Incorporation Of 3H-uridine Research Articles

EFFECTS OF ACTINOMYCIN D ON LOCALIZATION OF ACRIDINE ORANGE CHROMATIN INTERACTION COMPLEX IN RAT ASTROCYTOMA C6 CELLS

The purpose of this study was to examine the effects of actinomycin D on localization of acridine orange (AO) binding to DNA in rat astrocytoma C6 cells and to discuss briefly the significance of AO chromatin interaction products. Actinomycin D markedly inhibited 3H-uridine incorporation into RNA and the percentage of AO positive cells was reduced to approximately 40% of that of the untreated control cells, whereas no distinct decrease of 3H-thymidine incorporation was induced by actinomycin D. Electron microscopic radioautography combined with the AO ultracytochemistry revealed that silver grains indicating binding of 3H-actinomycin D are located mostly over the euchromatin portion near the segregated nucleolus and heterochromatin and that no or only a few AO chromatin complex was found in nuclei labeled heavily with 3H-actinomycin D. These results of the present study together with the results of the previous studies seem to indicate that AO might selectively bind to active or derepressed DNA template sites for DNA dependent RNA polymerase in the euchromatin portion of the cell nucleus.

Mode of action of the antimicrobial compound 5-bromo-5-nitro-1,3-dioxane (bronidox).

The mode of action of the antimicrobial agent, 5-bromo-5-nitro-1,3-dioxane (bronidox), was studied in detail for gram-positive and gram-negative bacteria, yeast and fungi. The studies included MIC testing, thiol inhibition of activity, intracellular leakage, oxygen consumption, incorporation of 3H-uridine, scanning electron microscopy, inhibition of enzyme activity (papain) and in vitro oxidation of thiols to disulfides. It appears that the primary mode of action of bronidox is the same as, or similar to, that of bronopol, i.e. the oxidation of essential protein thiol causing inhibition of enzyme activity and subsequent inhibition of microbial growth.

Eimeria tenella: parasite-specific incorporation of 3H-uracil as a quantitative measure of intracellular development.

An assay has been developed using parasite-specific incorporation of 3H-uracil to assess the intracellular growth of Eimeria tenella in vitro. As shown by both scintillation counts and autoradiography, 3H-uracil was incorporated specifically into intracellular parasites from the onset of infection and continued throughout development of the first generation schizonts. Mature schizonts and first generation merozoites did not continue to incorporate additional 3H-uracil, indicating that RNA synthesis had halted in these stages. Based on these findings, a semi-automated microscale uracil incorporation assay was developed to determine parasite viability. This method should be useful for biochemical studies with intracellular parasites and for screening compounds for anticoccidial activity. The ease, rapidity, and quantitative nature of this assay contrasts favorably with standard morphometric approaches of determining parasite development. In addition, parallel studies using host cell incorporation of 3H-uridine have been introduced as a method of determining whether antiparasitic activity is direct or indirect in relation to effects on the host cell.

Autoradiography of 3H-uridine incorporation in the normal early blastocysts of cattle

RNA synthesis in morphologically normal early bovine blastocysts collected 7 days after insemination was investigated using fine-structure autoradiography after 3H-uridine incorporation. The level of autoradiographic labelling is proposed as a criterion for assessing metabolic activity in the individual cells of the blastocyst. There was a detectable difference between the cells of the trophoblast and those of the inner cell mass (higher labelling), indicating a divergence of metabolic patterns according to blastomere differentiation. The occasional absence of nuclear labelling was correlated with cell degeneration. A low nuclear labelling was observed mainly in cells sequestered in the subzonal space or in the blastocoele cavity, respectively. Estimation of RNA synthesis in different blastomeres by fine structure autoradiography opens the way to a better interpretation of the relationship between morphology and metabolism during the development of early cow embryos.

Effect of diquat on cell growth and macromolecule synthesis in cultured pneumocytes.

