Effects of adrenalectomy and excess hydrocortisone on the incorporation of 35S-labelled cysteine in the hypothalamic-neurohypophyseal neurosecretory system of the rat

Summary The incorporation of ³⁵S-labelled cysteine in the hypothalamic-neurohypophyseal system was studied in normal and adrenalectomized rats and in rats treated with excess hydrocortisone. Labelled cysteine was intraperitoneally administered and grain counts were made of autoradiographs produced from sections of the supraoptic and paraventricular nucleus, median eminence and neurohypophysis of animals killed 45 min., 4 hours and 24 hours after administration of the labelled substance. On the whole, lower incorporation levels of the label were noted in the adrenalectomized rats, compared with the controls. In the rats treated with excess hydrocortisone, the grain counts at 45 min and 4 hours after injection were higher and those at 24 hours were lower than those of the controls.The findings are discussed, among other things, in terms of rate of uptake vs. time and related to previous reports on the cysteine uptake and neurosecretory activity of the hypothalamic-neurosecretory sytem.This study was financially supported by the Sigrid Jusélius Foundation, Helsinki, Finland and the National Research Council for Medical Sciences, Finland.     

Changes in RNA-, DNA- and proteinsynthetic activity during the formation of analfin processes in ethisterone-treated females of Oryzias latipes

The incorporations of uridine-³H, thymidine-³H, and leucine-³H were studied in the process-forming regions of the anal-fin rays of the ethisterone-treated females of Oryzias latipes. The activity of alkaline phosphatase was also studied. The increased incorporation of uridine-³H was detected between 12 and 24 hours of ethisterone treatment, attaining the maximum at 24 hours. The percentage of thymidine-³H labeled nuclei increased rapidly between 48 and 84 hours. The incorporation of leucine-³H was found to increase during the first 12 hours, attaining a constant level at 24 hours. An additional increase in incorporation of leucine-³H took place at 60 hours, the incorporation coming up to the maximum at 72 hours. In the horny substance secreted by the scleroblast mass, grains in the autoradiograph were detected at and after 72 hours. Alkaline phosphatase activity was manifested between 48 and 72 hours. These results seem to correspond to the histological changes, such as the appearance of the precursor cells of scleroblasts at 48 hours, the formation of scleroblast mass during the next 24 hours, and the initiation of horny substance secretion at 72 hours.     

State of the hypothalamo-hypophyseal neurocirculatory system of the gerbil, Rhombomys opimus, during dehydration

Big gerbils, inhabitants of desert, were found to have indices of an increased functional activity of HHNS: low content of neurosecretory material all over the system, relatively large volumes of neurosecretory cell nuclei and nucleoli, hyperemia. Under the condition of a seven-day dehydration, the neurosecretory material reduction in all links of HHNS, enlargement of neurosecretory cell nuclei and nucleoli, intensification of hyperemia were found. With the increase in dehydration time to 23 days a progressive reduction of neurosecretory material in cell processes and hypophysis are seen. However, the decrease in nuclear size takes place under the condition of continuing increase in nucleolar size both in supraoptic and paraventricular nuclei. Extremely intensive hyperemia of all regions of HHNS is seen. The activation of HHNS under the water-salt regime change, seen in big gerbils, is not as pronounced as in albino rats. This is, evidently, due to a specificity of the function and their osmoregulating system.     

