DNA synthesis in the nuclei of resting and proliferating cells of the NIH 3T3 line in monokaryons, homo- and heterodikaryons

Serum-deprived (0.5%) resting NIH 3T3 mouse fibroblasts were fused with stimulated cells taken at 2 hour intervals after changing the medium to the one containing 10% serum, and DNA synthesis was investigated in monokaryons, homodikaryons, and heterodikaryous using radioautography with double-labeling technique. The presence of the resting nucleus in the common cytoplasm has an inhibitory effect on the entry of the stimulated nucleus into the S period in the medium containing either 0.5 or 10% serum, but DNA synthesis continues. After a 24 hour stay in the common cytoplasm with resting nuclei the stimulated nuclei return into the state of rest. When resting cells are stimulated by 10% serum, their inhibitory effect on stimulated nuclei in heterodikaryons still persists for at least 2 hours following stimulation. Preincubation of resting cells with cycloheximide for 4 hours abolishes their ability to suppress DNA synthesis in stimulated nuclei. The data suggest that the resting cells produce an endogenous inhibitor of cell proliferation whose formation depends upon the synthesis of protein(s). When stimulated, cell can proliferate only upon decreasing the level of this inhibitor. The obtained results are consistent with the idea of a negative control of cell proliferation.     

Satellite cells of striated muscle after compression injury so slight as not to cause degeneration of the muscle fibres

Summary Electron microscopy of musculus bulbi rectus superior in the rat reveales increase in the number of. muscle satellite cells during the first 10–12 hours following compression injury so slight as not to cause degeneration of the muscle fibre. These satellite cells are situated close to the muscle nuclei and, in contrast to those in the intact rectus superior, they have a pale nucleus with a conspicuous nucleolus, sparse, pale cytoplasm containing a few small mitochondria, a mainly agranular endoplasmic reticulum and numbers of pinocytotic vesicles along their cell membrane. Later on, between 24 and 48 hours, the cytoplasm increases greatly in amount and extendes in long processess over a considerable length of the muscle fibre. An increase in the number of mitochondria and free ribosomes, the appearance of polysomes and great numbers of Golgi complexes and a decrease in the number of pinocytotic vesicles is noted. On the 4th day, some of the satellite cells have still further extended their cytoplasm beneath the muscle basement membrane, while others are apposed to the muscle fibre only with a minor part of their membrane. On the 9th day, only satellite cells comparable in number and structure with those in intact muscles are found.The possibility is discussed that the increase and subsequent decrease in the number of these satellite cells as well as the changes in their morphology at different periods after the injury reflect morphogenesis of satellite cells originating from the nuclei of the muscle fibre by mitotic division even after a trauma, too slight to cause any visible muscle injury. The observations are interpreted as giving new support to the hypothesis that muscle satellite cells may be of importance in posttraumatic muscle regeneration.     

Intracellular traffic of oligodeoxynucleotides in and out of the nucleus: effect of exportins and DNA structure

The delivery of oligodeoxynucleotides (ODNs) into cells is widely utilized for antisense, antigene, aptamer, and similar approaches to regulate gene and protein activities based upon the ODNs' sequence-specific recognition. Short pieces of DNA can also be generated in biological processes, for example, after degradation of viral or bacterial DNA. However, the mechanisms that regulate intracellular trafficking and localization of ODNs are not fully understood. Here we study the effects of major transporters of microRNA, exportin-1 (Exp1) and exportin-5 (Exp5), on the transport of single-stranded ODNs in and out of the nucleus. For this, we employed a fluorescent microscopy-based assay to quantitatively measure the redistribution of ODNs between the nucleus and cytoplasm of live cells. By measuring the fluorescent signal of the nuclei we observed that after delivery into cells via cationic liposomes ODNs rapidly accumulated inside nuclei. However, after removal of the ODN/liposome containing media, we found re-localization of ODNs from the nuclei to cytoplasm of the cells over the time course of several hours. Downregulation of the Exp5 gene by siRNA resulted in a slight increase of ODN uptake into the nucleus, but the kinetics of ODN efflux to the cytoplasm was not affected. Inhibition of Exp1 with leptomycin B somewhat slowed down the clearance of ODNs from the nucleus; however, within 6 hours most of the ODN were still being cleared form the nucleus. ODNs that could form intramolecular G-quadruplex structures behaved differently. They also accumulated in nuclei, although at a lesser extent than unstructured ODN, but they remained there for up to 20 hours after transfection, causing significant cell death. We conclude that Exp1 and Exp5 are not the major transporters of our ODNs out of the nucleus, and that the transport of ODNs is strongly affected by their secondary structure.     

