Incorporation Of 14C-uridine Research Articles

Beta-adrenoceptor stimulation attenuates the hypertrophic effect of alpha-adrenoceptor stimulation in adult rat ventricular cardiomyocytes

The study investigated whether beta-adrenoceptor antagonists augment the hypertrophic response of cardiomyocytes evoked by norepinephrine. In adult ventricular cardiomyocytes, stimulation of alpha- but not beta-adrenoceptors induces myocardial hypertrophy. Natural catecholamines, like norepinephrine, stimulate simultaneously alpha- and beta-adrenoceptors. We investigated whether beta-adrenoceptor stimulation interferes with the hypertrophic response caused by alpha-adrenoceptor stimulation. Adult ventricular cardiomyocytes isolated from rats were used as an experimental model. Hypertrophic parameters under investigation were stimulation of phenylalanine incorporation and protein mass, stimulation of 14C-uridine incorporation and RNA mass, and increases in cell shape. Norepinephrine (0.01 to 10 micromol/liter) increased concentration-dependent phenylalanine incorporation; pEC50 value was 5.9 +/- 0.1 (n = 8). The alpha1-adrenoceptor antagonist prazosin (0.1 micromol/liter) suppressed norepinephrine-induced increase in rate of protein synthesis. Conversely, propranolol (1 micromol/liter) and the beta1-adrenoceptor selective antagonists CPG 20712A (300 nmol/liter) or atenolol (1 micromol/liter) augmented increases in phenylalanine incorporation caused by norepinephrine. Addition of the beta2-adrenoceptor antagonist ICI 118,551 (55 nmol/liter) did not influence the hypertrophic effect of norepinephrine. Atenolol augmented the norepinephrine-induced increases of all hypertrophic parameters investigated (i.e., protein mass, uridine incorporation, RNA mass, cell volume, and cross-sectional area). In the presence of norepinephrine, inhibition of beta1-adrenoceptors increased the amount of protein kinase C-alpha and -delta isoforms translocated into the particulate fraction. The effect of pharmacological inhibition of beta1-adrenoceptors could be mimicked by Rp-cAMPS (adenosine-3', 5'-cyclic phosphorothiolate-Rp). The inhibitory effect of beta1-adrenoceptor stimulation on the alpha-adrenoceptor-mediated effect persisted in cardiomyocytes isolated from hypertrophic hearts of rats submitted to aortic banding. In isolated ventricular cardiomyocytes from rats, beta1-adrenoceptor stimulation attenuates the hypertrophic response evoked by alpha1-adrenoceptor stimulation.

In Vitro Temperature Sensitivity of DNA, RNA, and Protein Syntheses Throughout Puberty in Human Testis

To evaluate the optimal temperature for DNA, RNA, and protein syntheses in the prepubertal, pubertal, and postpubertal human testis, the levels of incorporation of 3H-thymidine, 14C-uridine, and 14C-leucine into cultured testicular tissue were studied at 28 degrees C, 31 degrees C, 34 degrees C, 37 degrees C, 40 degrees C, and 43 degrees C. The maximum level of 3H-thymidine incorporation in prepubertal testes occurred at 37 degrees C, whereas the maximum incorporation in the pubertal and postpubertal testes occurred at 31 degrees C. Incorporations of 14C-uridine and 14C-leucine in the three groups were temperature dependent (28 degrees C to 37 degrees C). DNA synthesis by germ cells in the pubertal and postpubertal testes was maximum at 31 degrees C and was impaired at body temperature (37 degrees C), whereas in the prepubertal testis it was temperature dependent with a maximum at 37 degrees C. RNA and protein synthesis in the three groups was temperature dependent at 28 degrees C to 37 degrees C, was depressed at 40 degrees C, and remarkably depressed at 43 degrees C.

