REFORMATION OF NUCLEOLI AFTER ETHIONE-INDUCED FRAGMENTATION IN THE ABSENCE OF SIGNIFICANT PROTEIN SYNTHESIS

The rat liver nucleolus, after fragmentation induced by ethionine treatment, has been found to undergo complete reformation by adenine in the presence of a dose of cycloheximide sufficient to cause inhibition of protein synthesis by 90–95%. In contrast, actinomycin D given along with adenine was followed by the appearance of a small compact mass containing only the fibrillar component with no evident granules. This structure resembled pseudonucleoli seen in the anucleolate mutant of Xenopus laevis or in certain early stages of amphibian oocytes. Actinomycin D administered 2 hr after adenine induced a segregation of the fibrillar and granular components of nucleoli similar to that induced in the normal nucleolus. The implications of these findings in relation to nucleolar organization are briefly discussed.     

THE DIFFERENTIAL SENSITIVITY OF CULTURED CHICK MESODERMAL CELLS TO ACTINOMYCIN D

Cells were isolated from the somite mesoderm and from the unsegmented (presomite) mesoderm of early chick embryos and exposed to actinomycin D in single cell culture. Actinomycin D inhibited proliferation in cell cultures derived from the unsegmented mesoderm, although the same concentrations of this antibiotic did not inhibit cultures derived from the somite mesoderm. This differential sensitivity parallels the regionally specific necrosis and degeneration observed in the unsegmented mesoderm of intact chick embryos exposed to actinomycin D. In culture, both cell types exhibited approximately the same permeability to labeled actinomycin D and showed comparable inhibition of RNA, DNA, and protein syntheses in the presence of the antibiotic. However, freshly isolated mesodermal cells from the somite region had a higher content of RNA than did cells from the unsegmented region, and the somite cells maintained a higher rate of macromolecular synthesis in untreated cultures.     

Nucleologenesis: Composition and fate of prenucleolar bodies

A time course study was conducted on nucleologenesis after release from a mitotic block in the presence and absence of actinomycin D to determine the composition and fate of prenucleolar bodies (PNBs). Prenucleolar bodies, whether naturally occurring or induced by actinomycin D treatment, stain with silver and contain phosphoproteins B23 and C23, two of the major proteins of the interphase nucleolus as determined by double label immunofluorescence with specific antibodies. The nucleolus is formed by fusion of PNBs, which subsequently reorganize and form internal fibrillar and peripheral granular regions. Actinomycin D prevents fusion of PNBs, which are then randomly dispersed throughout the nucleus but they still contain proteins B23 and C23. These results demonstrate that the nucleolus is formed by fusion of prenucleolar structures whose biochemical composition resembles the mature nucleolus, since PNBs contain at least two of the major nucleolar proteins.     

THE SYNTHESES OF TOTAL MACRONUCLEAR PROTEIN, HISTONE, AND DNA DURING THE CELL CYCLE IN EUPLOTES EURYSTOMUS

Increased alkaline phosphatase activity is induced in certain epithelial cell cultures by hormones with adrenal glucocorticoid activity or their analogues such as prednisolone (Δ^I-hydrocortisone). Enzyme induction occurs in two distinct phases. During the first 12 hr after the addition of prednisolone, there is a small increase in alkaline phosphatase levels. After 15 to 24 hr, the enzyme activity shows a sudden, marked linear rise, reaching a maximum at 60 to 80 hr. Puromycin blocks enzyme induction immediately, even when added during the period of rapid increase of enzyme. Actinomycin D blocks induction when added no later than 8 hr after the addition of prednisolone. On the other hand, Actinomycin D added during the phase of rapid enzyme induction has no effect for at least 12 hr. These findings suggest that de novo protein synthesis is involved in prednisolone induction of alkaline phosphatase and that the RNA messenger for this enzyme is relatively stable.     

