Effect of cytochalasin D on DNA synthesis in cultured cells

The effect of cytochalasin D (CD), an agent specifically destroying actin cytoskeleton, on DNA replication of cultured mouse embryonic fibroblasts (MEF) and BALB/3T3 strain cells was studied. Incubation of normal cells with CD resulted in progressive inhibition of DNA synthesis: in the first 16-20 h the percentage of cells pulse-labelled with 3H-thymidine was similar to that in control cultures, on day 8 the percentage of labelled cells was 7-8 times lower than in the control. The transfer of cells into fresh medium upon 8-day incubation in the presence of CD resulted in the recovery of DNA synthesis. Similar curves of DNA synthesis inhibition in the presence of CD and of DNA synthesis recovery in fresh medium were observed both in mononuclear and binuclear cells. Thus, CD-induced reorganization of actin cytoskeleton can have an abrupt but reversible disturbing effect on normal cell cycle.     

Unscheduled DNA synthesis in growing roots and stored embryos of Vicia faba after the action of maleic hydrazide and methyl methanesulphonate

Growing roots of Vicia faba were treated with MH for 5 h, washed for 2 h and exposed to 3H-thymidine (3H-TdR) for additional 2-h periods at 7 h, 24 h and 32 h after the onset of MH treatment, to label DNA. As the replicative DNA synthesis was suppressed by HU, an enhancement of 3H-TdR incorporation into nuclear DNA above the control, as determined by microautoradiography, was considered to be due to unscheduled DNA synthesis induced by the mutagen. A significantly higher incorporation of 3H-TdR into DNA of MH-treated roots occurred, when labelling was applied 7 h after the MH action, whereas at 24 h only slight and at 32 h no enhancement of DNA labelling above control was registered. A 3-14-day storage with 50% water content of V. faba seeds exposed to MH or MMS resulted in a recovery from mutagen-induced chromosomal damage and a significantly higher incorporation of 3H-TdR into nuclear DNA. This supports the hypothesis that recovery from MH- and MMS-induced chromosomal damage is mediated by excision repair during seed storage.     

Developmental changes in the expression of high mobility group chromosomal proteins

The high mobility group (HMG) chromosomal proteins may modulate the structure of distinct regions in chromatin, thereby affecting processes such as development and differentiation. Here we report that the levels of the HMG chromosomal proteins and their mRNAs change significantly during erythropoiesis. Erythroid cells from 5-day chicken embryos contain 2.5-10 times more HMG mRNAs than cells from 14-day embryos, whereas circulating cells from adult animals are devoid of HMG and most other mRNAs. Nuclear run-off experiments and Northern analysis of RNA from various developmental stages and from Percoll-fractionated cells indicate that the genes are transcribed in early cells of either the primitive or definitive erythroid lineage. The rate of synthesis of the various HMGs changes during erythropoiesis; in erythroid cells from 7-day embryos the ratio of HMG-14b or HMG-17 to HMG-14a is, respectively, 8 and 10 times lower than in 9-day erythroids. HMG-14a, the major chicken HMG-14 species, is synthesized mainly in primitive cells, while HMG-14b is preferentially synthesized in definitive cells. Thus, the change from primitive to definitive erythroid lineage during embryogenesis is accompanied by a change in the expression of HMG chromosomal proteins. Conceivably, these changes may affect the structure of certain regions in chromatin; however, it is not presently clear whether the switch in HMG protein gene expression is a consequence or a prerequisite for proper differentiation.     