The effects of diquat (DQ) on cell growth and macromolecule synthesis were investigated in cultured rat, feline and human. pneumocytes. DQ at 10(-3) M and 10 (-5) M showed time- and dose-dependent inhibition of cell growth for 7 days. The incorporation of 3H-thymidine, 3H-uridine and 14C-leucine into DNA, RNA and protein was reduced to 0-47% by 10(-3) M of DQ, and that of 14C-leucine into protein of A-549 cells was reduced to 67% by 10(-5) M of DQ. Among the cells examined, A-549 cells derived from human lung were the most resistant to the inhibitory effects of DQ on cell growth and macromolecule synthesis. On the other hand, human lung embryonic fibroblast cells and L-2 cells derived from rat lung were the most sensitive to the toxicity of DQ. These results indicate that the inhibitory effects of DQ on cell growth and on macromolecule synthesis are dependent on the concentrations of DQ administrated, and that species differences in the sensitivity to DQ toxicity may exist.

Effects of paraquat on macromolecule synthesis in cultured pneumocytes.

The effects of paraquat (PQ) on cell growth, DNA, RNA and protein synthesis and on superoxide dismutase (SOD) activity were investigated in cultured pneumocytes of type II cell origin and human embryonic fibroblast cells of the lung. The incorporation of 3H-thymidine, 3H-uridine and 14C-leucine into DNA, RNA and protein, respectively, were all reduced to 25%-70% of the control by PQ at 10(-3) M, but not at 10(-5) M nor 10(-7) M. The activity of SOD in the cells was increased to 130%-270% by 10(-3) M of PQ. Among the cells studied, A-549 cells, which were most resistant to the inhibitory effect of PQ on cell growth and the synthesis of DNA, RNA and protein had the highest induction of SOD activity by PQ. In contrast, L-2 cells in which the cell growth and the synthesis of nucleic acids and protein were most inhibited had the lowest induction of SOD activity by PQ. These results indicate that nucleic acids and protein synthesis are possible targets for lethal effects of PQ in the pulmonary cells, and that the specificity of PQ toxicity in pulmonary cell lines might be related to the ability of induction of SOD by PQ.

The Activation of Calcium/Phospholipid Dependent Protein Kinase and the Association with Interleukin-2 Production

The thymic leukemia cell line EL4 has been shown to produce the lymphokine Interleukin-2 (IL-2) following stimulation with phorbol ester (PMA). We investigated intracellular enzyme pathways triggered by phorbol stimulation using an EL4 cell line which responds to PMA with IL-2 synthesis (EL4r) and one which does not produce IL-2 following stimulation (EL4nr). By comparing these two cell lines we hoped to establish which enzyme activities were associated with IL-2 synthesis. The enzyme pathways studied included calcium/phospholipid dependent protein kinase (C-kinase) activity, the induction of polyamine synthesis, RNA, DNA and protein synthesis and finally IL-2 production. Our results indicate that both EL4 cell lines have a receptor for PMA, which can activate the C-kinase enzyme. Further, in both cell lines PMA activates the nuclear synthesis of polyamines as demonstrated by ornithine decarboxylase induction. Both RNA and protein synthesis measured by 3H-uridine and 3H-leucine uptake respectively appear comparable between EL4r and EL4nr. The only difference in cellular responsiveness between EL4r and EL4nr was in the 3H-thymidine uptake, and IL-2 production. IL-2 production or lack of production was established by 3H-uridine and 3H-thymidine incorporation as well as viable cell count using the IL-2 dependent cell line CTLL-2. We, therefore, conclude that EL4r and EL4nr cells show similar intracellular responses to phorbol ester except for 3H-thymidine uptake and detectable IL-2 production. Our results suggest that failure of PMA-stimulated EL4nr cells to produce IL-2 is either due to inability of this cell line to synthesize IL-2 or the production of defective IL-2. It is not due to failure of PMA to activate C-kinase or the subsequent nuclear events.

No loss of estrogenic or anti-estrogenic activity after demethylation of droloxifene (3-OH-tamoxifen).

The binding affinity of 3-OH-Tamoxifen (Droloxifene or DROL) and N-demethyl-droloxifene (ND-DROL) to the cystosolic estrogen receptor of rabbit uteri was 10 times higher than that of Tamoxifen. Both compounds exhibited similar stimulation (estrogenic effect) and inhibition (anti-estrogenic effect) of uterine growth of immature female rats. 3H-Uridine incorporation into the RNA of MCF-7 and ZR-75 cells as a measure of anti-estrogenic activity was equally inhibited by concentrations of 0.05-1.0 mumol/l of both compounds. Thus, the pharmacological properties of DROL were not changed by N-demethylation.