AN EXAMINATION OF THE EFFECTS OF ULTRACENTRIFUGATION ON THE ORGANELLES IN LIVING ROOT TIP CELLS

Bouck, G. Benjamin. (Yale U., New Haven, Conn) An examination of the effects of ultracentrifugation on the organelles in living root tip cells. Amer. Jour. Bot. 50(10): 1046–1054. Illus. 1963.—Excised pea roots were centrifuged at 20,000 g for various periods to determine the speed and order of stratification of living plant organelles; 20,000 g for 24 hr at 6 C seems to produce the most consistent layering with least damage to the organelles or to the root as a whole. The recovery of the cells was then followed to determine how fast and in what order the organelles achieve a random redistribution. Rough endoplasmic reticulum seems slow to stratify in its ultimate position at the centrifugal pole but is among the first of the organelles to reorient after centrifugation. Proplastids, large vacuoles, and the nucleolus are slow to recover from centrifuging, but after 8 hr of recovery only the nucleolus and very large vacuoles show indications of a previous layering. Within 12 hr after removal of the root from the centrifuge, growth (in length) of the whole root could be measured.     

THE EFFECTS OF ACTINOMYCIN D ON THE ULTRASTRUCTURE OF THE NUCLEUS OF THE AMPHIBIAN EMBRYONIC CELL

Observations have been made of ultrastructural modifications induced in the nuclei of differentiating amphibian embryonic cells cultured in the presence of Actinomycin D. Of particular interest are regions within the nucleus (regions otherwise rather empty) containing loose groupings of uniform threads having a diameter of around 200 A. These threads have been observed in continuous lengths up to 0.5 µ, and appear to be composed of subfilaments. It is suggested, after taking account of some recent work on lampbrush chromosomes, that these threads are lengths of uncoiled chromosome in a condition of heterosynthetic inhibition. It is further suggested that active and inactive portions of the genome may be distinguishable by the facility with which they can be induced to undergo this ultrastructural modification.     

水稻胚胎发育与萌发过程中凝集素的生物合成及其对转录与翻译抑制剂的敏感性

稻胚凝集素(RGL)在开花后7—13天之间合成活性很强,15天以后明显下降。7—13天之间RGL合成相当旺盛是由于这一时期编码RGL的mRNA得到迅速转录,而在开花后15—30天之间以及萌发4小时内RGL的合成主要是由胚分化发育期间(7—13天)所形成的mRNA所指导的。RGL主要在水稻胚胎发育过程中合成、积累,在萌发过程中几乎不表达,所以RGL是胚胎特异的蛋白质。     

ACTINOMYCIN D-INDUCED CHANGES IN GROWTH AND RIBONUCLEIC ACID METABOLISM IN THE GAMETOPHYTES OF BRACKEN FERN

Actinomycin D affected the morphological type of growth in the gametophytes of Pteridium aquilinum and the distribution of RNA and protein in their particulate fractions. Increasing concentrations of the drug progressively inhibited two-dimensional growth at the end of a period during which controls had formed typical two-dimensional plants. RNA was lost maximally from the nuclei-rich and ribosome-rich fractions of plants growing in a concentration of actinomycin D which inhibited two-dimensional growth. The magnitude of changes in protein content of the plants was less striking. Presence of actinomycin D in the medium also suppressed incorporation of uridine-H³ into cytoplasmic fractions of gametophytes. The possibility that two-dimensional growth in the gametophytes is under control of a newly synthesized messenger RNA, which is sensitive to actinomycin D, is discussed.     