Depressed DNA Synthesis in Vitro by Early Prepubertal Undescended Human Testis

To study the spermatogenic potential of the early prepubertal inguinal testis and the effect of temperature on spermatogenesis, the levels of incorporation of 3H-thymidine and 14C-uridine into the testicular tissue were studied at 31 degrees C and 37 degrees C in vitro and compared with those of scrotal (descended) testis. 3H-thymidine incorporation into the inguinal testicular tissue at 31 degrees C, which is close to normal testicular temperature, was significantly lower than that of the scrotal testis. There was no significant differences in 14C-uridine incorporation between inguinal and scrotal testis at that temperature. At 37 degrees C, which is close to normal body temperature, 3H-thymidine and 14C-uridine incorporation into the tissues of inguinal and scrotal testes was significantly higher than at 31 degrees C. It would appear that DNA synthesis of the inguinal testis is lower than that of the scrotal testis at 31 degrees C, and higher temperature, at least for a short while, did not contribute to the impairment of spermatogenic potential in early prepubertal inguinal testis.

Effectiveness of Hmg-Hcg Treatment for DNA/RNA Syntheses in Subfertile Human Testis In Vitro

To evaluate the effect of hMG-hCG treatment on spermatogenic impairment, DNA/RNA syntheses were measured in 25 patients with idiopathic male infertility by estimating the values of 3H-thymidine and 14C-uridine incorporation into the testicular tissue cultured with or without the hormonal treatment. Without the hormonal treatment, the levels of 3H-thymidine incorporation were in inverse correlation with the progress of spermatogenic impairment, whereas disturbances in responsiveness to the hormonal treatment directly correlated with spermatogenic impairment. Although the 14C-uridine incorporation increased both with and without the hormonal treatment, the values showed different patterns from those of 3H-thymidine incorporation. It would appear that the DNA synthesis in testicular tissue cultured with or without hMG-hCG treatment might predict the responsiveness to hormonal treatment for patients with idiopathic male infertility.

The role of intercellular contacts in the activation of B lymphocytes by anti-immunoglobulin antibodies.

It has been proposed that anti-Ig antibody activates B cells in a way analogous to the antigens, i.e., it delivers its signal by cross-linking and clustering the Ig receptors on the surface of one cell. However, the cross-linking of different B cells which may deliver to each other a signal has not been considered. Thus, we examined the effect of preventing cell contacts on the response of rabbit B cells to anti-Ig allotype antibody by using a solid agarose medium. First, we examined the 14C-uridine incorporation in liquid medium and in liquid or solid agarose in cultures stimulated with anti-Ig antibody (Ab). The response was high in liquid agarose or liquid medium but was absent when the agarose-containing medium was solidified at the start of the cultures. If the agarose was solidified 6 hr after the start, a good response was obtained. Moreover, if the cells were sedimented at the start before solidifying the agarose-containing medium, a good response was also obtained. Similar results were obtained when T cells were activated by Con A. To examine whether B cells require contacts with other B cells or with non-B cells, we examined their response to anti-Ig Ab in the absence of macrophages or T cells, and found that purified B cells from lymphoid organs or from thoracic duct responded well in the absence of T cells and/or macrophages. The response was also absent when the cells were cultured in agar at a "local" concentration close to 10(8) cell/ml. Also, concentrated supernatants of anti-Ig-stimulated cells did not increase the response to anti-Ig in solid agarose. These two last observations suggest that the lack of response in solid agarose is not due to a lack of diffusible factors or to a lack of feeder cells. Therefore, because the only difference between the cultures that responded to anti-Ig or Con A and those that did not was the distance between the cells, we concluded that the contact between B cells or between T cells is essential to their activation by their respective mitogens. We speculate that the anti-Ig Ab or the antigen cross-links B cells, which then provide each other with the activating signal or with one of the activating signals.

An affinity labeling of estrogen receptor. II. Synthesis and biological activity of 3-hydroxy-17 beta-(p-nitrophenyldithio)-1,3,5(10)-estratriene.