Proteins in nucleocytoplasmic interactions. 3. Redistributions of nuclear proteins during and following mitosis in Amoeba proteus

The behavior of nuclear proteins in Amoeba proteus was studied by tritiated amino acid labeling, nuclear transplantation, and cytoplasmic amputation. During prophase at least 77% (but probably over 95%) of the nuclear proteins is released to the cytoplasm. These same proteins return to the nucleus within the first 3 hr of interphase. When cytoplasm is amputated from an ameba in mitosis (shen the nuclear proteins are in the cytoplasm), the resultant daughter nuclei are depleted in the labeled nuclear proteins. The degree of depletion is less than proportional to the amount of cytoplasm removed because a portion of rapidly migrating protein (a nuclear protein that is normally shuttling between nucleus and cytoplasm and is thus also present in the cytoplasm) which would normally remain in the cytoplasm is taken up by the reconstituting daughter nuclei. Cytoplasmic fragments cut from mitotic cells are enriched in both major classes of nuclear proteins, i.e. rapidly migrating protein and slow turn-over protein. An interphase nucleus implanted into such an enucleated cell acquires from the cytoplasm essentially all of the excess nuclear proteins of both classes. The data indicate that there is a lack of binding sites in the cytoplasm for the rapidly migrating nuclear protein. The quantitative aspects of the distribution of rapidly migrating protein between the nucleus and the cytoplasm indicate that the distribution is governed primarily by factors within the nucleus.     

Colloidal gold labeling of intracellular ligands in dorsal root sensory neurons, visualized by scanning electron microscopy

We report a novel technique that combines high-resolution scanning electron microscopy (SEM) of intracellular structures with backscattered electron imaging (BEI) of colloidal gold-labeled intracellular ligands. Murine dorsal root ganglia were immersion-fixed, freeze-cleaved, labeled with gold complexes, and critical point-dried. Specimens were carbon-coated and viewed by BEI. They were then minimally sputter-coated with gold and previously identified cells relocated by secondary electron imaging (SEI). This permitted increased resolution of intracellular detail while gold particles remained detectable by BEI. Incubation with RNAse-gold and DNAse-gold complexes resulted in specific labeling of cytoplasm and nucleus, respectively. Immunolabeling of neurofilament (NF) and small nuclear ribonucleoproteins (snRNP) resulted in selective labeling of intracellular antigens. Nonspecific binding was abolished by use of 1% skin milk. Specifically, incubation with monoclonal anti-NF68 resulted in labeling of cytoplasm in 66% of neurons, notably of the large cells known to contain large amounts of NF. Satellite cells, which lack NF, showed low levels of background label. Human autoimmune anti-Sm serum recognizes snRNP particles, with the exception of the nucleolar U3 snRNP. Labeling with this serum resulted in specific labeling of 92% of nuclei, with only background labeling over nucleoli and cytoplasm. The results show that it is feasible to employ high-resolution SEM in conjunction with colloidal gold labeling to localize intracellular ligands in situ.     