K-Opioid Agonist Modulation of [3H]Thymidine Incorporation into DNA: Evidence for the Involvement of Pertussis Toxin-Sensitive G Protein-Coupled Phosphoinositide Turnover

Abstract: A body of evidence has indicated that μ-opioid agonists can inhibit DNA synthesis in developing brain. We now report that K -selective opioid agonists (U69593 and U50488) modulate [³H]thymidine incorporation into DNA in fetal rat brain cell aggregates in a dose- and developmental stage-dependent manner. K agonists decreased thymidine incorporation by 35% in cultures grown for 7 days, and this process was reversed by the K -selective antagonist, norbinaltorphimine, whereas in 21-day brain cell aggregates a 3,5-fold increase was evident. Cell labeling by [³H]thymidine was also inhibited by the K -opioid agonist as shown by autoradiography. In addition, U69593 reduced basal rates of phosphoinositide formation in 7-day cultures and elevated it in 21-day cultures. Control levels were restored by norbin-altorphimine. Pertussis toxin blocked U69593-mediated inhibition of DNA synthesis. The action of K agonists on thymidine incorporation in the presence of chelerythrine, a protein kinase C (PKC) inhibitor, or in combination with LiCl, a noncompetitive inhibitor of inositol phosphatase, was attenuated in both 7- and 21-day cultures. These results suggest that K agonists may inhibit DNA synthesis via the phosphoinositide system with a pertussis toxin-sensitive G protein as transducer. In mixed glial cell aggregates, U50488 increased thymidine incorporation into DNA 3.1-fold, and this stimulation was reversed by the opioid antagonist naltrexone.     

N^7＋重离子注入和贯穿后小麦种胚的期外DNA合成效应

用＾３ＨＴｄＲ掺入法研究了经Ｎ＾７＋重离子注入贯穿处理的８２５７９小麦和８８１２小麦种胚的ＤＮＡ合成动态。结果发现,未经Ｎ＾７＋重离子任何处理的两个小麦品种的对照种胚,在萌发早期（２０ｈ内）仅存在一个ＤＮＡ合成峰（于萌发的第１４ｈ）,而经过Ｎ＾７＋重离子注入和贯穿处理的小麦种胚则存在两个ＤＮＡ合成峰（分别于萌发的８－１０ｈ和１４－１６ｈ）,该种子经ＤＮＡ修复合成的抑制剂咖啡因处理后,第一个ＤＮＡ合     

Sensitivity of DNA synthesis to gamma-irradiation in Ehrlich ascites carcinoma cells in stationary and logarithmic growth phases

A study was made of the influence of gamma-radiation on DNA synthesis in cells of 3-day and 7-day Ehrlich ascites tumor cultures. DNA synthesis in cells of the 3-day culture was more sensitive to moderate radiation doses than those of the 7-day culture as was observed during the first 30 min after irradiation. After 3-hour postirradiation incubation, no appreciable difference was noted in radiosensitivity of DNA synthesis in the cells of the 3-day and 7-day cultures.     

Dynorphins Modulate DNA Synthesis in Fetal Brain Cell Aggregates

Abstract: Previously, opioid peptide analogues, β-endorphin, and synthetic opiates were found to inhibit DNA synthesis in 7-day fetal rat brain cell aggregates via κ-and μ-opioid receptors. Here dynorphins and other endogenous opioid peptides were investigated for their effect on DNA synthesis in rat and guinea pig brain cell aggregates. At 1 µ M , all dynorphins tested and β-endorphin inhibited [³H]thymidine incorporation into DNA by 20–38% in 7-day rat brain cell aggregates. The putative ε-antagonist β-endorphin (1–27) did not prevent the effect of β-endorphin, suggesting that the ε-receptor is not involved in opioid inhibition of DNA synthesis. The κ-selective antagonist norbinaltorphimine blocked dynorphin A or B inhibition of DNA synthesis, implicating a κ-opioid receptor. In dose-dependency studies, dynorphin B was three orders of magnitude more potent than dynorphin A in the attenuation of thymidine incorporation, indicative of the mediation of its action by a discrete κ-receptor subtype. The IC₅₀ value of 0.1 n M estimated for dynorphin B is in the physiological range for dynorphins in developing brain. In guinea pig brain cell aggregates, the κ-receptor agonists U50488, U69593, and dynorphin B reduced thymidine incorporation by 40%. When 21-day aggregates were treated with dynorphins, a 33–86% enhancement of thymidine incorporation was observed. Because both 7- and 21-day aggregates correspond to stages in development when glial cell proliferation is prevalent and glia preferentially express κ-receptors in rat brain, these findings support the hypothesis that dynorphins modulate glial DNA synthesis during brain ontogeny.     