Early changes in the synthesis of acidic nuclear proteins in human diploid fibroblasts stimulated to synthesize DNA by changing the medium

When resting WI-38 cells in a confluent monolayer were stimulated to proliferate by changing the medium, the incorporation of leucine-³H into nuclear acidic proteins was promptly stimulated, although its incorporation into total cellular proteins was unchanged or even decreased. Three fractions, all acidic by aminoacid analysis, were extracted from the nuclei: (1) ribonucleoproteins (RNP); (2) a fraction extractable with 0.15 M NaC1; and (3) a fraction tenaciously bound to the insoluble residue (residual fraction). A first increase occurred between one and three hours after stimulation in all three fractions. The synthesis of NaCl-soluble proteins then returned to control levels, while the synthesis of residual and RNP proteins remained high between 6 and 12 hours and increased even further at 18 hours, the peak of DNA synthesis. Pulse chase experiments indicated that the proteins synthesized in the first hour after stimulation have a turnover time of less than four hours, while the same fractions in non-proliferating cells were stable for at least 12 hours. 2-mercapto-1-(β-4-pyridethyl) benzimidazole, when added at the same time as the fresh medium, produced an inhibition of the increase in nuclear protein synthesis at one hour, but, if added at five hours after stimulation, it did not inhibit the increase in nuclear protein synthesis occurring at six hours. Actinomycin D (0.01 μg/ml) inhibited both the stimulation of DNA synthesis and the increases in nuclear acidic protein synthesis occurring at one and six hours after stimulation. These results seem to indicate that the serum factors responsible for the stimulation of WI-38 cells, after binding to cells, induce an early synthesis of acidic nuclear proteins which is sensitive to very low doses of actinomycin D. In turn, the newly synthesized proteins could in some way activate in the nuclei the genes that control DNA synthesis and cell division.     

EVIDENCE FOR CYTOPLASMIC DNA IN ROOT CELLS OF NICOTIANA

Sterile root cultures from Nicotiana tabacum were grown with H³-thymidine added to the medium for various intervals. Incorporation of the labeled nucleoside into nuclear DNA occurred in a fraction of the nuclei which increased with time. In addition, the cytoplasm of all cells incorporated enough tritium to be readily detected by autoradiography. The tritium was not removed by hydrolysis in 1 N HCl at 60°C for 10 minutes, but was removed by digestion in a DNase solution which also removed nuclear DNA. The amount of tritium in the cytoplasm increased during the first 2 hours, but did not appear to increase significantly during the following 5 hours. If the roots were transferred to unlabeled medium after 2 hours, the label was diluted faster than expected by growth without turnover of the labeled component. If FUdR was added to the unlabeled medium, the depletion occurred faster during the first 6 hours, but later appeared to level off so that at 10 hours these cultures did not differ from those incubated without FUdR. However, the addition of an excess of unlabeled carrier had no effect on the rate of depletion of the cytoplasmic label. Actinomycin D, which inhibited the incorporation of H³-cytidine into RNA in the root tips, had no effect on the incorporation of H³-thymidine into the cytoplasmic component. However, Mitomycin C or a high concentration of deoxyadenosine inhibited the incorporation of H³-thymidine into the cytoplasmic component as well as into the nuclear DNA. It is concluded that H³-thymidine is incorporated into a cytoplasmic fraction which has the characteristics of DNA, with a measurable rate of turnover. This fraction is synthesized regardless of whether or not the nucleus is synthesizing DNA. Although the function of cytoplasmic fraction is not yet known, it does not appear to be that of supplying precursors for the synthesis of the nuclear DNA.     

Effect of Block of Catecholamine Synthesis on the Functional State of Vasopressinergic Neurons of Hypothalamus in Dehydrated Rats