Onset of DNA replication in nuclei of proliferating and resting NIH 3T3 fibroblasts following fusion

NIH 3T3 mouse fibroblasts arrested in medium containing 0.5% serum were fused with stimulated cells taken at 2-h intervals after replacing the medium with one containing 10% serum, and DNA synthesis was studied in mono-, homo- and heterokaryons using radioautography with double-labelling technique. The presence of a resting nucleus in a common cytoplasm with a stimulated nucleus from the prereplicative period has an inhibitory effect on the entry of the stimulated nucleus into the S period in medium containing either 0.5 or 10% serum, but ongoing DNA synthesis continues. After a 24-h stay in a common cytoplasm with resting nuclei the stimulated nuclei return into the state of rest. When resting cells are stimulated by 10% serum, their inhibitory effect on stimulated nuclei in heterokaryons still persists, at least for 2 h following stimulation. Preincubation of resting cells with cycloheximide for 4 h abolishes their ability to suppress DNA synthesis in stimulated nuclei.The data suggest that resting cells produce an endogenous inhibitor of cell proliferation, whose formation depends upon the synthesis of protein. When stimulated, the cells can proliferate only after decreasing the level of this inhibitor. The results obtained are consistent with the idea of a negative control of cell proliferation.     

Studies of nucleic acid metabolism in cultured chick embryo cells infected with Mycoplasma gallisepticum

M. gallisepticum infection of cultured chick embryo cells led to a sharp reduction the rate of 3H-thymidine and 3H-uridine incorporation into DNA and RNA cells, and almost completely suppressed the transposition of uridine label from the nucleus into the cytoplasm, this pointing to the inhibition of escape of RNA synthesized de novo into the infected cells cytoplasm. As suggested, weak labeling of the cytoplasm after prolonged (about several hours) incubation of cultured cells with labeled urine could indicate infection of cell cultures with the mycoplasmae.     

The distribution of nuclear and cytoplasmic proteins in the blastoderm of the loach, Misgurnus fossilis L

The concentration of dry substance (protein) and the dry weight of nuclei, cytoplasm and cells from different blastoderm regions at the early blastula and midgastrula stages were determined by interferentional microscopy. It was shown that at the early blastula stage the dry weight of cells in the basal layer is higher than that in the outer layer. Although the protein concentration in the basal layer cells appears to be somewhat higher, differences in their dry weight are due primarily to the big volume of cytoplasm of the basal layer cells. By the midgastrula stage, the total (nucleus + cytoplasm) protein concentration increases (by 17% in the basal layer cells and by 9% in the outer layer cells) due to the increase of nuclear protein concentration. At the same time dry weight of these cells markedly decreases due to the decrease of their volumes in the process of cell divisions. At the midgastrula stage the epiblast cells have the highest dry weight due to the highest protein concentration in the cytoplasm and the biggest cell volume. The results obtained are discussed with respect to the data on the pattern of accumulation of newly synthesized protein in nuclei and cytoplasm with special reference to the duration of individual cell cycle phases.     

Expression of vitamin D receptors in the superior cervical ganglia of rats

We investigated the role of vitamin D in the sympathetic nervous system including the distribution of vitamin D receptors (VDR), 1α-hydroxylase and 24-hydroxylase (CYP24) in neuronal subpopulations and satellite glia in the superior cervical ganglia (SCGs) of rats using immunohistochemistry. VDR immunoreactivity was observed in the cytoplasm and nucleus of nearly all neurons in the SCG. Intensity of VDR fluorescence was significantly greater in the cytoplasm of neuropeptide Y (NPY) negative somata compared to NPY positive neurons. Immunoreactivity for 1α-hydroxylase also was observed in the cytoplasm of all neurons of the SCG, but the intensity of fluorescence was less in the nuclei. To the contrary, the immunoreactivity for CYP24 was stronger in the nuclei, although it was present at lower intensity also in the cytoplasm of neurons. VDR and 1α-hydroxylase immunofluorescence was observed in many non-neuron cells, except satellite glial cells, which exhibited weak CYP24 immunofluorescence. Expression of VDRs and key metabolizing enzymes indicated the importance of vitamin D in the autonomic nervous system and the ability of sympathetic neurons to activate and deactivate vitamin D for its autocrine and paracrine roles.     

