Incorporation Of 3H-uridine Research Articles

Alteration in RNA synthesis in the dorsal root ganglia of methylmercury-treated rats.

The effect of methylmercury in vivo on the incorporation of 3H-uridine in vitro into RNA of dorsal root ganglia of the rat was examined. Modified methods for the incubation of the tissue and extraction of RNA were applied to adequately determine the rate of RNA synthesis. Methylmercury significantly decreased the RNA content and RNA synthetic activity only in the symptomatic period, while uptake of the precursor into the acid-soluble pool remained unchanged. These results indicate that the previously reported inhibition of protein synthesis in dorsal root ganglia at an early phase of methylmercury intoxication was not due to impairment RNA synthesis in this tissue.

Effects of Progesterone on Nuclear Ribonucleoprotein Constituents of Endometrial Cells of the Rat

The effects of changes of progesterone concentration on 3H-uridine incorporation and on the volume or number of nuclear ribonucleoprotein particles of the rat endometrial epithelial cells are studied in vivo and in vitro. All progesterone treatments are carried out after estradiol priming and their results are compared with estradiol-treated controls sampled from the same uterus or the same pool of isolated cells. Progesterone induces the same changes, in the parameters studied, administered in vivo or in vitro. Nuclear volume undergoes small but significant increases between 1/2 and 8 h of treatment. The total number of extranucleolar ribonucleoprotein structures as well as the incorporation of 3H-uridine on non-nucleolar areas are also increased by progesterone treatments. However, the numerical density of perichromatin granules does not change during these experiments. The nucleolar volume and the incorporation of 3H-uridine in this structure are increased after an early diminution. These results indicate that progesterone induces small increases in the rate of transcription in the nucleolus and in non-nucleolar genes. The constancy of the density of perichromatin granules suggests that progesterone, in contrast to estradiol, does not affect the ratio transcription/transportation of RNA to the cytoplasm.

Immunofluorescence localization of DNA:RNA hybrids in Drosophila melanogaster polytene chromosomes.

Sites of transcriptional activity in the whole set of Drosophila melanogaster polytene chromosomes have been localized by means of fluorescent antibodies against DNA:RNA hybrid molecules and compared with results on 3H-uridine incorporation obtained earlier. The majority of large and small puffs with intensive 3H-uridine incorporation demonstrate bright fluorescence. Moreover, bright fluorescence is also observed for a large number of small puffs though the intensity of 3H-uridine incorporation is low. Some prominent puffs with high levels of 3H-uridine incorporation show weak fluorescence. Condensed bands, as a rule, do not show fluorescence. The regions that look like interbands under the light microscope are not real interbands, but consist of minibands visible only in the electron microscope (EM). However, a region that has been previously studied by EM and proven to be a real interband between two thick dark bands (100B3-100B4-5) showed fluorescence. These data support previous suggestions indicating a substantial contribution of transcriptional products from small puffs and interbands to the whole transcriptional system of polytene chromosomes.

RNA and protein synthesis of irradiated Ehrlich ascites tumour cells. I. In vivo investigations related to the cell cycle.

The effects of roentgen irradiation on the incorporation of 3H-uridine and 14C-leucine into RNA and protein and the RNA and protein contents of in vivo growing Ehrlich ascites tumour cells were studied. The results were related to changes in the composition of cells in cell cycle and compared with the synthesis of RNA and protein in cell material from various parts of the cell cycle obtained by means of elutriator centrifuging. The incorporation expressed by the ratio between acid insoluble/acid soluble activity was unchanged for RNA during the observation period up to 24 hours after a dose of 5.0 Gy. The ratio for protein was markedly decreased between 4 and 24 hours. This decrease was partly due to a decrease of the pool size of leucine as studied by changing the amounts of 14C leucine used. From these studies, the existence of at least two pools, an expandable and a non-expandable fixed pool can be concluded. There were no differences in the decrease of protein-synthesis between cells from the various parts of the cell cycle. The RNA and protein contents of the irradiated cells from various parts of the cell cycle corresponded to those of non-irradiated cells except for G1/early S-phase cells at 15 and 24 hours after irradiation. Possible reasons for this discrepancy are discussed.

Quantitative analysis of in situ hybridization methods for the detection of actin gene expression.