Nepro is localized in the nucleolus and essential for preimplantation development in mice

We generated knockout (KO) mice of Nepro, which has been shown to be necessary to maintain neural progenitor cells downstream of Notch in the mouse developing neocortex by using knockdown experiments, to explore its function in embryogenesis. Nepro KO embryos were morphologically indistinguishable from wild type (WT) embryos until the morula stage but failed in blastocyst formation, and many cells of the KO embryos resulted in apoptosis. We found that Nepro was localized in the nucleolus at the blastocyst stage. The number of nucleolus precursor bodies (NPBs) and nucleoli per nucleus was significantly higher in Nepro KO embryos compared with WT embryos later than the 2‐cell stage. Furthermore, at the morula stage, whereas 18S rRNA and ribosomal protein S6 (rpS6), which are components of the ribosome, were distributed to the cytoplasm in WT embryos, they were mainly localized in the nucleoli in Nepro KO embryos. In addition, in Nepro KO embryos, the amount of the mitochondria‐associated p53 protein increased, and Cytochrome c was distributed in the cytoplasm. These findings indicate that Nepro is a nucleolus‐associated protein, and its loss leads to the apoptosis before blastocyst formation in mice.     

VEGETALIZATION OF SEA URCHIN LARVAE INDUCED WITH cAMP PHOSPHODIESTERASE INHIBITORS

Treatment of sea urchin embryos with cAMP phosphodiesterase (PDE)-inhibitors such as caffeine (4×10⁻³ M), theophylline (8×10⁻³M), or nicotinamide (10⁻²M), at the morula stage for only a couple of hours, yields vegetalized larvae. Most of the embryos treated with these reagents before the morula stage develop to blastulae filled with mcsenchyme-like cells. Almost all embryos at the blastula stage develop normally even if they are treated with a PDE-inhibitor for a considerable period. The rate of ³H-valine incorporation into protein in the morulae is reduced by caffeine and theophylline, but does not decrease in the presence of nicotinamide. Actinomycin D cancels the vegetalizing effect of PDE-inhibitors on the morulae.     

The effect of actinomycin D on RNA synthesis and nucleolar ultrastructure in the liver of rats treated with fluorouracil

Summary Rats were given cytidine-³H and 10 min later 50 mg fluorouracil. They were killed after 25 hours. Actinomycin D was given at various times before sacrifice. The collapse of the nucleolus and the segregation of its components, seen in rats sacrificed one hour after administration of actinomycin D only, was prevented by prior treatment with fluorouracil. In rats treated with fluorouracil and given actinomycin 12 or 20 hours prior to death, there was a more or less pronounced collapse of the nucleolus but no typical segregation of its components. Radioautographs of livers from untreated rats or rats given actinomycin only at the times mentioned, and killed 25 hours after administration of cytidine-³H, were labelled mainly over the cytoplasm. Radioautographs from rats, treated with fluorouracil only, or fluorouracil plus actinomycin, showed labelling over the nucleoli, but depressed labelling over the cytoplasm. Biochemical analysis of RNA labelling showed high ribosomal peaks in untreated rats and rats treated with actinomycin only. Rats treated with fluorouracil, or fluorouracil plus actinomycin showed no labelling of the 29S and 18S ribosomal peaks. The results indicate that fluorouracil blocks or delays the formation of ribosomal RNA and that the inhibition, at least in part, takes place in the nucleolus.This work was supported by grants from the Swedish Medical Research Council (Project K68-12X-623-04), the Swedish Cancer Society (Project 6831), the Medical Faculty of Uppsala and the Swedish Society for Medical Research.     

Differences in the fine structure and chemical constitution of nucleolar bodies and the true nucleolus in Xenopus laevis embryos