In vitro activity of alkylating agents on human tumors as measured by a short-term antimetabolic assay.

The activity of some alkylating agents widely used in clinical practice was studied by an in vitro antimetabolic assay, which evaluates the interference of drugs on the incorporation of 3H-thymidine and 3H-uridine in short-term cultures of human tumors. The effect of CCNU, chlorambucil, cisplatin, melphalan, prednimustine, procarbazine and the pro-active derivatives of cyclophosphamide and ifosfamide, 4-hydroperoxycyclophosphamide and 4-hydroperoxyifosfamide, was tested on non-Hodgkin lymphomas, germ cell testicular tumors, breast and ovarian cancers. Similar effects were generally produced by the drugs on both DNA and RNA precursor incorporation, and the in vitro response rates were similar to those clinically obtained for the same tumor types by using the same drugs in monochemotherapy. The effects of different alkylating agents on the individual tumors were not significantly associated, except for the analogues 4-hydroperoxycyclophosphamide and 4-hydroperoxyifosfamide, for which a significant agreement rate was observed. No evidence of acquired resistance to 4-hydroperoxycyclophosphamide and cisplatin after clinical treatment with the same drug was found in samples from ovarian cancer and non-Hodgkin lymphoma, but there was a definite trend in germ cell testicular tumors to lower sensitivity to cisplatin in previously treated patients.

Sensitivity of Preimplantation Mouse Embryos to Abrin

The effect of the plant toxin abrin on preimplantation mouse embryos in vitro was studied. Two-cell embryos from C57BL/6J or B6CBA/F1/Bom female X B6CBA/F1/Bom male mice were exposed to abrin for 72 hrs in vitro, in medium containing abrin in concentrations ranging from 0.1 to 10,000 pg/ml. Two-cell mouse embryos were also exposed to corresponding concentrations of ricin in vitro. The effect of abrin was evaluated by daily morphological observation of the embryos up to the blastocyst stage and by measuring DNA, RNA and protein synthesis by radioactive precursor incorporation. The effect of ricin was evaluated by morphological observation of the embryos up to the blastocyst stage. The results showed that 2-cell mouse embryos were highly sensitive to abrin, 1 pg/ml being the lowest concentration of the toxin that significantly inhibited the development of 2-cell embryos to the blastocyst stage. With ricin statistically significant inhibition of blastocyst formation was obtained at a concentration of 10 pg/ml. The ID50 for abrin was calculated to be 24 pg/ml and for ricin 47 pg/ml. A dose-dependent lag period was observed before the effect of abrin or ricin on the formation of blastocysts became evident. Incorporation of 3H-thymidine, 3H-uridine and 14C-leucine into DNA, RNA and protein, respectively, was also inhibited by abrin after different time intervals in culture.

DNA, RNA, and Protein Synthesis by Porcine Oocyte-Cumulus Complexes during Expansion1

The experiments described in this report were designed to determine three biosynthetic functions of oocyte-cumulus complexes during expansion. The events investigated were DNA, RNA, and protein synthesis during a 24-h in vitro culture; these were determined by 3H-thymidine, 3H-uridine, and 3H-leucine incorporation into oocyte-cumulus complexes, respectively. The quality of proteins produced was also determined by slab-gel electrophoresis. Results indicated that, during follicle-stimulating hormone-induced cumulus expansion, total DNA synthesis was significantly (P less than 0.05) reduced whereas RNA synthesis remained unchanged. Overall protein synthesis was markedly increased (P less than 0.05), with one major band (Mr = 22,000) and two minor bands (Mr = 19,500 and 78,000) being produced during expansion.

Reliability of an in vitro short-term assay to predict the drug sensitivity of human breast cancer.

The feasibility and reliability of an in vitro assay that evaluates drug interference on nucleic acid precursor incorporation were investigated on 135 previously untreated locally advanced breast cancers. The assay, which was carried out on tumor fragments incubated for 3 hours with drugs, proved to be feasible on a sufficiently high percentage of biopsy specimens (70%) for routine clinical use. In vitro drug activity evaluated with this assay appeared to reproduce the clinical patterns of sensitivity of the tumor type as well as of the individual tumors. In fact, in vitro response rates to conventional agents resembled the clinical response rates reported for the same agents used in monochemotherapy. From a retrospective--correlative study carried out on 41 patients treated in vitro and in vivo with the same drugs (Adriamycin [doxorubicin] and vincristine), in vitro effect of Adriamycin on 3H-uridine incorporation appeared significantly correlated with clinical response (overall agreement, 78%; P = 0.0032) with specific agreements of sensitivity and resistance of 75% and 81%, respectively.

