In situ studies on the effect of acid extraction on the DNA template activity of mature avian erythrocytes.

Exogenous E. coli RNA polymerase was used to determine the in situ DNA template activity of ethanol/acetone fixed avian erythrocytes. No RNA polymerase-catalyzed incorporation of 3H-UTP was detected in mature avian erythrocytes while simultaneously fixed avian lymphocytes did exhibit incorporation of 3H-UTP. Nuclei of mature erythrocytes which were subjected to treatments known to remove histones showed dramatic increases in RNA polymerase-catalyzed incorporation of 3H-UTP. The chromatin of treated cells was presumed to be more accessible to RNA polymerase as determined by the increase in RNA polymerase-catalyzed incorporation of 3H-UTP. Incubation of acid-treated nuclei in poly-L-lysine prior to incubation with RNA polymerase failed to inhibit the incorporation of 3H-UTP. Possible mechanisms for the inactivation of avian erythrocyte nuclei are discussed.     

Effect of etimizol on the RNA synthesizing activity of brain cell nuclei during the learning process in rats

A study was made of the effect of the central nervous system stimulant ethymizol on RNA-polymerase activity of cell nuclei of the rat cerebral cortex and hippocampus during learning with different reinforcements. Ethymizol stimulated the incorporation of 3H-UTP in the TCA-insoluble fraction of hippocampal nuclei during training of the animals in the avoidance response to the light in a Y-shaped mize and training in the reaction of spontaneous alternation of food reinforcements in a complex mize, and decreased the ratio brain cortex/hippocampus RNA-polymerase activity. The data obtained are discussed from the standpoint that the action of ethymizol is consequent on the activation of the genome of nervous cells.     

Bone marrow ribonucleic acid polymerase. Effect of testosterone on nucleotide incorporation into nuclear RNA.

The incorporation of 3H-UTP into RNA by isolated rat bone marrow nuclei is stimulated by testosterone. This effect is hormone and tissue specific. Using alpha-amanitine and different ionic strength conditions it was found that testosterone enhances preferentially RNA polymerase I activity. The sedimentation pattern of RNA isolated from bone marrow nuclei shows that the synthesis of RNA species within the 14-30 S range is mainly stimulated by the hormone.     

Changes in the transcription capacity of the chromatin of nerve and liver cells exposed to morphine and opioids

Morphine stimulates in vitro 3H-UTP incorporation into the nuclei of neurons of the superior cervical ganglion and into the hepatocyte nuclei of some rodents. This effect is dose-dependent and partially inhibited by naloxone. A synthetic analog of enkephalins, D-Ala2-Gly-ol5-enkephalin (DAGO), and beta-endorphin stimulate the incorporation of RNA precursors into pre-rRNA. Possible mechanisms of morphine action are discussed.     

表皮生长因子对细胞核转录活性的直接作用

表皮生长因子（ＥＧＦ）对纯化的鼠胚成纤维细胞（Ｃ_３Ｈ_１０Ｔ_１／２Ｃ１８）胞核~３Ｈ－ＵＴＰ掺入有明显的促进作用。这一作用在ＥＧＦ与细胞核共同培养９０ｍｉｎ时达到高峰。该作用是ＥＧＦ浓度依赖性的。ＥＧＦ对β射线转化的上述细胞的纯化细胞核,亦同样有促进转录作用。本实验所用的细胞核经光镜,电子显微镜观察,细胞膜、细胞浆标志酶检测及放射性同位素示踪法鉴定,未检出有核外成份的污染。本实验结果为膜受体信号传递途径以外的ＥＧＦ对核直接作用途径的存在提供了证据。     

A comparison of bacterial RNA-polymerase RNA synthesis on polytene Drosophila chromosomes with transcription in living cells