The relationship of DNA synthesis to protein accumulation in the cell nucleus

The relationship between DNA synthesis and protein accumulation in cell nucleus and cytoplasm has been investigated by the use of a combination of ultramicrointerferometric and ultramicrospectrophotometric methods. 5-Fluoro-2'-deoxyuridine (FUdR) inhibited DNA synthesis, resulting in inhibition of cell proliferation in G-1 and early S-phase. However, synthesis and accumulation of protein continued in the presence of FUdR, as indicated by a 54% increase in the average dry mass value per individual cell during 18-hour exposure to FUdR; due primarily to protein accumulation in the cytoplasm, the average cytoplasmic dry mass increased by as much as 85%, while the dry mass of the nucleus increased by only 21%. The dry mass values of individual nuclei were well-correlated to the nuclear DNA content throughout the period of exposure to FUdR. In contrast to the continued accumulation of protein in the cytoplasm during inhibition of DNA synthesis, protein accumulation in the nucleus was inhibited. When cells were released from inhibition of DNA synthesis by the addition of 2'-deoxythymidine, the nuclear DNA content and nuclear dry mass increased in near-synchrony, there being some evidence that DNA synthesis was initiated somewhat prior to initiation of increase in nuclear dry mass. Thus, it appears that DNA synthesis (or an increase in nuclear DNA content) is intimately related to the regulation of protein accumulation in the nucleus.     

Fractionation of nuclei from brain by zonal centrifugation and a study of the ribonucleic acid polymerase activity in the various classes of nuclei

1. The nuclei of the cells of the whole rat brain have been fractionated in a B-XIV zonal rotor with a discontinuous gradient of sucrose. Five fractions were obtained. Zone (I) contained neuronal nuclei (70%) and astrocytic nuclei (23%). Zone (II) contained astrocytic nuclei (81%) and neuronal nuclei (15%). Zone (III) contained astrocytic nuclei (84%) and oligodendrocytic nuclei (15%). Zone (IV) contained oligodendrocytic nuclei (92%) and zone (V) contained only oligodendrocytic nuclei. 2. The content of DNA, RNA and protein per nucleus was determined for each zone. Although the amount of DNA per nucleus is constant (7pg) the RNA varies from 4.5 to 2.5pg/nucleus and the protein from 38 to 17.6pg/nucleus. The neuronal nuclei have the greatest amounts of protein. The oligodendrocytic nuclei have the least content of RNA and protein. 3. The effects of pH, ionic strength, and Mg(2+) and Mn(2+) concentration on the activity of the nuclear system for synthesis in vitro of RNA have been investigated for unfractionated nuclei. From these studies a standard set of conditions for the assay of nuclear RNA polymerase has been established. 4. The activity of the RNA polymerase in each of the zonal fractions has been determined in the presence and in the absence of alpha-amanitin. Zone (II) is the most active, followed by zone (I). The nuclei of zones (IV) and (V) have comparable activity, which is 40% of that of zone (II). 5. The extent of incorporation of each of the four labelled nucleoside triphosphates by the nuclei from each zone has been measured. These values have been used to calculate the base composition of the RNA synthesized in vitro in each class of nucleus. 6. The effect of changes in the condition of assay of RNA polymerase in the different classes of nuclei has been investigated. Significant differences in the response to concentrations of metal ions and ammonium sulphate have been observed. 7. Homopolymer formation in each zone of brain nuclei has been determined. The extent of formation of the four homopolymers roughly parallels the RNA polymerase activity.     

3H-fucose incorporation into glycoproteins of toad bladder epithelial cells.

