rats were fed with a diet containing 1, 2 or 4% di-(2-ethylhexyl)-phthalate (DEHP), a peroxisome-proliferating agent. Others were fed with normal diet as controls. Both groups were sacrificed at varying intervals from 3 days to 4 weeks. The livers were either removed and fixed in glutaraldehyde and osmium tetroxide or fixed in glutaraldehyde, incubated in a diaminobenzidine (DAB) medium, postfixed, embedded in Epon, and sectioned. Other tissues were incubated in Eaglés MEM containing either [³H]thymidine or [³H]uridine, fixed, embedded in Epon, sectioned, and radioautographed. Specimens were observed in a Hitachi H-700 electron microscope.The number of peroxisomes showing DAB reactivity increased in DEHP-fed animals as compared with normal controls In radioautograms of normal rats labelled with [³H]thymidine, no silver grains were, observed, whereas grains were observed over some nuclei, mitochondria and peroxisomes of DEHP-fed animals. In contrast, radioautograms of tissue labelled with [³H]uridine revealed a few grains in nuclei and mitochondria or endoplasmic reticulum of normal rats, although grains appeared in nuclei, mitochondria, endoplasmic reticulum and peroxisomes of DEHP-fed animals more frequently.From these results, it is concluded that [³H]thymidine and [³H]uridine were incorporated in the proliferating peroxisomes, suggesting that nucleic acid synthesis had taken place.     

Nucleic Acid Metabolism in the Developing Primary Leaf and Senescing Coleoptile of Germinating Oats

Nucleic acid metabolism in the coleoptile and primary leaf tissues of the germinating oat (Avena sativa L.) seedling was studied. The concentrations of the different species of nucleic acid present at various stages of development were determined and the amounts of each compared. All species of nucleic acid in the coleaptile increased as the tissue elongated; but, with the onset of senescence all species decreased, especially rRNA. Exposing dark grown coleoptiles to light did not modify their capacity to synthesize nucleic acids. In the rapidly developing leaf, all species of nucleic. acid increased throughout early germination. A general enhancement in the synthesis of all species of nucleic acids resulted when dark-grown-leaves were exposed to light. Furthermore, the tRNA/DNA ratio remained constant in both tissues during development, whereas the rRNA/DNA ratio changed.     

Effect of second-leaf removal or kinetin treatment on the nucleic acid metabolism of senescing first seedling leaf of barley

1. Changes in nucleic acid metabolism in first seedling leaves of barley plants during aging (from 7 to 27 days) were followed, and the effect of continual removal of the second leaf and basal meristem or of treating the first leaf with 20p.p.m. kinetin on these changes was examined. During aging of the first seedling leaves the ribosomal RNA, DNA and soluble RNA declined, with ribosomal RNA showing the most rapid fall. This was, however, accompanied by increased incorporation of ³²P into RNA, which reached its peak on the fifteenth day. 2. Second-leaf removal partially suppressed first-leaf senescence as judged by retarded chlorophyll and nucleic acid decline and by a decreased extent of RNA labelling. Treatment with kinetin, however, did not prove effective. 3. No significant differences in the sucrose-gradient pattern of ³²P-labelled nucleic acids or in the ³²P-labelled nucleotide composition of RNA fractions during aging or during the two treatments were noted, except for a decrease in CMP content of soluble RNA during aging. 4. The results demonstrate that important changes in RNA metabolism are associated with leaf senescence.     

The development of polyploidy in two classes of rat liver nuclei

Two classes of nuclei from livers of Sprague-Dawley rats were isolated, one pelleting in 2.3 M sucrose (H nuclei) and the second class sedimenting through 1.6 and 1.8 M sucrose and banding at the 1.8/2.3 M sucrose interface (L nuclei) of a three-step discontinuous gradient. In younger animals, the L nuclear fraction was the major fraction, but the percentage of nuclei found in the L fraction decreased as the animals grew. Nuclear ploidy was determined by flow microfluorometry using propidium iodide as a DNA stain. Both the H and L nuclear fractions contained diploid, tetraploid and octaploid nuclei; but the degree of polyploidy was greater in the H fraction. Concomitant with the change in distribution of nuclei between the H and L fractions with increasing age was a progressive increase in the degree of polyploidy in the H fraction. Polyploidy did not increase linearly with age in the H nuclear fraction but increased in cycles marked by large changes in the numbers of nuclei found in H and L nuclear fractions. By 12 weeks of age, 4n-H nuclei were the largest single population of nuclei in rat liver. These observations suggested that the shift of liver nuclei from the L fraction to the H fraction was associated with the development of polyploidy and with the differentiation of hepatocytes.     

Is there "Metabolic" DNA in the Mouse Seminal Vesicle?