Drosophila apurinic/apyrimidinic DNA endonucleases. Characterization of mechanism of action and demonstration of a novel type of enzyme activity

Two species of apurinic/apyrimidinic (AP) endonuclease have been purified approximately 400-fold from extracts of Drosophila embryos. AP endonuclease I, which flows through phosphocellulose columns, has an apparent subunit molecular weight of 66,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas AP endonuclease II, which is retained by phosphocellulose, has a subunit molecular weight of 63,000. The molecular weight determinations were made possible in part by the finding that both Drosophila enzymes, along with Escherichia coli endonuclease IV, cross-react with an antibody prepared toward a human AP endonuclease (Kane, C. M., and Linn, S. (1981) J. Biol. Chem. 256, 3405-3414). The nature of phosphodiester bond breaks produced by the two partially purified AP endonucleases from Drosophila have been investigated. Nicks introduced into partially depurinated PM2 DNA by Drosophila AP endonuclease I did not support DNA synthesis by E. coli DNA polymerase I, whereas nicks created by AP endonuclease II were able to support DNA synthesis, but at a rate far less than that observed for nicks introduced by E. coli endonuclease IV. The priming activity of DNA incised by either of the Drosophila enzymes can be enhanced, however, by an additional incubation with E. coli endonuclease IV, which is known to cleave depurinated DNA on the 5'-side of an apurinic site. These results suggest that the Drosophila enzymes cleave depurinated DNA on the 3'-side of the apurinic site. This suggestion was strengthened by the observation that the combined action of AP endonuclease II and E. coli endonuclease IV resulted in the removal of [32P]dAMP from partially depyrimidinated [dAMP-5'-32P,uracil-3H]poly(dA-dT). Taken together, these results propose that Drosophila AP endonuclease II produces 3'-deoxyribose and 5'-phosphomonoester nucleotide termini. Conversely, the absolute inability to detect priming activity for DNA cleaved by AP endonuclease I alone suggested a different mechanism, possibly the formation of a deoxyribose-3'-phosphate terminus. When apurinic DNA cleaved by AP endonuclease I was subsequently treated with bacterial alkaline phosphatase, DNA synthesis was now detected at levels similar to that observed for AP endonuclease II alone. Additionally, DNA nicked by AP endonuclease I was susceptible to 5'-end labeling by polynucleotide T4 kinase without prior phosphomonoesterase treatment. These results suggest that AP endonuclease I forms deoxyribose 3'-phosphate and 5'-OH termini upon cleaving depurinated DNA.     

Influence of DNA synthesis inhibition on the coordinate expression of core human histone genes during S phase

Core histone mRNA metabolism has been examined in S phase HeLa cells recovering from DNA synthesis inhibition by 1 mM hydroxyurea. Using cloned human histone genes as probes for histone mRNA quantitation, the response to and recovery from DNA synthesis inhibition is shown to depend on the position of the cell with respect to the initiation of DNA replication. The incorporation of 3H-uridine into multiple histone mRNAs in recovering cells does not exceed preinhibition levels, and as this incorporation is maximal in early S phase, the synthesis of core histone mRNA is apparently related to the ordered replication of the genome. The total histone mRNA present in interrupted S phase cells after recovery is not significantly different from that present in control cells, and a temporal and functional coupling between histone mRNA levels and the relative rate of DNA synthesis is maintained in perturbed cells.     

Positive inotropic action of acetylcholine on the heart ventricles of the chick embryo

Acetylcholine increased twitch tension in the whole ventricle or in ventricular strips from 2-10-day chick embryos. The effect of acetylcholine was mediated by muscarinic receptors, since it was prevented by atropine, but not by tubocurarine or propranolol. Prostigmine significantly increased the sensitivity to acetylcholine in the strips from 7-day embryos, being almost ineffective in the strips from 3-day embryos. The decrease in acetylcholine sensitivity in the developing chick embryo is presumably associated with the increase in cholinesterase activity of the myocardial tissue.     