Electron microscope autoradiography was used to detect the incorporation of 3H-fucose into glycoproteins of toad bladder epithelial cells. After short exposure to 3H-fucose, without a chase period, the Golgi regions of all four cell types were labeled. When exposure to 3H-fucose was followed by chase periods (1,3,4 and 6 hours) the apical and basal-lateral plasma membranes of granular cells were heavily labeled. Apical granules and the cytoplasm of granular cells were also labeled, suggesting that they both provide the means for glycoprotein transfer from the Golgi to the plasma membranes. The heaviest labeling in mitochondria-rich cells, after the 1- and 3-hour chase periods, was over the apical tubules, although the apical and basal-lateral plasma membranes were also heavily labeled. After 4- and 6-hour chases, the labeling of the apical tubules decreased, whereas the labeling of the plasma membranes increased, strongly suggesting that in these cells apical tubules play a major role in the transfer of glycoproteins from the Golgi to the plasma membrane. Our results demonstrate that the route of 3H-fucose incorporation into plasma membrane glycoproteins and the rate of glycoprotein synthesis and breakdown are not the same in the two major epithelial cell types in toad bladder.     

An ether stressor increases REM sleep in rats: possible role of prolactin

Sleep alterations after a 1-min exposure to ether vapor were studied in rats to determine if this stressor increases rapid eye-movement (REM) sleep as does an immobilization stressor. Ether exposure before light onset or dark onset was followed by significant increases in REM sleep starting approximately 3-4 h later and lasting for several hours. Non-REM (NREM) sleep and electroencephalographic slow-wave activity during NREM sleep were not altered. Exposure to ether vapor elicited prolactin (Prl) secretion. REM sleep was not promoted after ether exposure in hypophysectomized rats. If the hypophysectomy was partial and the rats secreted Prl after ether exposure, then increases in REM sleep were observed. Intracerebroventricular administration of an antiserum to Prl decreased spontaneous REM sleep and inhibited ether exposure-induced REM sleep. The results indicate that a brief exposure to ether vapor is followed by increases in REM sleep if the Prl response associated with stress is unimpaired. This suggests that Prl, which is a previously documented REM sleep-promoting hormone, may contribute to the stimulation of REM sleep after ether exposure.     

Effects of bromocriptine and alpha-flupenthixol on sleep in REM sleep deprived rats.

Administration of bromocriptine mesylate (5 mg/kg, i.p.), a dopamine receptor stimulant, to rats which were deprived of REM sleep for 24 hours resulted in a significant increase in wakefulness as well as significant reduction of REM sleep during the first 5 hours of EEG recording. These effects were completely abolished by pretreatment with α-flupenthixol (0.2 mg/kg, i.p.), a dopamine receptor blocker. The loss of REM sleep has not been regained during the next 25 hours of EEG recording suggesting that the stimulation of dopamine receptors reduced REM sleep without causing subsequent REM rebound. These data raise questions on the negative dopamine control of REM sleep and on the potential use of dopamine stimulants in clinical situations characterized by excessive REM or by REM sleep dysfunction (narcolepsy).     

Development of REM sleep drive and clinical implications.

Rapid eye movement (REM) sleep in the human declines from approximately 50% of total sleep time ( approximately 8 h) in the newborn to approximately 15% of total sleep time (approximately 1 h) in the adult, and this decrease takes place mainly between birth and the end of puberty. We hypothesize that without this developmental decrease in REM sleep drive, lifelong increases in REM sleep drive may ensue. In the rat, the developmental decrease in REM sleep occurs 10-30 days after birth, declining from >70% of total sleep time in the newborn to the adult level of approximately 15% of sleep time during this period. Rats at 12-21 days of age were anesthetized with ketamine and decapitated, and brain stem slices were cut for intracellular recordings. We found that excitatory responses of pedunculopontine nucleus (PPN) neurons to N-methyl-D-aspartic acid decrease, while responses to kainic acid increase, over this critical period. During this developmental period, inhibitory responses to serotonergic type 1 agonists increase but responses to serotonergic type 2 agonists do not change. The results suggest that as PPN neurons develop, they are increasingly activated by kainic acid and increasingly inhibited by serotonergic type 1 receptors. These processes may be related to the developmental decrease in REM sleep. Developmental disturbances in each of these systems could induce differential increases in REM sleep drive, accounting for the postpubertal onset of a number of different disorders manifesting increases in REM sleep drive. Examination of modulation by PPN projections to ascending and descending targets revealed the presence of common signals modulating ascending arousal-related functions and descending postural/locomotor-related functions.     