Effect of catecholamines (CA) on the functional state of vasopressin (VP)-ergic neurons of hypothalamus at their stimulation produced by dehydration (salt diet and water deprivation) was studied in in vivo experiments on adult male Wistar rats. Quantitative assessment of VP-immunopositive substance and digoxigenin-labeled VP mRNA (hybridization in situ) in neurosecretory cells of supraoptic (SON) and paraventricular (PVN) nuclei was performed using measurements of optical density of the stained substance in perykaria and a computer digital television analyzer with PhotoM software. Hybridization in situ technique allowed evaluating intensity of VP synthesis, while comparison of the amount of VP mRNA and VP-immunoreactive substance in neurons of SON and PVN made it possible to evaluate release of VP from perykaria. In PVN, repeated saline administration (0.25 ml per 100 g weight) and severe dehydration led to activation both of synthesis and of release of VP from cell perikarya. Use of -methyl-p-tyrosine, an inhibitor of catecholamine (CA) synthesis on the background of dehydration was not accompanied by changes of the functional state of VP-ergic neurons of PVN as compared with dehydrated animals. No changes in functional state of VP-ergic neurosecretory cells in SON were found after saline administration, whereas dehydration activated synthesis and release of VP from perykaria, like in VP-ergic neurons of PVN. Inhibition of CA synthesis on the background of dehydration led to activation of VP release by SON neurons without affecting the level of VP synthesis. The data obtained indicate that CA is able to suppress the VP release from neurons of SON, which is produced caused by activation of the VP-ergic system under conditions of dehydration.     

In Vitro RNA Synthesis by Rat Liver and I Leukemic Nuclei. Isolation of Undegraded RNA

Incubation of nuclei from rat liver or human leukemic cells in the presence of ³H-UTP² and other factors results in th incorporation of label into a material precipitable by acid, alcohol or ether. This materials is isolated by phenolsds extraction, is sensititve to ribonuclease digestion and presumed to be RNA.

The addition of Cu++ to the incubation system is necessary to inhibit RNA breakdown and allows the isolation of undegraded RNA without interefering with th incorporation of radiosactivity. The time patterns of labl incorporation by the two nuclei preparations are different. Whereas label incorporation by th two nuclei preparations are different. Whereas labelincorporation by liver nuclei continues to increase up to 60 minutes, incorporation by th leukemic nuclei is high during the first 10 minutes and continues at a slower rate up to 45 minutes of incubation. further, th two nuclei preparations also synthesize diferent RNA species. While liver nuclei synthesize RNA sedimenting at 4.5S and 7S to 13S, leukemic nuclei synthesize a heterogeneous, polydisperse type of RNA.     

Epifluorescent microscopic studies on the mechanism of preferential destruction of chloroplast nucleoids of male origin in young zygotes ofChlamydomonas reinhardtii

Summary The studies on the kinetics of nucleoid destruction reported here showed that destruction of chloroplast nucleoids (ct nucleoids) of male origin began to occur at about 30 minutes after mixing of male (mt^–) and female (mt⁺) gametes. The timing of initiation of the destruction differed among zygotes but usually occurred during 50–120 minutes after mixing. About 10 minutes was required for complete digestion of the ct nucleoids. UV irradiation on young zygotes or addition of an RNA-synthesis inhibitor, actinomycin D, to the incubation medium during the first 0–30 minutes after mixing almost completely inhibited the incorporation of³H uridine into the cell nuclei and the preferential destruction without inhibiting cell nuclear fusion. These results suggest that soon after mating,de novo RNA synthesis is concerned for the preferential destruction of ct nucleoids. To determine in which of the two cell nuclei in the zygotes the RNA is synthesized, each gamete (mt^–, mt⁺) was irradiated with UV and mated with unirradiated gametes of opposite mating type. This treatment of the male gametes had no effect on the incorporation of³H uridine into cell nuclei and the preferential destruction of ct nucleoids but UV irradiation of female gametes almost completely inhibited the incorporation of³H uridine into cell nuclei and the preferential destruction of ct nucleoids. Similar phenomena occurred in other crosses. The UV effect was photoreactivated in about 50% by white light, suggesting that the UV target is DNA. Thus, RNA synthesized in the cell nucleus of female origin soon after mating may be responsible for the preferential destruction of ct nucleoids of male origin     

Increase in the number of colloidal droplets in the hypothalamus of Natrix maura caused by dehydration

The influence of osmotic stress on the number of colloid droplets in the magnocellular neurosecretory nuclei of the water snake Natrix maura, has been studied. Five experimental groups of five specimens each, were submitted to dehydration by immersion in sea water for several periods of time (3 to 60 h). The number of colloid droplets, identified by histochemical procedures, was counted in serial sections of the retrochiasmatic nuclei. The study of the mean of each group revealed that the amount of colloids increased with the time of permanence in the hyperosmotic environment (48 h elicited the greatest response). As a conclusion, dehydration seems to activate the hypothalamic magnocellular neurosecretory nuclei of N. maura and, consequently, increases the production of colloid droplets.     