We have implemented an efficient, quantitative approach for the optimization of in situ hybridization using double-stranded recombinant DNA probes. The model system studied was actin mRNA expression in chicken embryonic muscle cultures. Actin and control (pBR322) probes were nick-translated with p32 labeled nucleotides, hybridized to cells grown on coverslips, and quantitated in a scintillation counter. Cellular RNA retention was monitored via the incorporation of H3-Uridine into RNA prior to cell fixation. Over a thousand samples were analyzed, and among the technical variables examined were the fixation protocol, proteolytic cell pretreatment, the time course of hybridization, saturation kinetics, hybridization efficiency, and effect of probe size on hybridization and network formation. Results have allowed us to develop a reproducible in situ hybridization methodology which is simpler and less destructive to cellular RNA and morphology than other protocols. Moreover, this technique is highly sensitive and efficient in detection of cellular RNAs. Lastly, the rapid quantitative approach used for this analysis is valuable in itself as a potential alternative to filter or solution hybridizations.

The inhibitory effects of β-momorcharin on endometrial cells in the mouse

β-Momorcharin purified from the seeds of Momordica ucharantia L. suppressed the decidual response of pseudopregnant mouse uterus to mechanical stimulus. In the β-momorcharin-treated uterus, stromal cells failed to decidualise, uterine glands distended, and a diminished cell proliferation was found in the endometrium and myometrium. β-Momorcharin also inhibited the biosynthetic activity of the cultured endometrial cells. The rate of incorporation of 3H-thymidine, 3H-uridine and 3H-leucine was all depressed by β-momorcharin. The termination of the early pregnancy in the mouse may have resulted from an inhibitory effect of the abortifacient protein on the differentiating endometrium.

Developmental changes in chromatin of skeletal muscle of rats: acetylation of chromosomal proteins and transcription

In vivo acetylation of chromosomal proteins and RNA synthesis were studied in the skeletal muscle of 3-30 day old developing rats. The levels of acetylated histones and nonhistone chromosomal (NHC) proteins are high at day 3 and decrease as development progresses. Spermine has no significant effect on acetylation. Incorporation of 3H-Uridine into RNA of both nuclear and cytoplasmic fractions is also maximum at day 3 and plateaus by day 14. Nuclear RNA synthesis following acetylation of chromosomal proteins is greatly stimulated in all the ages studied, whereas that in cytoplasmic RNA occurs only in 3 day old rats. Such modifications during early development may bring about conformational and functional changes in the chromatin and contribute significantly to the process of terminal differentiation of skeletal muscle cells.

Structure, development, and cytochemical properties of the nucleolus-associated "round body" in rat spermatocytes and early spermatids.

The "round body," a spherical structure typically associated with a nucleolus in male germ cells of the rat, has been examined in the electron microscope using routine and cytochemical methods to determine its structure, composition, and mode of development. Cytochemical analysis indicates that the round body includes neither nucleic acid nor lipid, but is composed of nonhistone protein which appears in the form of 1.6-nm-wide fibrils. Development begins in late leptotene, when a single round body appears in each spermatocyte as an irregular spheroid located along the inner surface of the nuclear envelope. During subsequent stages of the meiotic prophase, the round body leaves the nuclear envelope, becomes a regular sphere, and gradually enlarges from a diameter of 0.4 micron in leptotene to 1.6 micron in diplotene. Concurrently, lacunae appear within its substance and enlarge. At each maturation division, the amount of round-body material is decreased by about half, presumably because the constituent proteins are dissociated at metaphase, distributed between the two daughter cells at telophase, and reconstituted into half-sized round bodies. As spermiogenesis proceeds, the round body shrinks gradually and disappears at step 8. Soon after its appearance at leptotene, the round body becomes associated with and is surrounded by the pars granulosa of one of the nucleoli. Moreover, 3H-uridine incorporation into nucleolar RNA is high as long as the size of the round body increases, but is low or absent when it decreases. It is possible, therefore, that the round body exerts some control on nucleolar activity in meiotic cells.

Electron microscopic localization of acridine orange binding to DNA within rat astrocytoma C6 cells.

Combination of autoradiography and ultracytochemical acridine orange method in rat astrocytoma C6 cells is described. Electron microscopic examination revealed that AO binds to DNA exclusively within the euchromatin portion of the cell nucleus. X-ray microanalysis showed that AO reaction products contain lead, uranium, and osmium. These heavy metals seem to contribute to electron density of the AO reaction products. The percentage of AO positive cells was 51.8 +/- 1.9%, whereas average labeling index of C6 cells incubated with 3H-thymidine for 30 minutes was 25.6 +/- 1.7%. About 82% of 3H-thymidine-labeled cell nuclei were AO positive and the remaining 18% were free of AO reaction products. On the other hand, about 27% of AO positive cells were not labeled with 3H-thymidine. However, most of C6 cell nuclei with active 3H-uridine incorporation showed AO chromatin reaction products. The results suggest that visualization of AO chromatin reaction products might be related to RNA synthesis rather than DNA synthesis in the cell nuclei.