This study was designed to answer the question: Is H³-thymidine uptake by nuclei of the mouse seminal vesicle evidence for DNA synthesis and mitosis, or does it signify some "metabolic" function of DNA unrelated to chromosome duplication? Mice were given an intraperitoneal injection of H³-thymidine. Six hours later Feulgen squashes of the seminal vesicle epithelium were made and covered with autoradiographic stripping film. The silver grains above labeled nuclei were counted, and the Feulgen dye contents of these same nuclei were determined photometrically after removal of the grains from the emulsion. Unlabeled nuclei were also measured. The dye contents of non-radioactive nuclei form a unimodal distribution, indicating that polyploidy is absent from this tissue. The radioactive nuclei fall into two groups. In the first, the average dye content is the same as that of the cold nuclei (2C). In the second, the values range from 2C to 4C. In the 2C to 4C group the grain count is proportional to the dye content, showing that incorporation is correlated with synthesis. The radioactive 2C nuclei arose by mitosis during the course of the experiment. This is shown by the following facts: (1) They frequently occur in pairs. (2) They average smaller than unlabeled 2C nuclei. (3) Their average grain count is approximately half that of the 4C nuclei. (4) Labeled division figures are found. (5) A mitotic rate estimated from the number of labeled 2C nuclei accords reasonably well with one based on the number of observed mitoses. Since the incorporation of thymidine accompanies DNA synthesis and precedes mitosis, there is no reason to postulate a special "metabolic" DNA in this tissue.     

Effect of different conditions of light on the nucleic acid fractions in cotyledons ofChenopodium rubrutn L.

The nucleic acid fractions in cotyledons of young Chenopodium rubrum plants exposed to continuous light, continuous darkness and short (8 h) day, respectively and labelled with³²P 24 h prior to harvesting were studied by means of chromatography on MAK columns. Some parameters of cotyledon growth (dry weight, cotyledon area, occurrence of mitoses) were also investigated. The changes in the nucleic acid fractions agreed with the dynamics of cotyledon growth. In continuous light the content of all fractions increased. The radioactivity of DNA and s-RNA did not undergo any great changes and only r-RNA increased. The specific activity of r-RNA increased slightly, that of soluble RNA and DNA was reduced. In continuous darkness the content of all the fractions did not undergo any great changes. The radioactivity as well as the specific activity of all fractions decreased. In short day the content of the nucleic acid fractions did not change conspicuously. Only the specific activity of s-RNA increased in a noticeable way while the radioactivity of r-RNA and the specific activity of DNA decreased and this of r-RNA did not change. The changes in nucleic acid metabolism were partially connected with changes in³²P uptake which depended upon light conditions but they were not merely a consequence of this fact. Obviously, there also exists a more direct relationship between nucleic acid synthesis and growth.     

Analysis of bases of rat-liver nucleic acids after administration of the carcinogen dimethylnitrosamine

Dimethylnitrosamine is metabolized to form an alkylating intermediate, which may have significance for its carcinogenic action. However, certain other compounds that are known to be highly mutagenic, including nitrous acid and hydroxylamine, might also be formed. Owing to the general reactivity of these compounds, it would be difficult to detect their formation in the intact animal. Instead, the nucleic acids of carcinogen-treated animals were examined for products of reaction with nitrous acid and hydroxylamine, i.e. for deamination of adenine and guanine, and formation of N(6)-hydroxycytosine, respectively. A double-labelling technique was used to detect very small amounts of the abnormal bases. The purine moieties of DNA in adult rat liver were labelled either with (14)C or with (3)H, by treating the neonatal animals with [(14)C]formate or with [(3)H]formate, and then allowing a period for normal growth. During this time, in liver, the labels were largely lost by metabolic turnover from cell components other than DNA. The pyrimidine moieties in DNA were labelled by treating the neonatal animals with [(14)C]orotate. The purine constituents of RNA of adult rat liver were labelled by repeated administration of [(14)C]- or [(3)H]-formate to the adult rats. The [(14)C]nucleic acid-labelled rat could then be treated with the carcinogen, and the [(3)H]nucleic acid-labelled animal could be used as a control. By this means the experimental and control tissues could be homogenized together in a single preparation, and the nucleic acids from the two tissues could be isolated, hydrolysed and analysed in a single sample. It was therefore possible to have an internal control for artifacts due to changes taking place in the nucleic acid bases during the experimental procedures. With this technique, the formation in vivo of 7-methylguanine in rat-liver DNA and RNA after administration of dimethylnitrosamine was confirmed, and no evidence was found for the formation of xanthine, hypoxanthine, N(6)-hydroxycytosine, or any other abnormal base.