Nuclear-cytoplasmic relationships in human cells in tissue culture. III. Auto-radiographic study of interrelation of nuclear and cytoplasmic ribonucleic acid

The movement of ribonucleic acid (RNA) from nucleus to cytoplasm has been studied, by autoradiographic techniques, in cells of the human amnion grown in tissue culture. Cells were exposed to cytidine-H(3) for 1 hour after which time only the RNA of the nuclei was labelled. After this 1 hour exposure the cells were placed in a medium containing an excess amount of unlabelled cytidine. Periodically, cells from this medium were fixed. Autoradiographs showed that there was a progressive movement of the label from nucleus to cytoplasm, such that after 24 hours essentially all the label was in the RNA of the cytoplasm. A study of the incorporation of the cytidine-H(3) in deoxyribonucleic acid (DNA), in the same population of cells at the same times, indicated that the presence of excess amounts of unlabelled cytidine almost instantaneously inhibited further utilization of cytidine-H(3). It is concluded that RNA moves from nucleus to cytoplasm as a complex polynucleotide structure.     

Cell proliferation in the Rana catesbeiana auditory medulla over metamorphic development

During metamorphic development, bullfrogs (Rana catesbeiana) undergo substantial morphological, anatomical, and physiological changes as the animals prepare for the transition from a fully-aquatic to a semi-terrestrial existence. Using BrdU incorporation and immunohistochemistry, we quantify changes in cell proliferation in two key auditory brainstem nuclei, the dorsolateral nucleus and the superior olivary nucleus, over the course of larval and early postmetamorphic development. From hatchling through early larval stages, numbers of proliferating cells increase in both nuclei, paralleling the overall increase in total numbers of cells available for labeling. Numbers of proliferating cells in the superior olivary nucleus decrease during the late larval and deaf periods, and significantly increase during metamorphic climax. Proliferating cells in the dorsolateral nucleus increase in number from hatchling to late larval stages, decrease during the deaf period, and increase during climax. In both nuclei, numbers of proliferating cells decrease during the postmetamorphic froglet stage, despite increases in the number of cells available for label. Newly generated cells express either glial- or neural-specific phenotypes beginning between 1 week and 1 month post-BrdU injection, respectively, while some new cells express gamma-aminobutyric acid within 2 days of mitosis. Our data show that these auditory nuclei dramatically up-regulate mitosis immediately prior to establishment of a transduction system based on atmospheric hearing.     

Nuclear-Cytoplasmic Relationships in Human Cells in Tissue Culture : III. Autoradiographic Study of Interrelation of Nuclear and Cytoplasmic Ribonucleic Acid

The movement of ribonucleic acid (RNA) from nucleus to cytoplasm has been studied, by autoradiographic techniques, in cells of the human amnion grown in tissue culture. Cells were exposed to cytidine-H³ for 1 hour after which time only the RNA of the nuclei was labelled. After this 1 hour exposure the cells were placed in a medium containing an excess amount of unlabelled cytidine. Periodically, cells from this medium were fixed. Autoradiographs showed that there was a progressive movement of the label from nucleus to cytoplasm, such that after 24 hours essentially all the label was in the RNA of the cytoplasm. A study of the incorporation of the cytidine-H³ in deoxyribonucleic acid (DNA), in the same population of cells at the same times, indicated that the presence of excess amounts of unlabelled cytidine almost instantaneously inhibited further utilization of cytidine-H³. It is concluded that RNA moves from nucleus to cytoplasm as a complex polynucleotide structure.