The effect of inhibition of protein synthesis on some biochemical and functional activities of the brain in chick embryos]

By means of radioautographic and biochemical methods, it has been shown that cycloheximide administration to the brain in a dose of 76 mug significantly decreases protein synthesis within 30 min, the decrease being maximal after 2 hours. RNA synthesis in neuronal cytoplasm is blocked within 4 hours. Changes in biopotentials of the forebrain and midbrain structures were observed already 15-20 minutes after cycloheximide treatment. Application of cycloheximide (12.5-25 mug) to the area of vascular brannching of the foetal membranes of a 17-day embryo inhibits motor activity within 20 min.; in 28-day embryos, this effect is observed at a dose of 75 mug. Therefore, inhibition of protein synthesis in the brain results in sharp changes of the electrical activity of the brain and inhibits motor activity of the chick embryo.     

Remodeling of donor nuclei, DNA-synthesis, and ploidy of bovine cumulus cell nuclear transfer embryos: effect of activation protocol

The purpose of this study was to investigate the effects of two activation protocols on nuclear remodeling, DNA synthesis during the first cell cycle, chromosome segregation after first mitosis and development to blastocyst of embryos produced by somatic nuclear transfer. Pronuclear formation was significantly higher when activation lasted 5 hr compared to 3 hr for both ethanol-cycloheximide and ionomycin-bohemine treatment. However, the presence of a single nucleus was significantly higher in embryos activated for 3 hr in bohemine. Initiation of DNA synthesis was delayed in ethanol-cycloheximide group, however, after 12 hr labeling 100% of embryos synthesized DNA in both groups. Embryos activated with ethanol-cycloheximide developed to blastocysts at a significantly higher rate than those activated with ionomycin-bohemine. Analysis of 2-cell embryos with DNA probes for chromosome 6, 7, and 15 by fluorescence in situ hybridization showed that at least 50% of NT embryos were of normal ploidy independent of the activation stimulus. The results presented in this study show differences between the protocols compared on the nuclear events during the first cell cycle and on the development to blastocyst. Mol. Reprod. Dev. 59: 371-379, 2001.     

The effect of activation of Mammalian oocytes on remodeling of donor nuclei after nuclear transfer

Activation of bovine oocytes by experimental procedures that closely mimic normal fertilization is essential both for intracytoplasmic sperm injection and for nuclear transfer (NT). Therefore, with the goal of producing haploid activated oocytes, we evaluated whether butyrolactone I and bohemine, either alone or in combination with ionomycin, are able to activate young matured mammalian oocytes. Furthermore, the effect on the patterns of DNA synthesis after pronuclear formation as well as changes in histone H1 kinase and MAP kinase activities during the process of activation were studied. Our results with bohemine show that the specific inhibition of cyclin-dependent kinases (CDKs) in metaphase II bovine oocytes induces parthenogenetic activation in a dose dependent manner (25, 50, and 100 microM, respectively), either alone (3%, 30%, and 50%) or in combination with ionomycin (30%, 70%, and 87.5%). The effect of two activation protocols on nuclear remodeling, DNA synthesis during the first cell cycle, chromosome segregation after first mitosis, and development to blastocyst of embryos produced by somatic nuclear transfer were studied. Pronuclear formation was significantly higher when activation lasted 5 h compared to 3 h for both ethanol-cycloheximide and ionomycin-bohemine treatment. Initiation of DNA synthesis was delayed in ethanol-cycloheximide group, however, after 12-h labeling 100% of embryos synthesized DNA in both groups. Analysis of two-cell embryos with DNA probes for chromosome 6, 7, and 15 by fluorescence in situ hybridization showed that at least 50% of NT embryos were of normal ploidy, independent of the activation protocol.     