Inhibition of DNA synthesis by isolated liver nuclei from frog virus 3 infected mice

Incorporation of [³H] dTTP into acid insoluble material by liver nuclei from mice infected for 3 hours with 5 × 10⁸ PFU of Frog Virus 3 was markedly decreased. The incorporation was noticeably stimulated by the addition of calf thymus DNA. It has been verified that the inhibition of incorporation by the nuclei from infected animals was not related to an increase of deoxyribonuclease activity and that the product of the reaction showed the properties of DNA.     

Time-lapse cinematography study of the germinal vesicle behaviour in mouse primary oocytes treated with activators of protein kinases A and C

Previous studies have shown that early embryos contain information that can alter the developmental fate of adjacent cells and transferred nuclei. In this report we show that a specific combination of cells from early murine embryos, a single blastomere from an eight-cell embryo placed under the zona pellucida with a two-cell embryo, results in a difference in incorporation of ³H-uridine and expression of two protein bands between the chimeric treatment group and the nonchimeric controls, a single blastomere from an eight-cell embryo in a separate zona pellucida and a two-cell embryo. The incorporation of ³H-uridine in the chimeric group and nonchimeric control group was significantly different at 45 hours after chimerization (P < .02). A stage-specific protein band (52k) on a polyacrylamide gel detected with fluorography was found to be qualitatively different (present more often; P < .01) and another stage-specific protein band (48k) was found to be quantitatively different (more protein; P equals; .07) in the chimeric treatment vs. the nonchimeric controls at 45 hours after chimerization. These results suggest communication between the cells resulting in a change in their incorporation of uridine and protein synthetic profiles.     

INCORPORATION OF TRITIATED THYMIDINE IN THE CELLS OF CAENORHABDITIS BRIGGSAE (NEMATODA) REARED IN AXENIC CULTURE

In the rhabditid nematode Caenorhabditis briggsae the incorporation of thymidine-H³ has been studied by autoradiography after Feulgen staining, with animals maintained under axenic conditions in a medium of only partly defined composition. Labeling has been followed in adults left in the presence of thymidine-H³ for periods of from ½ to 24 hours, as well as in adults reared from larvae in the presence of the tritiated nucleoside. A massive incorporation is found in the nuclei of the gonads and intestine; also a less intense particulate cytoplasmic incorporation is clear in certain cells, especially those of the intestine. In general, all labeling has proved to be sensitive to DNase, but resistant to RNase. The label's stability has been tested by the transfer of adults into a medium containing "cold" thymidine. They remain there for up to 48 hours. A transfer for 24 hours results in a considerable decrease in the intensity of nuclear and cytoplasmic labeling; a stay of 48 hours leads to its complete disappearance from non-dividing (intestinal) as well as dividing (gonadal) nuclei. A phenomenon of DNA turnover is envisaged and discussed as a possible physiological attribute of C. briggsae.     

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.     

The paramedial neurosecretory cells of the suboesophageal ganglion of the cricket,Teleogryllus commodus (Walk.)

Summary Ovariectomy, performed immediately after the final hatch, caused a reduction of stainable (neurosecretory?) material in the paramedial neurosecretory cells (PNC) (A-type) of the suboesophageal ganglion in 10 day-old females of Teleogryllus commodus (Walk.). A concomitant increase in nuclear volume and in the incorporation of ³⁵S-cysteine indicates increased synthesis of neurosecretory material. From these findings it is concluded that more stainable material is secreted in the cerebral neurohaemal organ after Ovariectomy. A functional relationship between the PNC and the ovaries is suggested.     