The incorporation of 3H-uridine in different regions of polytene chromosomes in live cells of the Drosophila melanogaster salivary glands was compared with the incorporation of 3H-UTP in the same regions under the incubation of cytological preparations of these chromosomes with the E. coli RNA polymerase. The label distribution by regions was compared with the DNA content in them. Individual regions of chromosomes differ by 3H-uridine incorporation in live cells to a much greater extent than by 3H-UTP incorporation in vitro under the incubation with a non-homologous enzyme. RNA synthesis in an exogenous enzyme depends on the DNA content in different chromosome regions to a much greater extent than RNA synthesis in vivo. The correlation of label distribution after 3H-uridine incorporation in live cells and after RNA synthesis in vitro on the preparations by the bacterial RNA polymerase is, correspondingly, very low. This enzyme forms, however, RNA's on puffs 2-3 times more actively than on the same regions in non-puffing state but this difference is dozens of times greater in live cells. RNA synthesis in vitro is, thus, non-specific and does not correspond practically to the intensity of RNA synthesis on the same chromosome regions in live cells. At the same time, as in live cells, the E. coli enzyme synthesizes twice more RNA on the single X-chromosome of males (1X2A) than on each of X-chromosomes of diploid (2X2A) and triploid (3X3A) females or superfemales (3X2A), whereas in intersexes (2X3A) X-chromosomes display intermediate template activity. Thus, RNA synthesis by a heterologous enzyme in vitro does not differ by this index from the synthesis in live cells. It is suggested that differences in the template activity of X-chromosomes in vitro depending on the sex index (X : A) are due to different degree of DNP condensation in these chromosomes. In spite of differences in the degree of condensation, the male X-chromosome binds on the fixed preparation approximately the same amount of thymus histone F1 carrying fluorochrome as each of two female X-chromosomes. Hence, there is no sharp difference between the male and female X-chromosomes by the number and length of DNA regions accessible for interaction with exogenous proteins. On the basis of the data obtained, a hypothesis about two levels and, respectively, two mechanisms of control gene activity in animal chromosomes is considered. The first mechanism is, supposedly, based on decondensation of DNP appears to result in that the same proteins-regulators in the same amount activate corresponding genes in X-chromosome in males twice more strongly than in females.     

Deoxyribonucleic acid-dependent ribonucleic acid polymerase activity in purified trachoma elementary bodies: effect of sodium chloride on ribonucleic acid transcription

Highly purified trachoma elementary bodies (T'ang strain), incubated in the presence of the four nucleoside triphosphates [Mg(2+), Mn(2+), 2-mercaptoethanol, tris(hydroxymethyl)aminomethane buffer (pH 7.5)] were found to incorporate (3)H-uridine triphosphate (UTP) into ribonucleic acid (RNA) molecules. Eighty-seven per cent of the labeled molecules were sensitive to ribonuclease treatment. In vitro RNA synthesis was almost completely inhibited by actinomycin D. Rifampin was also inhibitory, but allowed some initial RNA synthesis before complete inhibition occurred. When the reaction mixture lacked Mn(2+), trachoma elementary bodies synthesized, for a limited period, high-molecular-weight RNA species (23 to 24S, 16 to 17S, and 10 to 11S). Addition of 0.2 m NaCl to the same reaction mixture stimulated and prolonged (3)H-UTP incorporation into the same radioactive RNA species. Addition of 0.001 m Mn(2+) instead of NaCl also stimulated (3)H-UTP incorporation but prevented the synthesis of the high-molecular-weight RNA species.     

Inhibition of proliferation of MCF-7 breast cancer cells by a blocker of Ca2+-permeable channel

In MCF-7 breast cancer cells, insulin-like growth factor-1 (IGF-1) increased the calcium-permeability of the cells by activating a voltage-independent calcium-permeable channel. IGF-1 also induced oscillatory elevation of cytoplasmic free calcium concentration in these cells. An anti-allergic compound, tranilast, reduced the calcium-permeability augmented by IGF-1 in a dose-dependent manner and blocked the oscillatory elevation of cytoplasmic free calcium concentration. Tranilast did not affect early intracellular signals activated by IGF-1, including receptor autophosphorylation, activations of Ras, mitogen-activated protein kinase and phosphatidylinositol 3-kinase. Tranilast inhibited increases in [³H]-thymidine incorporation, DNA content and cell number induced by IGF-1. The ID₅₀ for [³H]-thymidine incorporation and DNA content were about 10 μM. The inhibitory effect of tranilast was reversible, and cell viability was not affected. Treatment with tranilast increased the number of cells in the G₁ phase suggesting that this compound induced G₁ arrest. Tranilast also reduced the phosphorylation of the retinoblastoma protein. These results indicate that tranilast inhibits the IGF-1-induced cell growth in MCF-7 cells by blocking calcium entry.     