GT3 Synthesis in the Proximal Golgi Occurs in a Compartment Different from Those for GD3 and GM3 Synthesis

Abstract: Previous studies from this laboratory have shown that synthesis of GT3, the precursor of c series gangliosides, occurs in proximal Golgi compartments, as has been shown for the synthesis of GM3 and GD3, the precursors of a and b series gangliosides, respectively. In this work we studied whether the synthesis of GM3, GD3, and GT3 occurs in the same or in different compartments of the proximal Golgi. For this, we examined in retina cells (a) the effect of monensin, a sodium ionophore that affects mostly the trans Golgi and the trans Golgi network function, on the metabolic labeling of glycolipids from [³H]Gal by cultured cells from 7- and 10-day chick embryos and (b) the labeling in vitro of endogenous glycolipids of Golgi membrane preparations from 7-day embryos incubated with UDP-[³H]Gal. In (a), 1 µM monensin produced a twofold accumulation of radioactive glucosylceramide and a decrease to ～50 and 20% of total ganglioside labeling in 7- and 10-day cells, respectively. At both ages, monensin produced a threefold accumulation of radioactive GM3 and an inhibition of >90% of GT3, GM1, GD1a, and GT1b synthesis. GD3 synthesis was inhibited ～30 and 70%, respectively, in 7- and 10-day cells. In (b), >80% of the [³H]Gal was incorporated into endogenous glucosylceramide to form radioactive lactosylceramide. About 90% of [³H]Gal-labeled lactosylceramide was converted into GM3, and most of this in turn into GD3 when unlabeled CMP-NeuAc was also present in the incubation system. Under the same conditions, however, <5% of labeled GD3 was converted into GT3. Golgi membranes incubated with CMP-[³H]NeuAc incorporated ～20% of [³H]NeuAc into endogenous GT3, and this percentage was not affected by 1 µM monensin. These results indicate that synthesis of GT3 is carried out in a compartment of the proximal Golgi different from those for lactosylceramide, GM3, and GD3 synthesis. Results from the experiments with monensin point to the cis/medial Golgi as the main compartment for coupled synthesis of lactosylceramide, GM3, and GD3 and to the trans Golgi as the main compartment for synthesis of GT3.     

Behavior of M-phase synchronized blastomeres after nuclear transfer in cattle

M-phase synchronized bovine blastomeres were used to study the effect of nuclear-cytoplasmic synchronization on the developmental potential after nuclear transfer (NT). The capacity of nocodazole and benomyl to reversibly synchronize blastomeres from embryos in M-phase was evaluated. Nocodazole reversibly arrested bovine embryos at the studied stages and induced high rates of M-phases in morulae and compact morulae. In contrast, benomyl was less efficient than nocodazole to synchronize in M-phase. After transfer of an M-phase blastomere, premature chromatin condensation was the prevalent finding 1 hr post-fusion (hpf). Condensed chromosomes non-arranged in the equatorial plate (1-3 hpf) that acquired an organized structure over time (3-7 hpf) were subsequently observed. Anaphase-telophase structures were predominantly recorded at 4-9 hpf. About 50% of the embryos activated at both 3-4 and 6-7 hpf extruded a polar body-like structure 5 hr after activation, but this was not observed in embryos activated immediately after fusion. A significantly lower activation rate was observed for oocytes activated 3-4 hpf compared to those activated 6-7 hpf. However, the ability to undergo first cleavage was significantly lower in the latter group. Reconstructed embryos activated immediately after fusion showed no difference in the rate of activation compared to those activated 6-7 hpf, although the cleavage rate was higher. DNA synthesis was observed at a significantly higher rate in embryos activated both immediately and 3-4 hpf that did not extrude a PB-like structure than in those activated 3-4 hpf that extruded a polar body-like structure. Under the conditions tested M-phase donor cells cannot be properly remodeled after NT in cattle to trigger normal embryonic development. Our observations of chromatin structures together with DNA synthesis suggest that the failure in the development may be due to improper chromatin remodeling of mitotic nuclei after NT, which may result in chromosomal abnormalities incompatible with normal embryo development.     