In vitro effects of insulin on macromolecular events in newt limb regeneration blastemata.

This work provides data demonstrating a stimulatory effect of insulin on macromolecular events occurring in cultured regeneration blastemata and demonstrates a synergistic interdependence between nerves and insulin in newt limb regeneration. The current experiments provide evidence for the following: (1) Insulin is paramount for expression of the mitogenic effect of nerves on cultures blastemata. (2) Insulin stimulates the incorporation of (3H)uridine into the acid-insoluble fraction of blastemal homogenates, but it does not alter the turnover rate of incorporated labeled uridine. (3) Insulin also stimulates the incorporation of 35SO4 and (3H)leucine into both chondroitinase-sensitive and chondroitinase-resistant blastemal proteoglycans. (4) Insulin increases the uptake of radiolabeled precursors by the blastemata, namely, (3H)leucine, (3H)uridine, 35SO4, (3H)alpha-aminoisobutyrate, and (3H)2-deoxy-D-glucose. The importance of insulin in the regulation of newt limb regeneration is discussed.

Intranuclear defect in beta-globin mRNA accumulation due to a premature translation termination codon

We have analyzed a cloned beta O-thalassemia (beta O-thal) gene from a patient doubly heterozygous for hemoglobin Lepore and beta O- thalassemia. Studies of 3H-uridine incorporation into beta-globin mRNA in this patient's erythroblasts suggested an intranuclear defect in both beta and Lepore (delta beta) mRNA synthesis, as did S1 nuclease analysis of nuclear RNA. However, the nucleotide sequence of the beta O- thal gene revealed only a single base change in codon 39 (CAG----UAG), which created a premature translation termination codon. The 5′ flanking sequence, including transcription promotor boxes and the mRNA initiation (CAP) site, were normal. The unexpected effect of this mutation on intranuclear beta-mRNA synthesis in vivo was studied by insertion of the cloned gene into a plasmid expression vector and transfection into tissue culture (COS-1) cells. beta-Globin mRNA produced by the transfected cells was assessed by S1 nuclease analysis. The beta O-39 thalassemia gene generated five- to tenfold less beta- mRNA than a normal beta-gene in both nuclear and cytoplasmic RNA, simulating the results observed in vivo. Moreover, the small amount of beta O-39 mRNA produced was as stable as normal beta-mRNA during an actinomycin D chase, ruling out rapid cytoplasmic turnover as a cause of the reduced accumulation. Cotransfection of the beta O-39 thalassemia gene with a mutant tyrosine suppressor tRNA gene resulted in restoration of the beta O-39 mRNA accumulation to near-normal levels. On the basis of these results, we suggest that the low levels of beta-mRNA known to exist in the common form of beta O-thalassemia, beta O-39 thalassemia, result from a lesion in transcription, or early posttranscriptional processes; the defect appears to be corrected by restoration of proper translational potential to the mutant mRNA, at least in a gene transfer-expression system in tissue-culture cells.

Intranuclear Defect in β-Globin mRNA Accumulation Due to a Premature Translation Termination Codon

We have analyzed a cloned beta O-thalassemia (beta O-thal) gene from a patient doubly heterozygous for hemoglobin Lepore and beta O-thalassemia. Studies of 3H-uridine incorporation into beta-globin mRNA in this patient's erythroblasts suggested an intranuclear defect in both beta and Lepore (delta beta) mRNA synthesis, as did S1 nuclease analysis of nuclear RNA. However, the nucleotide sequence of the beta O-thal gene revealed only a single base change in codon 39 (CAG----UAG), which created a premature translation termination codon. The 5' flanking sequence, including transcription promotor boxes and the mRNA initiation (CAP) site, were normal. The unexpected effect of this mutation on intranuclear beta-mRNA synthesis in vivo was studied by insertion of the cloned gene into a plasmid expression vector and transfection into tissue culture (COS-1) cells. beta-Globin mRNA produced by the transfected cells was assessed by S1 nuclease analysis. The beta O-39 thalassemia gene generated five- to tenfold less beta-mRNA than a normal beta-gene in both nuclear and cytoplasmic RNA, simulating the results observed in vivo. Moreover, the small amount of beta O-39 mRNA produced was as stable as normal beta-mRNA during an actinomycin D chase, ruling out rapid cytoplasmic turnover as a cause of the reduced accumulation. Cotransfection of the beta O-39 thalassemia gene with a mutant tyrosine suppressor tRNA gene resulted in restoration of the beta O-39 mRNA accumulation to near-normal levels. On the basis of these results, we suggest that the low levels of beta-mRNA known to exist in the common form of beta O-thalassemia, beta O-39 thalassemia, result from a lesion in transcription, or early posttranscriptional processes; the defect appears to be corrected by restoration of proper translational potential to the mutant mRNA, at least in a gene transfer-expression system in tissue-culture cells.