Characteristics of DNA replication in isolated nuclei initiated by an aprotinin-binding protein

Isolated cell nuclei were used as the source of template DNA to investigate the role of a cytosolic aprotinin-binding protein (ADR) in the initiation of eukaryotic DNA replication. Computerized image cytometry demonstrated that the DNA content of individual nuclei increased significantly following incubation with ADR-containing preparations, and the extent of DNA synthesis is consistent with that allowed by the limiting concentration of dTTP. Thus, dTTP incorporation into isolated nuclei represents DNA synthesis and not parent strand repair. We found that dTTP incorporation into the isolated nuclei is dependent on DNA polymerase α (a principal polymerase in DNA replication) but that DNA polymerase β (a principal polymerase in DNA repair processes) does not play a significant role in this system. Finally, neither aprotinin nor a previously described cytosolic ADR inhibitor can block the replication of nuclease-treated calf thymus DNA, while both strongly inhibit replication of DNA in isolated nuclei. This result, coupled with the relative ineffectiveness of nuclease-treated DNA compared with nuclear DNA to serve as a replicative template in this assay, argues against a significant contribution from repair or synthesis which initiates at a site of DNA damage. These data indicate that ADR-mediated incorporation of ³H-dTTP into isolated nuclei results from DNA replicative processes that are directly relevant to in vivo S phase events. © 1993 Wiley-Liss, Inc.     

Influence of polyhydroxysteroids on [Ca(2+)](i)

Recently, we have shown that two biologically active, disulfated polyhydroxysteroids from the Pacific brittle star Ophiopholis aculeata stimulate Ca(2+) influx into different cell types. In the present study, 45Ca(2+) and two fluorescent calcium probes, quin-2/AM and fura-2/AM, were employed to investigate the course and amplitude of calcium signals induced in different mouse cells using an radio-isotope, spectrofluorimetry, and microcytofluorimetry techniques. The cytotoxic and hemolytic effects were not observed for both steroids at the wide range of concentrations. Steroids did not influence [3H]-uridine incorporation in a variety of cells. The investigated steroids stimulated a rapid increase in cytosolic Ca(2+) content in Ehrlich mouse carcinoma cells, mouse spleen lymphocytes, and mouse peritoneal macrophages in the concentration range 1-100 microg/ml on a dose-dependent basis. Blockers of L-type calcium channels, such as verapamil, diltiazem, nifedipine (1 x 10(-7)M), and 1mM EGTA, inhibited this process and reduced the response of cells to steroid application. The stimulatory effect of steroids on human fibroblast proliferation and mouse macrophage lysosome activity was observed also. It is suggested that the investigated compounds may act as Ca(2+)-agonists and increase Ca(2+)-transport across cell membranes.     

Separate effect of hydroxyurea on the initiation and elongation of DNA synthesis in BHK cells

Summary BHK21/C₁ cells, starved for 30 h in serum deficient medium and treated for 15 h with 1 mm hydroxyurea (HU) in order to obtain a synchronous cell population in the G₁/S-boundary, incorporate a residual proportion of ³H-thymidine (dThd). This residual incorporation is due to semiconservative synthesis and may not be reduced by increasing the drug concentration without affecting the reversion capacity of the cells proportionally. As shown by autoradiographic analysis, the residual DNA synthesis does not correspond to ³H-dThd incorporation within a small number of resistant cells, but is located in the nuclei of a high proportion of cells with reduced density of silver grains. After treatment with 0.05 mm HU, however, the incorporation of ³H-dThd increases considerably over the control values. The determination of the radioactivity incorporated by µg DNA corresponding to nuclei in S phase indicates that this concentration of HU is also able to reduce the rate of DNA polymerization. Kinetic data on the appearance of this increased ³H-dThd incorporation and on the accumulation of labelled nuclei in cells growing at random and labelled continuously with the radioactive DNA precursor indicate that HU stimulates the cells to enter the S phase. The reported results are consistent with a mechanism of action of HU which affects initiation and elongation of DNA chains separately.     