Effects of sodium azide on replicative and repair DNA synthesis in barley embryos

Sodium azide inhibited protein synthesis and replicative DNA synthesis at doses used for mutation induction (0.1–1 mM) in barley embryos. During subsequent treatment for 24 h using lower doses of azide, partial recovery from this inhibition was observed, especially in the case of replicative DNA synthesis. Contrary to expectation, cysteine acted as a weak enhancer of the inhibitory effect of azide on DNA replication. Under conditions of minimal replicative DNA synthesis, DNA-repair synthesis was detected after the azide action as measured by the BND-cellulose method. Benazzmide — an inhibitor of poly(ADP-ribose)-polymerase — stimulated the azide-dependent repair synthesis approximately 2-fold.     

The effects of aphidicolin and α-amanitin on DNA synthesis in preimplantation mouse embryos

The effects of aphidicolin and α-amanitin on DNA synthesis by preimplantation mouse embryos were studied. It was found that both blastocyst and 8-cell embryos showed marked inhibition of ³H-thymidine incorporation into DNA by aphidicolin at concentrations of 20–50 μg/ml. However, aphidicolin did not inhibit the conversion of morula embryos to blastocyst embryos, although aphidicolin-treated blastocysts lost their blastocoel and collapsed into a compact form after prolonged exposure to the drug. Both 8-cell and blastocyst embryos were found to be susceptible to inhibition of DNA synthesis by α-amanitin.     

Degradation and repair of rat hepatoma cell DNA following exposure to gamma rays and methylnitrosourea]

Single-strand breaks induced in DNA of ascitic hepatoma cells by gamma-rays and N-methyl-N-nitrosourea (MNU), resp., may be effectively repaired. Double-strand breaks of DNA from MNU-treated hepatoma cells are also effectively repairable in vivo. Only a small part of double-strand breaks induced by gamma-rays in DNA of these cells is repaired in the postradiation period. The combined action of gamma-rays and MNU on the hepatoma cells causes a complete inhibition of repair of DNA and its further degradation. Under these conditions, inhibition of the repair of DNA synthesis and repression of DNA polymerase I activity is observed.     

Effect of polyamine limitation on DNA synthesis and development of mouse preimplantation embryos in vitro

In-vitro treatment of preimplantation mouse embryos with spermine and spermidine biosynthesis inhibitor, methylglyoxal-bis-(guanylhydrazone) (MGBG), arrested embryo development at the 8-cell or morula stage. In addition, the embryo DNA synthetic rate, as measured by [3H]thymidine incorporation, was strongly inhibited. The inhibition of blastocyst formation and DNA synthesis by MGBG was readily reversible by an exogenous supply of spermine and/or spermidine to the culture medium. DL-alpha-Methylornithine or DL-alpha-difluoromethylornithine (alpha-DFMO), inhibitors of putrescine biosynthesis, had no effect on embryos cultured for 1 or 2 days, but on the 3rd day embryo DNA synthesis was significantly depressed in the presence of alpha-DFMO. These observations suggest that, during early development of the preimplantation mouse embryo, spermine and spermidine are involved in regulation of embryo growth and DNA synthesis. They may also indicate a role of putrescine at a later stage of mouse embryo development.     

The inotropic action of adrenergic agonists on the heart ventricles of the chick embryo]

The effects of adrenergic drugs on the twitch tension of the electrically driven (1.2-1.5 Hz) ventricular preparations from 2-20-day old chick embryos and hatched chicks were studied. Agonists evoked positive inotropic responses of 3-day embryonic ventricles and of ventricles from older animals. 2-day embryonic ventricles were unresponsive. 5-day embryonic ventricles were most sensitive to agonists (EC50 value of adrenaline = 4.5 x 10(-9) M), while ventricles from 14-20-day old embryos had a minimal sensitivity (1-2 x 10(-9) M), while ventricles from 14-20-day old embryos had a minimal sensitivity (1-2 x 10(-7) M). The order of agonists activity (isoproterenol greater than noradrenaline greater than adrenaline much greater than phenylephrine) and the high potency of propranolol as antagonist of adrenaline indicate that responses are mediated with beta-adrenoceptors. The role of GTP-binding protein for the regulation of adrenoreactivity in embryonic chick heart during ontogenesis is discussed.