3-Hydroxy-17β-(p-nitrophenyldithio)-1, 3, 5(10)-estratriene (Reagent A), an affinity labeling reagent for estrogen receptor, was prepared by iodine oxidation of an alkaline mixture of 3-hydroxy-1, 3, 5(10)-estratriene-17β-thiol and p-nitrothiophenol in 50% ethanol, and was isolated in a pure state. The structure was confirmed by the physical as well as spectral properties. The biological activity of the compound was tested by the measurement of the effects of 3H-thymidine incorporation into deoxyribonucleic acid (DNA) and on 14C-uridine incorporation into ribonucleic acid (RNA) in a human breast cancer cell line, MCF-7. There were increases of about 2-fold in thymidine incorporation and of about 1.1- to 2-fold in uridine incorporation in cells in-cubated in 10-7M Reagent A compared with the control.

Phagocytosis measured as inhibition of uridine uptake: a method that distinguishes between surface adherence and ingestion

Polymorphonuclear leucocytes selectively inhibited the incorporation of 14C-uridine by intracellular staphylococci. Within specified limits, the amount of radiolabel incorporated by extracellular staphylococci was related to bacterial concentration. The incorporation of labelled uridine can thus be exploited to assay the extent to which association between staphylococci and polymorphonuclear leucocytes reflects surface adherence as opposed to ingestion. A comparison of the new method with a conventional viable-count determination of leucocyte-associated bacteria shows it to be comparable in efficiency when non-immune serum is used as opsonin and superior when specific opsonin is used.

Effects of the rat renotropic system on 14C-uridine incorporation into RNA and RNA precursors

We used the incorporation of 14 C-uridine into RNA of incubating kidney fragments from normal control rats to evaluate RNA metabolism. Sera from unilaterally nephrectomized rats (uni) obtained 20 hrs post-operatively stimulate 14 C-uridine incorporation into RNA significantly more than sera from sham-operated rats (sham). Differently, sera from uni and sham rats have little influence on specific activities of endogenous uridine nucleotide pool in renal fragments. Renal extracts were obtained by homogenizing kidneys in saline. Extracts from kidneys of uni and sham rats 20 hrs post operation depress incorporation markedly, and each depresses to a similar extent, but kidney extracts dilute the specific activities of uridine pools. Correcting for the latter dilution demonstrates that kidney extracts alone have little effect on 14 C-uridine incorporation into RNA. We then followed the results when these sera and extracts were combined. Compared to fragments incubating in sham sera and sham extracts, substitution of uni extracts or both uni extracts and uni sera enhances 14 C-uridine into renal RNA, whether or not results are corrected for changes in the specific activities of the uridine pools. We conclude that after uninephrectomy there is a concurrent elevation in circulating renotropin and a tissue activating factor in the remaining kidney. The tissue factor can only form an excitor to 14 C-uridine incorporation into RNA when serum is present. The rat renotropic system that enhances incorporation of 3 H-thymidine into DNA also can stimulate 14 C-uridine incorporation into renal RNA.

Paragonial proteins of Drosophila melanogaster adult male: In vitro biosynthesis

Abstract When paragonia were incubated for 8 hr in simplified Robb's medium a continuous increase in incorporation of 14 C-amino acids into TCA precipitable proteins was found. The increase is linear over the first 2 hr. Copulation provides the stimulus for an alteration in rate of in vitro incorporation: a rapid and clear increase occurs. A similar increase in rate of incorporation of 14 C-uridine into RNA and 3 H-ethanolamine into ‘paragonial substance’ could be found. The pattern of incorporation of 14 C-amino acids into proteins, as determined by polyacrylamide gel electrophoresis, remains qualitatively unaltered. Results from transplantation experiments of single paragonia suggest that the stimulation, at least in part, is neuronal or humoral mediated. In vitro incubations with actinomycin D, under conditions of 90% inhibition, resulted in an increased rate of incorporation of 14 C-amino acids into proteins.