Numerous bodies resembling nucleoli, named “prenucleolar bodies”, were seen in the interphase nucleus of Xenopus laevis embryos between stages 7 and 11 of Nieuwkoop and Faber (1956) but not at stage 12. These bodies are composed of thick strands, 200 A in diameter, and apparently differ from the fibrillar component of the true nucleolus which consists of thin fibrils, 50 A in diameter. The granular component of the true nucleolus consists of fibers and granules which are both also 150–200 A in diameter, but which differ in chemical nature from the prenucleolar bodies. The granular component and fibrillar component are readily digested by RNase with or without pretreatment with trypsin, while the prenucleolar body is only digested with RNase after pretreatment with trypsin. This suggests that the prenucleolar body consists of strands of RNA coated with protein. At stage 9, another type of nucleolus-like body is formed, which is larger (2–2.6 μ in diameter) than the prenucleolar body (0.2–1 μ) and consists of thin fibrils of 50 A. This body resembles the fibrillar component of the true nucleolus in the size of the elemental fibrils as well as in its susceptibility to actinomycin D, RNase and trypsin. It seems to be a precursor of the true nucleolus and for this reason was named the “primary nucleolus.” From stage 9 to stage 10, each nucleus in the presumptive ectodermal and mesodermal areas contains 2 primary nucleoli together with multiple prenucleolar bodies. At stage 12, the prenucleolar body is not seen at all, but a new type of nucleolus-like body appears. There are usually 2 of these bodies in each nucleus, and they consist of 2 components: a network of 50 A fibrils, and a group of strands, 150–200 A in diameter, containing some granule-like elements. The former has the same susceptibility to actinomycin D, RNase and/or trypsin as the fibrillar component of the definitive nucleolus and the primary nucleolus, while the latter has the same susceptibility as the granular component of the definitive nucleolus. Thus, this body may     

The participation of the embryonic genome during early cleavage in the rabbit

Actinomycin D and the mushroom toxin α-amanitin similarly inhibit ribonucleic acid synthesis in the rabbit zygote. Actinomycin D also causes an immediate arrest of cleavage, whereas α-amanitin allows limited further development. The decreasing rate of amino acid incorporation caused by continuous exposure of cleaving rabbit embryos to α-amanitin suggests that a relatively homogeneous embryonic RNA is involved in the support of early protein synthesis and is turning over with a half-life of approximately 24 hr, or three cell generation times.     

Scattering of the silver-stained proteins of nucleolar organizer regions in Ishikawa cells by actinomycin D.

Scattering of the silver-stained proteins of nucleolar organizer regions (Ag-NOR proteins) was produced by actinomycin D in Ishikawa cells. Scattering of Ag-NOR proteins was found only in cells treated with actinomycin D and various other agents had no effect. Scattering was dose-dependent up to 10(-2) micrograms/ml of actinomycin D, but it was not found at higher concentrations that caused marked inhibition of total DNA and RNA synthesis. Actinomycin D (10(-2) micrograms/ml) caused the following changes: (i) nucleolar segregation and (ii) emergence of dense fibrillar bodies in the nucleoplasm. Ag-NOR proteins were observed on the fibrillar centers and surrounding fibrillar components in control nucleoli, on the fibrillar and amorphous zones in segregated nucleoli, and on the dense fibrillar bodies emerging in the nucleoplasm. The scattering of Ag-NOR proteins was due to the argyrophilic nature of the dense fibrillar bodies. Actinomycin D (10(-1) micrograms/ml) also caused similar morphological alterations in the nucleolus and nucleoplasm, but Ag-NOR proteins were observed only on nucleolar remnants.     

NUCLEAR MEMBRANES OF CULTURED MAMMALIAN CELLS IN THE PERIOD PRECEDING DNA SYNTHESIS

When kidney cells are cultured directly from the rabbit, the nuclear membranes undergo a change that can be measured as an increase in electrophoretic mobility. The change appears to begin immediately upon culture and is maximal 2 hours later, after which the mobility remains constant at the elevated level. Actinomycin D and p-fluorophenylalanine, but not EDTA or ionizing radiation, suppress the increase in nuclear electrophoretic mobility. With synchronously growing L cells, no change was detected in nuclei from cells taken during various parts of the division cycle.     