The mitogenic principle of [formula omitted][formula omitted] lipoprotein: B-lymphocyte mitogenicity of the synthetic analogue Palmitoyl-tetrapeptide (PAM-SER-SER-ASN-ALA)

N-Palmitoyl-(S)-seryl-(S)-seryl-(S)-asparaginyl-(S)-alanine (Palmitoyl-tetrapeptide) is an analogue of the N-terminal part of the lipoprotein from the outer membrane of Escherichia coli . It was prepared by chemical synthesis and tested for biological activity in in vitro lymphocyte culture systems. In spleen cells of the inbred mouse strains C3H HeJ , C3H/He/Bom/nunu, and Balb c , the compound exhibited stimulatory activity towards B-lymphocytes comparable to the effect of native lipoprotein, as measured by the incorporation of 3H-thymidine and 3H-uridine, and by a hemolytic plaque assay. The B-lymphocyte tumor cell line BCL1 was also activated by the compound. The results demonstrate, that the N-terminal tetrapeptide moiety of lipoprotein, linked to a lipophilic molecule, constitutes by itself a novel B-lymphocyte mitogen.

Altered biochemical responses by rat Sertoli cells and peritubular cells cultured under simulated diabetic conditions.

The purpose of the present study was to determine whether the secretion of androgen-binding protein and lactate by 16-day-old rat Sertoli cells is altered when these cells are cultured in simulated diabetic conditions. Incorporation of 3H-uridine into RNA by peritubular cells cultured in simulated diabetic conditions was also studied. The cells were exposed to various concentrations of glucose, beta-hydroxybutyrate, sodium bicarbonate (to alter the pH of the medium), mannitol (to influence the osmolarity of the medium), or a combination of these compounds. All of the metabolic parameters when tested alone or in combination were capable of increasing lactate secretion by Sertoli cells above control values. Basal secretion of androgen-binding protein, however, was not influenced by an individual component or when all components were tested together. FSH-stimulated levels of androgen-binding protein secretion was depressed only when the Sertoli cells were exposed to all the components simultaneously. Incorporation of uridine by peritubular cells was decreased by exposing the cells to butyrate or mannitol, while no effect was observed with glucose treatment. These results indicate that Sertoli cell and peritubular cell function can be directly altered by several specific metabolic parameters associated with diabetes.

Prediction of the response to chemotherapy in acute leukemia by a short-term test in vitro.

To detect sensitivity or resistance of leukemic cells to chemotherapy prior to treatment, a short-term incubation method was employed. Blast cells from the peripheral blood or bone marrow of adult patients presenting with different forms of acute leukemia were analyzed for in vitro responsiveness to anticancer drugs in terms of suppression of 3H-uridine incorporation into cellular nucleic acids. The following agents were tested over a wide range of concentrations: Adriamycin, cytosine arabinoside, thioguanine, 6-mercaptopurine, prednisone, and 4-hydroperoxycyclophosphamide. Retrospectively, the in vitro data were compared to the clinical response of the patients to polychemotherapy. In the majority of the patients, in vitro cytotoxic effectiveness of Adriamycin (doxorubicin) and cytosine arabinoside reflected the in vivo situation. The levels of in vitro inhibition that could distinguish between drug-sensitive and drug-resistant diseases appeared to be 30% for Adriamycin and 20% for cytosine arabinoside. No correlation was found between the Adriamycin effect in vitro and the proliferative state (rate of 3H-thymidine incorporation) of the leukemic cell population. Serial in vitro sensitivity testing during the course of disease of various patients proved the ability of the test system to detect acquired resistance to chemotherapeutic agents. Therefore, the assay might serve as a reliable tool in the selection of effective chemotherapy in individual patients suffering from acute leukemia.

Effects of cannabinoids on macromolecular synthesis in isolated spermatogenic cells.