Increased phosphorylation of nuclear substrates for rat brain protein kinase C in regenerating rat liver nuclei.

Protein phosphorylation catalysed by rat brain protein kinase C (PKC) has been studied in nuclei isolated from normal and regenerating rat liver. Histone H1 and a 40,000 molecular weight protein were hyperphosphorylated at all the explored regeneration times, ranging from 3 to 22 h after partial hepatectomy. Phosphorylation of the two substrates was totally dependent on calcium and lipids and was abolished by low concentration of staurosporine. The observed early change of phosphate content of histone H1 and of the 40,000 molecular weight protein on the time scale of liver regeneration suggests that PKC might be involved in the initial nuclear events leading to cell proliferation.     

Growth and differentiation of primary tracheal epithelial cells in culture: regulation by extracellular calcium

Growth and differentiation of primary monkey tracheal epithelial (MTE) cells maintained on collagen gel substrata were studied in a defined serum-free culture medium containing 0.03 to 3.0 mM extracellular calcium. Cell attachment efficiency (40-60%) was not altered by different calcium levels. Growth of primary MTE cells on collagen gel substrata, which was vitamin A dependent, was enhanced 50% in the medium supplemented with high calcium (greater than 0.3 mM). High calcium medium also increased cell-cell interactions, formation of desmosomes, and multi-cell layering. The relative content of mucous cells, which were identified by a mucin-specific monoclonal antibody and the presence of mucus-secreting granules at the ultrastructural level, was greater in the high-calcium medium. Furthermore, the secretion of mucin into the medium, determined either by an ELISA or by the incorporation of 3H-glucosamine into mucous glycoprotein fractions, was also increased more than 5-fold in media containing high calcium content (greater than 0.6 mM). In contrast, MTE cells cultured in low calcium medium (less than 0.15 mM) were squamous-like with prominent tonofilaments, and their secretory product was mainly hyaluronate. These results demonstrate that media containing a high calcium content promote conducting airway epithelium to express mucous cell differentiation, while media with low calcium content promote squamous cell differentiation.     

心肌细胞核Ca^2＋库特点及其调节的离体研究

To investigate the regulation of Ca2+ in the isolated cardiac nuclei from rats which may illuminated the mechanism of nuclear calcium transport system. Elocity and isopyknic gradient centrifugation were employed to fractionate rat cardiac nuclei. Then fluo-4 confocal microscopy techniques was used to verify the changes of nuclear Ca2+. There are calcium-dependent Ca2+ uptake in the cardiac nuclear obtained from normal rats. The accumulation Ca2+ of cardiac nuclei in vitro from the incubating medium were not consistent with free [Ca2+] in incubating medium. The nuclear envelope was initially loaded with Ca2+ (1 mmol/L ATP and approximately 100 nmol/L Ca2+), Adequate Ca2+ loading was next confirmed by imaging the nuclear envelope and nucleoplasm. Exposure of Ca2+ -loaded nuclei to IP3, ryanodine or ryanodine + thapsigargin, respectively, resulted in a rapid and transient elevation of nucleoplasmic Ca2+ free concentration, this effects were abolished by pretreatment of cardiac nuclei with Ca2+ -ATPase inhibitor thapsigargin. Thapsigargin and IP3 receptor antagonist heparin induced nucleoplasmic Ca2+ free concentration decrease. Fluorescence experiments indicated that both ryanodine receptors and Ca2+ -ATPase were distributed in the outer layer of nuclear envelope, and inositol 1,4,5-trisphosphate receptors mainly dispersively localized at inner layer of nuclear envelope. The present study demonstrates that nuclear calcium were regulated by free Ca2+, IP3 and ryanodine. The results suggested calcium transport system might be present in the myocardial nuclei, the myocardial nuclei might served as one of calcium pools in myocardial cell.     