Accumulation of RNA in blastocysts during embryonic diapause and the periimplantation period in the western spotted skunk

The in vivo incorporation of 3H-uridine into RNA was studied in delayed implanting and activated blastocysts obtained from 33 western spotted skunks. 3H-uridine was incorporated into RNA by all blastocysts; however, significantly more label was incorporated as blastocyst diameter increased. Activated blastocysts with diameters of 1.6 mm or greater on average incorporated 65 times more 3H-precursor in 5 hr than diapausing blastocysts with diameters of 1.1 mm or less. Polyadenylated RNA was likewise synthesized by delayed implanting and activated skunk blastocysts; however, the proportion of polyadenylated RNA synthesized by the former was greater than in the latter (43.9% vs. 27.5%). Our data suggest that the transition from embryonic diapause to fully activated blastocysts first occurs gradually for several days before entering a 1-2-day period of rapid development characterized by an abrupt increase in RNA accumulation.

Evidence for the mode of antischistosomal action of hycanthone

Evidence is presented which supports the hypothesis that the mode of action, or a slight variant thereof, suggested by Hartman and Hulbert (11) to account for the mutagenic effects of hycanthone (HC) is the mechanism whereby HC exerts its antischistosomal activity. HC is metabolically activated to a reactive ester which, upon dissociation, alkylates DNA. If resistant schistosomes are unaffected because they cannot convert HC to a reactive ester they should be killed upon direct exposure to an appropriately esterified drug. Hycanthone N-methylcarbamate (HNMC) was synthesized and shown to bind to DNA and also alkylate 4-(p-nitrobenzyl)-pyridine. When tested with schistosomes kept in vitro, HNMC caused an irreversible inhibition of 3H-uridine incorporation not only in sensitive. S. mansoni (as HC does) but also in HC-resistant and inmature S. mansoni worms and S. japonicum worms which are only transiently inhibited by HC. After in vitro contact with HNMC for 1 h both sensitive and resistant schistosomes died in three weeks if either kept in culture or re-transplanted into the host animal. Mice infected with HC-resistant schistosomes showed a drastic worm reduction after in vivo HNMC administration.

RNA content of normal and axotomized retinal ganglion cells of rat and goldfish

The responses of rat and goldfish retinal ganglion cells to axotomy were examined by a quantitative cytochemical method for RNA and by morphometric measurement 1-60 (rat) and 3-90 (goldfish) days after interruption of one optic nerve or tract intracranially. Unoperated control animals were studied also. The RNA content of axotomized neurons of rat fell 7-60 days postoperatively. Additionally, atrophy of the axotomized somas occurred. Over time, neuronal atrophy approximately paralleled the loss of RNA, and mean cell area and RNA content were reduced by about 25% 60 days after axotomy. Incorporation of 3H-uridine by axotomized neurons declined also. Axotomized retinal ganglion cells of goldfish behaved differently from those of the rat and showed increases in RNA content, most conspicuously 14-60 days postoperatively. Enlargement of axotomized fish neurons occurred but was less proportionately than concomitant increases in RNA content. The nonaxotomized ganglion cells of goldfish displayed statistically significant increases in size and RNA content 14-49 days after unilateral optic nerve or tract lesions. In contrast, alterations in rat retinal ganglion cells contralateral to interruption of one optic nerve were of limited and questionable significance. The contrasting reactions to axotomy by the retinal ganglion cells of these two vertebrates, one of which regenerates optic axons and one of which does not, may support the proposition that the somal response to axon injury has an important bearing upon the success or failure of CNS regeneration.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of 3H-uridine incorporation and messenger RNA synthesis in human monocytes activated to secrete alpha interferon or monocyte-derived fibroblast growth factor.