Toxicity of Platinum (IV) Salts for Cells of Pulmonary Origin

The acute toxicity of tetravalent platinum was studied in vitro by use of rabbit alveolar macrophages and human lung fibroblasts (strain WI-38). Alveolar macrophages were exposed in tissue culture for 20 hr to platinum dioxide (PtO2) or platinum tetrachloride (PtCl4). There was no evidence of dissolution of PtO2 and no decrease in viable cells at concentrations as high as 500 mug/ml. PtCl4 was soluble in the macrophage system and after a 20-hr exposure, resulted in loss of viability in 50% of the cells originally present at a concentration of 0.30mM (59 mug Pt/ml). After a 20-hr exposure, rapidly growing human lung fibroblasts were rendered nonviable by PtCl4 at comparable concentrations. A decrease in total cellular ATP was observed at lower concentrations in macrophages and fibroblasts along with a reduction in phagocytic activity of macrophages as compared to controls. With the fibroblasts, a 50% decrease in incorporation of 14C-thymidine was observed after a 22-hr exposure to PtCl4 at a concentration of 0.007mM; higher concentrations were required to inhibit the incorporation of 14C-uridine and 14C-leucine. Time-course studies indicated that the inhibition of 14C-thymidine incorporation was nearly complete (90%) after 7 hr in the presence of 0.06mM PtCl4. Under the same conditions, there was little inhibition (15%) of 14C-leucine incorporation and moderate inhibition (50%) of 14C-uridine incorporation. Higher concentrations of PtCl4 were required to inhibit 14C-thymidine incorporation into the acid-soluble fraction than were required to inhibit incorporation into the acid-precipitable fraction. Hence, the preferential inhibition of DNA synthesis by PtCl4 may result from an impairment of the incorporation process.

Toxicity of platinum (IV) salts for cells of pulmonary origin.

The acute toxicity of tetravalent platinum was studied in vitro by use of rabbit alveolar macrophages and human lung fibroblasts (strain WI-38). Alveolar macrophages were exposed in tissue culture for 20 hr to platinum dioxide (PtO2) or platinum tetrachloride (PtCl4). There was no evidence of dissolution of PtO2 and no decrease in viable cells at concentrations as high as 500 mug/ml. PtCl4 was soluble in the macrophage system and after a 20-hr exposure, resulted in loss of viability in 50% of the cells originally present at a concentration of 0.30mM (59 mug Pt/ml). After a 20-hr exposure, rapidly growing human lung fibroblasts were rendered nonviable by PtCl4 at comparable concentrations. A decrease in total cellular ATP was observed at lower concentrations in macrophages and fibroblasts along with a reduction in phagocytic activity of macrophages as compared to controls. With the fibroblasts, a 50% decrease in incorporation of 14C-thymidine was observed after a 22-hr exposure to PtCl4 at a concentration of 0.007mM; higher concentrations were required to inhibit the incorporation of 14C-uridine and 14C-leucine. Time-course studies indicated that the inhibition of 14C-thymidine incorporation was nearly complete (90%) after 7 hr in the presence of 0.06mM PtCl4. Under the same conditions, there was little inhibition (15%) of 14C-leucine incorporation and moderate inhibition (50%) of 14C-uridine incorporation. Higher concentrations of PtCl4 were required to inhibit 14C-thymidine incorporation into the acid-soluble fraction than were required to inhibit incorporation into the acid-precipitable fraction. Hence, the preferential inhibition of DNA synthesis by PtCl4 may result from an impairment of the incorporation process.

The activation of cultured epididymal tubules by androgens

We have studied the influence of androgens, added to the media in which rat epididymal tubules were cultured, upon RNA synthesis. The presence of 1 × 10−5 M dihydrotestosterone (DHT) in the medium during a 3 day culture period induced a 66% increase in the incorporation of 14C-uridine into acid insoluble material. This effect could also be elicited by 5α-androstane-3α, 17β-diol, a potent androgen, but neither by estradiol-17β nor corticosterone or progesterone. Furthermore, it was blocked by the simultaneous presence of cyproterone acetate in the medium, thus stressing the specific nature of the androgenic stimulation. Twelve and 36 h of exposure to DHT (1 × 10−6 M) induced an acceleration in the synthesis of 28S RNA accompanied by minor stimulations of the synthesis of 18S and 4S RNA. Androgens also had a pronounced affect on the aggregation of ribosomes into heavy polyribosomes which showed increased activity in protein synthesis. These results are interpreted as indirect evidence for the synthesis of messenger RNA.