THE MODE OF ACTION OF VITAMIN D

1. The purpose of this review article is to re-evaluate and integrate many of the observations related to the physiological effects of vitamin D, using as a working hypothesis the concept that the vitamin may be acting analogously to a steroid hormone in terms of its ability to interact with genetic information and ultimately elicit a physiological response. Prior to this time the problem of the mechanism of action of vitamin D has primarily been approached from the point of view that the vitamin was acting as a cofactor for some specific enzymic reaction. 2. The physiological activities of vitamin D are integrated with those of parathyroid hormone to provide a homeostatic control for the regulation of primarily calcium and secondarily phosphate metabolism. It is proposed that the role of vitamin D in this homeostatic control mechanism is older and more fundamental than that of parathyroid hormone. The interaction of vitamin D on skeletal calcium metabolism may have evolved before the effects of the vitamin on intestinal calcium absorption. 3. There are several physiological defects of calcium metabolism—rickets, osteo-malacia, vitamin D-resistant rickets and idiopathic hypercalcaemia—all of which may be a consequence of an aberration in one or another of the interlocking steps of the vitamin D-dependent and calcium-dependent homeostatic control mechanism. 4. The most thoroughly established action of vitamin D in vivo is to promote or facilitate the intestinal absorption of calcium. Although the exact biochemical details of this process are not available, this may involve vitamin D-mediated synthesis of the appropriate enzyme systems or the alteration of membrane structure necessary for calcium absorption. It is not yet unequivocally established whether calcium absorption is an energy-dependent active transport process or is a passive carrier-mediated or simple diffusion process. 5. The exact action of vitamin D on bone metabolism is not as well established, but the primary effect of the vitamin is likely to mediate bone resorption. The vitamin D-dependent activities of the cell in both the intestine and bone are to absorb calcium and transfer it to the blood. 6. No direct effects of vitamin D on intestinal absorption of phosphate have been found. Furthermore the validity of a vitamin D-mediated renal reabsorption of phosphate is questioned, for the major effects of vitamin D are cation oriented. If the renal effects of vitamin D are true, it is postulated that the mechanism of action of the vitamin here on the anion, phosphate, is fundamentally different from its cation oriented mechanism. 7. There is a lag in the action of vitamin D on the vitamin mediated: (a) transport of calcium both in vivo in rats and chicks, and in vitro with everted intestinal slices; (b) the apparent increased permeability of intestinal mucosa; (c) increased levels of citric acid in serum or bone; (d) the increased incorporation of radioactive inorganic phosphorus into intestinal mucosa phospholipids. As shown by the use of radioactive vitamin D, this lag is not due to a lack of the vitamin in the target organs. 8. Whereas large, unphysiological doses of radioactive vitamin D localize in all tissues and all subcellular fractions, small physiological doses of radioactive vitamin D localize predominantly in the nucleus of the intestinal mucosa. The amount of vitamin D localized in the nucleus would appear to be too low for the vitamin to function as a cofactor, and is more indicative of an interaction on or with deoxy-ribonucleic acid. 9. Actinomycin D, an inhibitor of DNA-directed RNA synthesis, inhibits the action of vitamin D in mediating intestinal calcium absorption and bone resorption. Vitamin D also stimulates messenger-RNA synthesis in intestinal mucosa within 1/2 hr. of vitamin treatment. Vitamin D may play a crucial role, along with parathyroid hormone and calcium, in a DNA, gene-dependent, homeostatic control mechanism for cal, ium metabolism. In this system the vitamin D molecule has certain very specific structural requirements which are probably a reflection of the specificity of its receptor molecule, rather than structural requirements for a cofactor-enzyme relationship.     

3H-URIDINE INCORPORATION IN THE PREMITOTIC STAGE OF RHIZOID CELL DIFFERENTIATION IN PTERIS VITTATA L.

The premitotic rhizoid stage (corona stage) of P. vittata gametophytes was pulsed in radioactive uridine for 5, 15, or 30 min and the data analyzed quantitatively by autoradiography. After 5 min, only the nucleoplasmic compartment is labelled significantly, suggesting that this short pulse is insufficient time for labelled precursor to be fixed in the nucleolus or to be transported from the nucleus to the cytoplasm. Since after 15 and 30 min, all compartments are labelled, with the greatest proportional increase over the nucleolus, it is concluded that cytoplasmic labelling is nuclear in origin and that nucleolar RNA activity is relatively high in this stage.