Effects of delta 9-tetrahydrocannabinol (THC), cannabinol (CBN) and cannabidiol (CBD) (1, 3 or 5 microM for 2 h) on macromolecular synthesis were investigated in homogeneous populations of pachytene spermatocytes and round spermatids obtained from CD-1 mice. Incorporation of 3H-uridine into the acid-insoluble fraction was used as an index of RNA synthesis. Treatment of pachytene spermatocytes with 5 microM THC, CBN and CBD reduced radioactive incorporation by 81%, 62% and 67%, respectively, compared to vehicle (dimethylsulfoxide) controls. Treatment of round spermatids with 5 microM THC, CBN and CBD reduced radioactive incorporation by 54%, 61% and 63%, respectively, compared to controls. In addition to reduction of radioactive incorporation into the acid-insoluble fraction, there was also reduction in the acid-soluble fraction not comparable in magnitude to the reduction in the acid-insoluble fraction. Pachytene spermatocytes and round spermatids isolated from THC-treated mice (10 and 20 mg/kg) showed no reduction in macromolecular synthesis compared to cells isolated from vehicle-treated mice. Suppression of macromolecular synthesis induced by in vitro cannabinoid treatment is discussed in terms of membrane-mediated interference with cell metabolism.

Methylxanthines inhibit nucleologenesis in binucleate cells

Roots of Zea mays L. were treated for 2 hr with 0.1% solutions of caffeine, isobutylmethylxanthine (IBMX) or 8-ethoxycaffeine (8-EOC) or with 0.01% colchicine. These treatments produced two types of marked cells in which to study nucleologenesis in early interphase, i.e. binucleate cells and mononucleate tetraploid cells. Nucleoli were stained with Fast Green or azure B. The mean sum of nucleolar volumes was similar, at 2 hr, in binucleate and mononucleate tetraploid cells but after a 6 hr recovery period the mean nucleolar volumes had increased much more in tetraploid cells than in binucleate cells. It is suggested that methylxanthines inhibit nucleologenesis: this parallels the inhibition of both cell and nuclear growth by these drugs. Roots of Pisum sativum L. were treated with 0.1% caffeine to induce binucleate cells and incorporation of 3H-uridine in 1 hr was determined. Treatment with 0.01% colchicine for 2 hr before exposure to 3H-uridine resulted in a 21% increase in mean grain count per binucleate cell: colchicine stimulates RNA synthesis in binucleate cells.

Effects of Coal Tar Dyes on Viability, and RNA and Protein Syntheses in Isolated Rat Hepatocytes

The effects of Amaranth, Ponceau 3R, Sunset Yellow FCF, and Rose Bengal on viability and RNA and protein syntheses were studied in suspensions of viable rat hepatocytes. Liver parenchymal cells were enzymatically prepared from normal adult rats by perfusion of the liver with buffer solution of 0.05% collagenase, followed by shaking of liver slices in the same solution. The cell yield was 60.5±15.8×106cells/g liver and the cell viability index was 87.0±2.4%. When the isolated hepatocytes were incubated in the presence of coal tar dyes at a concentration of 1mM for 4hr, a decrease in the cell viability was observed. In particular, the Rose Bengal-treated fraction showed a rapid decrease of the cell viability in comparison with other dye-treated fractions. 3H-Uridine incorporation into RNA was stimulated in the presence of Amaranth or Ponceau 3R. On the other hand, none of the coal tar dyes tested in this study had any effect on 14C-isoleucine incorporation into protein. These results suggest that the system of isolated hepatocytes may be a good model for studying the mechanism of action of toxic chemicals.

Modulation of glucocorticoid effect in thymus of adrenalectomized-diabetic rat.

Cellular receptors to glucocorticoids and the effect of dexamethasone and corticosterone on 3H-uridine incorporation into the acid-insoluble fraction were studied in thymocytes from adrenalectomized-diabetic rats and adrenalectomized ones. An increase in the dissociation constant of the hormone-receptor plot was observed when adrenalectomized-diabetic animals were compared to adrenalectomized controls. Meanwhile decreasing concentrations of dexamethasone and corticosterone below receptors saturation, were less active as inhibitor of 3H-uridine incorporation into RNA by thymocytes from adrenalectomized-diabetic than from adrenalectomized ones.

Viability of Stored Pollen Grains ofMalus DomesticaBorkh as Evaluated by the Incorporation of Labelled Nucleic Acid Precursors

SUMMARYViability of stored pollen grains of Malus domestica Borkh, cultivars «Golden Delicious» and «Starkrimson», is expressed as: 1) percentage of labelled generative and vegetative cells after feeding with 3H-thymidine and 3H-uridine; 2) percentage of germinated pollen grains. The ability to incorporate nucleic acid precursors decreases during storage; the vegetative cytoplasm response to storage is slower than the nuclear response, in respect to both 3H-thymidine and 3H-uridine incorporation. The germination ability is not influenced by the investigated storage. No relationship between the capacity of incorporating nucleic acid precursors and pollen grains germination is therefore detectable.