Neurosecretory and microstructural changes in the hypothalamus of rats following administration of lithium chloride and 3H-thymidin

Adult male rats were intraperitoneally administered aqueous solution of lithium chloride (LiCl). Studies, including neurosecretory and microstructural changes within particular neurocytes in supraoptic (NSO) and paraventricular nuclei (NPV) were performed on hypothalamic sections. In the experimental rats the administered LiCl increased the level of GOMORI-positive neurosecretory material both in supraoptic and paraventricular nuclei. Great amounts of the neurosecretory material were markedly conspicuous in the above areas after 20 days of LiCl administration. Investigations carried out on cellular nuclei of particular neurocytes showed a significant enlargement of the nuclei, and statistical calculations revealed that, in comparison with the basic control, the difference was essentially significant (p less than 0.001). 3H-thymidin administration to the rats which had previously been on LiCl for 20 days demonstrated also that within supraoptic nuclei the incorporation of the isotope in cellular nuclei took a faster course than in control animals.     

Effect of Ethrel and Ceratocystis fimbriata on the Synthesis of Fatty Acids and 6-Methoxy Mellein in Carrot Root

The rate of incorporation of ¹⁴C from acetate-1-¹⁴C into fatty acids by carrot root discs, 18 hours after inoculation with Ceratocystis fimbriata, was 9-fold greater than that in freshly cut discs. The rate in discs treated with water or Ethrel was 3-fold greater. The rate of incorporation of ¹⁴C from glucose-U-¹³C into fatty acids was 3-fold greater 18 hours after any of the above treatments. The rate of ¹⁴C incorporation from malonate-2-¹⁴C into fatty acids 24 hours after inoculation with C. fimbriata or treatment with water was 25 and 60%, respectively, of that in freshly cut discs. Linoleic acid was the principal fatty acid in carrot root, but incorporation of ¹⁴C from acetate-1-¹⁴C into the acid was low until 18 hours after inoculation with C. fimbriata or treatment with Ethrel. Turnover rates of the fatty acids appeared low and were similar for all treatments.     

The effect of castanospermine on the synthesis of synaptic glycoproteins by rat brain slices

Slices were prepared from rat forebrains and the incorporation of [³H]mannose and [³⁵S]methionine into proteins and glycoproteins determined. The incorporation of methionine continued to increase for up to 8 hours whereas mannose incorporation was maximal between 2 and 4 hours and declined thereafter. Glycopeptides prepared by pronase digestion of [³H]mannose-labeled glycoproteins were digested with endoglucosaminidase H (endo H) and analysed by gel filtration. The major endo H-sensitive oligosaccharide eluted in a position similar to standard Man₈GlcNAc. In the presence of castanospermine, which inhibits glucosidase I, the first enzymatic step in the processing of N-linked oligosaccharides, a new endo H-sensitive glycan similar in size to standard Glc₃Man₉GlcNAc₂ accumulated. Synaptic membranes (SMs) were isolated from slices which had been incubated with either [³H]mannose or [³⁵S]methionine in the presence and absence of castanospermine. In the presence of inhibitor the relative incorporation of [³H]mannose into high-mannose glycans of synaptic glycoproteins was increased. The incorporation of newly synthesized, [³⁵S] methioninelabeled, Con A-binding glycoproteins into SMs was not affected by the addition of inhibitor. Many of the glycoproteins synthesized in the presence of castanospermine exhibited a decreased electrophoretic mobility indicative of the presence of altered oligosaccharide chains. The results indicate that changes in oligosaccharide composition produced by castanospermine had little effect on the subsequent transport and incorporation of glycoproteins into synaptic membranes.To whom to address reprint requests.     