Human monocytes are multifaceted cells with a wide range of immunoregulatory functions and distinct secretory products. This manuscript reports on initial attempts to identify specific early macromolecular synthetic events associated with various types of human monocyte activation by observing the patterns of RNA synthesis displayed by human monocytes that are exposed to well characterized activating stimuli. It was found that muramyl dipeptide (MDP), an activator of monocyte-derived fibroblast growth factor (MD-FGF) release from monocytes, also stimulates a reproducible increase in human monocyte total 3H-uridine incorporation and cytoplasmic messenger RNA (mRNA) synthesis at 4 h following activation. In contrast, polyriboinosinic acid:polyribocytidilic acid (poly I:C), an excellent stimulator of monocyte alpha interferon (IFN alpha) release, did not cause a change in either 3H-uridine incorporation or cytoplasmic mRNA production at any of the time points tested. Poly I:C was also found to be a poor stimulator of MD-FGF release. Conversely, MDP did not stimulate any detectable IFN release from human monocytes. The discrepancy between the patterns of macromolecular synthesis observed in human monocytes activated to secrete MD-FGF as compared with IFN indicates that divergent postactivation control mechanisms may be operative at the RNA level in the monocyte following activation of these two distinct functions.

Effect of gonadotropin releasing hormone on ventral prostate of rat.

Testosterone induced increase in ornithine decarboxylase (ODC) activity was inhibited by simultaneous treatment with gonadotropin releasing hormone (GnRH) and its analogue in the ventral prostate of rat. Inhibition of 3H-uridine, 3H-phenylalanine and 3H-leucine incorporation into TCA precipitable material was also inhibited by GnRH in the dihydrotestosterone (DHT) treated animals. These studies further confirm that GnRH acts directly on ventral prostate and causes inhibitory effects.

Effects of aphidicolin and α-amanitin on visualization of acridine orange binding to DNA in rat astrocytoma C6 cells

The purpose of this study was to examine effects of aphidicolin and alpha-amanitin on visualization of acridine orange (AO) binding to DNA in rat astrocytoma C6 cells and to discuss briefly the significance of AO chromatin interaction products. Aphidicolin inhibited DNA synthesis but percentage of AO positive cells was approximately 60% of that of the untreated control cells. alpha-Amanitin caused a slight inhibition of RNA synthesis and 3H-uridine incorporation in the treated cells was about 64% of that of the untreated cells, whereas a distinct decrease of the number of AO positive cell nuclei was observed. The results suggest that activity of RNA polymerase II and mRNA synthesis is mainly concerned in visualization of AO chromatin interaction products.

Differentiation of nuclear structure during the sexual cycle in Tetrahymena thermophila: I. Development and transcriptional activity of macronuclear anlagen

During the postzygotic period of the sexual cycle (conjugation) in the ciliated protozoan Tetrahymena, transcriptionally active somatic macronuclei develop from descendants of the germinal micronuclei, which are mostly transcriptionally inactive. This nuclear differentiation process is accompanied by the amplification of ribosomal genes, genome polyploidization, and reorganization. In this context, the developmental sequence of macronuclear anlagen represents an interesting system for the study of finestructural aspects of nuclear differentiation in combination with an analysis of the onset and location of transcriptional activity. From our data, the differentiation of the fine structure of macronuclear anlagen can be divided into three distinct stages. During the first stage, the anlagen appear very electron lucent and exhibit a loose, fine fibrillar substructure (achromatic stage). 3H-Uridine incorporation was low, and the autoradiographic label was evenly distributed over the nuclear areas. During the second stage, specific nuclear regions showing arrays of small, condensed flocculent patches could be seen. In such areas, primary nucleolar structures are obviously formed; these structures initially only consist of fibrillar components and show very little or no transcriptional activity. During the last stage of differentiation, the macronuclear anlagen attain a fine structure similar to that exhibited by macronuclei in starved vegetative cells. The fibrillogranular substructure becomes more dense, and typical heterochromatic chromatin bodies appear. The multiple extrachromosomal nucleoli are exclusively located at the periphery of the nuclei and organized as either single units or nucleolar aggregates containing a cortex of granular components. The autoradiographic label was dense and clearly concentrated over the nucleoli. The combined fine-structural and autoradiographic data indicates that, during the first stage of macronuclear differentiation, only nonribosomal RNA transcription occurs and that there is a lag phase between the first appearance of nucleolar structures and the expression of the amplified ribosomal genes.

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

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