ATP浓度和缺氧暴露对大鼠脑线粒体RNA和蛋白质体外合成的影响

本文探讨介质中ATP浓度和急,慢性缺氧暴露对大鼠脑线粒体内RNA和蛋白质合成的影响。用差速离心法分离正常和低压舱模拟4000m高原急性连续缺氧暴露3d和慢性连续缺氧暴露40d大鼠脑线粒体,用体外无细胞（cell-free in vitro)^3H-UTP和^3H-Leucine掺入法分别测定线粒体RNA和蛋白质合成活性,结果显示,大鼠急性缺氧暴露后大脑皮质线粒体RNA体外合成活性降低40%,蛋白质合成活性降低60%;慢性缺氧暴露后线粒体RNA和蛋白质合成活性分别为对照的72%和76%;ATP对正常大鼠脑线粒体RNA以及蛋白质的体外合成活性的影响均呈双相性,大于或小于1mmol/L均可产生不同程度的抑制效应,结果提示,缺氧可在转录和翻译两个水平上影响脑线粒体mtDNA的表达,而慢性缺氧暴露时,线粒体半自主性功能的改善可能是机体对缺氧适应的细胞机制之一;ATP对脑线粒体内转录和释放活性的调节是一种经济有效的反馈调节方式。     

1α,25-Dihydroxyvitamin D3 rapidly increases nuclear calcium levels in rat osteosarcoma cells

1α,25-Dihydroxyvitamin D₃ increases intracellular calcium in rat osteoblast-like cells that possess the classic receptor (ROS 17/2.8) as well as those that lack the classic receptor (ROS 24/1), indicating that a separate signalling system mediates this rapid nongenomic action. To determine the intracellular sites of this calcium increase, cytosolic and nuclear fluorescence (340 nm/380 nm ratio) were measured in Fura 2AM loaded ROS 17/2.8 cells using digital microscopy. Within 5 min, cytosolic fluorescence increased by 29% (P < 0.05) and nuclear fluorescence by 30% (P < 0.01) after exposure to 1α,25-dihydroxyvitamin D₃ (20 nM). This effect was blocked by the inactive epimer 1β,25-dihydroxyvitamin D₃. In an individual cell, cytosolic and nuclear fluorescence increased gradually after 1, 3, and 5 min exposure to vitamin D. Nuclei were then isolated from ROS 17/2.8 cells to directly measure the hormone's effect on nuclear calcium. The calcium content of Fura 2AM loaded nuclei was not affected by increasing the calcium concentration in the incubation buffer from 50 nM to 200 nM. After 5 min, 1α,25-dihydroxyvitamin D₃, 20 nM, increased the calcium of isolated nuclei in medium containing 50 nM calcium and 200 nM calcium. 1β,25-dihydroxyvitamin D₃, 20 nM, had no effect on nuclear calcium but blocked the 1α,25-dihydroxyvitamin D₃ induced rise in the isolated nuclei. The results indicate that the nuclear membrane of the ROS 17/2.8 cells contain calcium permeability barriers and transport systems that are sensitive to and specific for 1α,25-dihydroxyvitamin D₃. 1α,25-Dihydroxyvitamin D₃ rapidly increases nuclear calcium levels in both intact cells and isolated nuclei suggesting that rapid nongenomic activation of nuclear calcium may play a functional role in osteoblastic activity.     

Method of determining the concentration of glycogen, DNA, 3H-thymidine label and dry weight in one and the same cell]

A method is proposed for determination of glycogen, DNA, 3H-thymidine incorporation and dry weight in the same cell, the technique being based on successive discovery and measuring of each of these indices. Cells are obtained from animals, previously injected with 3H-thymidine, to be charted on preparation, made pictures and measured in square units. Then on preparations embedded into glycerine or vaseline oil, the optical path difference of rays for the nucleus and cytoplasm of selected cells is measured with the interferencial microscope. This is followed by the fluorescent PAS reaction and the content of glycogen is registered microfluorimetrically in the same cells. Preparations after that are treated with a freshly prepared water solution of 0.025% borohydride sodium, stained with the routine or fluorescent Feulgen reaction, and DNA content is determined in the same cells in which glycogen and delta delta were previously measured. The stained nuclei are photographed, their areas are measured and the dry weight of the nucleus and cytoplasm of marked cells is calculated from the values of the nuclear areas and of delta delta. Eventually the preparations are covered by emulsion and exposed, and 3H-thymidine-containing nuclei are determined, the index of marked nuclei and the marking intensity over the nucleus are calculated. As a result, a precise and reliable determination of glycogen, DNA, dry weight and 3H- or 14C-thymidine incorporation is made in either of the marked cell.