INCORPORATION OF C14-LABELED MALEIC HYDRAZIDE INTO THE ROOT-TIP CELLS OF ALLIUM CERNUUM,VICIA FABA,AND TRADESCANTIA PALUDOSA

Allium cernuum, Vicia faba, and Tradescantia paludosa were treated by root immersion in maleic hydrazide (1 mM/liter) labeled with C¹⁴ (C¹⁴-MH) for 1 hour to determine the location within the cell to which MH moves during various periods of time after treatment. Root tips were fixed 24 hours, 48 hours, 72 hours, and 3 weeks after treatment. Autoradiographs of root tips squashed 24 to 72 hours after fixation showed that C¹⁴-MH was distributed throughout the nuclei and was particularly concentrated in the nucleoli. The nucleolar localization of the chemical was transitory, fixations made 3 weeks after treatment showing well labeled nuclei many of which completely lacked label in the nucleoli. The chromosomes seen in mitotic divisions of all three species had the same amount of label in euchromatic as heterochromatic areas. Since the chemical was not accumulated preferentially in heterochromatic areas, it seems likely that the reported specificity of MH for the breakage of heterochromatin can not be due to preferential heterochromatic incorporation.     

Studies on the incorporation of ( 14 C)amino acids into protein by isolated rat brain nuclei

—Various parameters of the in vitro incorporation of [¹⁴C]amino acids into protein by cell nuclei isolated and purified from rat brain and liver were investigated. Nuclei purified through 2.2 m sucrose solution were capable of amino acid incorporation in vitro; and washing procedure to eliminate hypertonic sucrose before incubation was essential since sucrose in high concentration was inhibitory. Microbial contamination was found to be a serious source of error and the use of sterile conditions for incubation were necessary to obtain reproducible and valid results. Using completely sterile conditions, Na ⁺, K⁺, RNase, DNase, puromycin, cycloheximide and chloramphenicol were without any effect on the ability of brain and liver nuclei to incorporate labelled amino acids into protein. Results of time-course and preincubation experiments revealed that some factors essential for amino acid incorporation pass out of the nucleus into the medium. In addition, approximately 15 per cent of the labelled nuclear proteins with higher specific radioactivity was recovered in the incubation medium. Incorporation of [¹⁴C]leucine was proportional to the concentration of labelled amino acid and to the number of nuclei, and it is suggested that carefully controlled conditions of incubation are essential to obtain valid comparisons between different types of nuclei in terms of their relative abilities to incorporate amino acids in vitro. No evidence was obtained indicating isotope dilution phenomenon in these experiments. Whether or not in vitro incorporation of amino acid by nuclei represents protein synthesis is discussed.     

A cell-free assay system for the analysis of changes in RNA synthesis during the development of Xenopus laevis.

Conditions were established for the maximal synthesis of RNA by Xenopus cultured cell nuclei. These differed from those for mammalian nuclei in having a lower K⁺ optimum. The Xenopus nuclei showed all three RNA polymerase activities and processed rRNA to 28 S and 18 S species. Extracts of full-grown oocytes stimulated the rate of RNA synthesis 2.5-fold and caused it to continue linearly for at least 6 hr. This full effect could be produced by preincubation of the nuclei with oocyte extract, followed by their reisolation and assay under standard conditions, provided that the four ribonucleotide triphosphates were present during the preincubation. The stimulatory factor(s) were mainly present in the cytoplasm of the oocyte. They produced quantitatively identical stimulations of RNA synthesis in hamster nuclei. The overall stimulatory effect of cell extracts disappears in the egg, remains absent through cleavage, but reappears at the late blastula stage. This corresponds to the changes in RNA synthesis believed to occur in early development. The extracts affect polymerases I and III, but not II to a significant extent. They also stimulate the incorporation of [γ-³²P]ATP and GTP into RNA, though to a lesser extent than the incorporation of [³H]UTP. The egg extract inhibits γ-³²P incorporation. There therefore seems to be some effect on the initiation of new chain synthesis, but its magnitude is uncertain, and the effect could be indirect.