Influence of nerve impulse on enzyme synthesis in skeletal muscle

Activity levels, distribution and isozyme patterns of the enzymes in denervated skeletal muscle are approaching the levels and types characteristic of embryonic muscle. Regeneration of the nerve fibres and nerve endings in denervated fast and slow skeletal muscles resulted in changes in these muscles so that they resembled corresponding intact muscles in their enzyme profiles. Actinomycin D prevented the short-term enhancement of G-6-P DH and transformation of its isozyme composition caused by denervation of the muscle. Denervation of the muscle or electrical stimulation of the distal nerve stump cause changes in the rates of histone acetylation, in template activity of DNP and 14 C-uridine incorporation to RNA in isolated nuclei. These results indicate that nerve impulses regulate the process of transcription.

Stimulation of uridine incorporation in isolated bone cells by parathyroid hormone and cyclic AMP.

Treatment of isolated bone cells with purified bovine parathyroid hormone (5 ng/ml-2 ¼g/ml) augmented the incorporation of 214C-uridine into acid-soluble RNA precursor pools and into RNA. Stimulation developed within 15 min and gradually declined after 30 min. A synthetic N-terminal 1–34 amino acid fragment of PTH was one-half as potent as native hormone. A 2–34 amino acid fragment, oxidized native hormone, and several basic polypeptides were inactive. PTH enhanced the labeling of cellular UDP and UTP pools appreciably and increased that of UMP slightly but failed to modify the total radioactivity of cellular free uridine, pointing to uridine or UMP phosphorylation (or both) as the site(s) of PTH action. Stimulation of 2–14C-uridine incorporation was not associated withalterations in total cell ATP levels. The effect of PTH on uridine metabolism appeared during simultaneous treatment of bone cells with actinomycin D, indicating that it was independent of RNAsynthesis. Exogenous (Bt)2 cAMP (0.1–1.0 min) an...

Incorporation of uridine into ribonucleic acid in rat brain slices after Δ 9-tetrahydrocannabinol and cannabis

Abstract Slices from rat hypophysis, hypothalamus, brain cortex and cerebellum were incubated in vitro in the presence of 14 C-uridine, and the incorporation of label into RNA was measured. Incorporation was linear with time and, in the hypophysis, inhibited significantly if the animals had been treated with 1.5 ml/kg of actinomycin D 90 min prior to killing. Rats were then given 20 mg/kg and 40 mg/kg of Δ 9 -tetrahydrocannabinol ip, dissolved in propylene glycol, and killed 90 min later. Although these doses produced changes in behavior and hypothermia, they did not affect in vitro incorporation of 14 C-uridine into RNA in the slices, nor did crude cannabis resin (75 mg/kg, po).

The role of nuclear proteins in RNA synthesis

Abstract When epithelial cells from baby mice are cultured on a solid substrate in vitro, DNA synthesis and cell division take place in a majority of the cells 24 to 48 h after inoculation. This cell proliferation is regularly preceded by a sequence of nuclear changes likely to be involved in gene activation during the process of growth-transformation. These nuclear changes, which in the present investigation were studied by quantitative cytochemical methods, are characterized by: (1) increased binding of acridine orange (AO) very soon after cell attachment, reflecting initial changes of the deoxyribonucleoprotein (DNP) complex; (2) accumulation of proteins in the cell nucleus; (3) dispersion of the nuclear chromatin; (4) increased rate of 14C-uridine incorporation. The initial DNP change, reflected by the increased AO-binding, was per se not a sufficient factor for stimulation of 14C-uridine incorporation. An increased 3H-uridine incorporation was only observed when protein began to accumulate in the nucleus and when the chromatin changed from a condensed to a more dispersed state. The rate of 14C-uridine incorporation was, during the whole process of growth transformation, found to be directly proportional to the amount of protein that had accumulated in the nucleus. However, the initial DNP change was a necessary prerequisite for the subsequent accumulation of protein in the nucleus. Attachment of the cells to the solid substrate was found to be necessary in expressing this sequence of nuclear events. Furthermore, in areas on the glass slide where many cells had attached, these nuclear changes occurred more rapidly.

Effects of serotonin on strain L mouse cells in culture

Abstract The growth of cultured mouse cells, strain L, in a strictly synthetic medium is depressed by serotonin. Beginning with concentrations of serotonin in the medium of 5 × 10 −4 M and higher, the mitotic burst which characterizes the logarithmic phase is quelled and at 3 × 10 −3 M serotonin completely eliminates growth. Under these conditions the utilization of glucose and the concomitant production of lactic acid are enhanced but the percent conversion of glucose to lactate is not altered. These alterations may arise as a secondary response to the depression of the rate of growth of L cells or, in part, from actions of serotonin on other areas of the metabolism of the cells. Incorporation studies with 14 C-leucine indicate that serotonin does not act as a general protein synthesis inhibitor. Serotonin appears to differentially affect the transport of labelled nucleotide precursors into the cells and their metabolism once within. While the incorporation of 14 C-uridine in both the acid-soluble and -insoluble fractions of the cells is slightly decreased, the incorporation of 3 H-thymidine into the acid-soluble pool is greatly increased without a significant alteration in the incorporation into the acid-insoluble fraction. These alterations may be associated with changes in the specific activity of intracellular nucleotides.

The effect of ethylene on the respiration, ethylene production, RNA and protein synthesis for apples stored in low oxygen and in air

Abstract A comparison is made between the respiration rate and ethylene production of the whole fruit and the respiration rate, ethylene production, incorporation of 14 C-uridine into an RNA fraction and of 14 C-valine into a protein fraction of peel disks prepared from the fruit from Bramley's Seedling apples stored at 12° in air and in 3% oxygen. Results show that the respiration and ethylene production of the whole fruit is closely reflected in the behaviour of the peel disks in air, in low O 2 and on transfer from low O 2 to air. Ethylene appears to be the key to the increased rate of respiration and the other parameters including the development in the disks of a malate decarboxylating system (the malate effect) which appears to be a coupled system involving malic enzyme, pyruvate decarboxylase and alcohol dehydrogenase (NADPH 2 -dependent). While oxygenous ethylene has a temporary stimulatory effect on the various systems investigated when applied in 3% O 2 , autostimulation of ethylene production with attendant physiological action does not appear possible in low O 2 . Both production and physiological action of ethylene appear to require relatively high concentration of O 2 for their full operation.

The relationship between ethylene and the synthesis of RNA and protein in ripening apples

The stimulation by ethylene of the respiration of whole fruits and peel discs prepared from them is followed using Worcester Pearmain and Cox's Orange Pippin apples. RNA and protein synthesis in relation to ethylene stimulation is investigated in terms of incorporation of 14C-uridine and 14C-valine into RNA and protein fractions in the discs of peel. Application of ethylene (40–60 ppm) to the preclimacteric fruit induces increased ethylene production in the fruit; uridine incorporation into RNA rises to a peak followed by a peak in valine incorporation into protein, while a malate decarboxylating system develops in the ripening fruit.

Effects of inhibitors of protein synthesis on structure and function of the crypts of the small intestine

Summary Studies conducted with TZA and several different doses of CHM suggest a possible relationship between the proliferation of the intestinal crypt cells and the degree to which their protein biosynthetic capacity is interrupted. An initial inhibition of protein synthesis of less than 80% has either no effect or induces only a partial decrease in the number of dividing cells, while an inhibition in excess of 80% results in a complete abolition of mitotic activity. Similarly, a return of protein synthesis to 20% of control levels appears to be sufficient to support the re-entry of G2 cells into division. Autoradiograms indicate that S phase cells can also continue their progression into the post-synthetic phases of the cell cycle despite a considerable suppression of DNA replication. Despite the marked inhibition of protein and DNA synthesis by CHM, the incorporation of 14C-uridine into RNA was enhanced. This stimulation was more pronounced when protein synthesis had returned to approximately 15–20% of controls. Electron microscopic studies provided some additional insight into the mechanism by which CHM exerts its antimitotic effect and, in addition, revealed that no cytological abnormalities developed in CHM-treated animals. This latter observation indicates that the intestinal crypt cells can tolerate profound aberrations in their metabolic and functional activities without undergoing degenerative changes.