Incorporation Of Orotic Acid Research Articles

Enhancement of the properties of biosourced poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by the incorporation of natural orotic acid

The aim of this study is to use natural orotic acid (OA) as a sustainable, environmentally friendly additive to improve the crystallization, rheological, thermal, mechanical, and biodegradation properties of bacterially synthesized poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB). OA was found to be an efficient nucleating agent for P34HB, and dramatically enhanced both non-isothermal and isothermal crystallization rates. The incorporation of OA increased nucleation density and decreased spherulite size, but had little effect on the crystalline structure. The rheological properties of the P34HB were greatly improved by the solid filler OA, particularly when a percolation network structure was formed in the blends. The thermal stability of P34HB was strongly enhanced, as exemplified by the ~23 °C increase in the onset thermal decomposition temperature (To) for the blend loaded with 5 wt% OA compared to that of pure P34HB. Moreover, the yield strength and elongation at break of P34HB containing 0.5 wt% OA increased by 25% and 119%, respectively. The most intriguing result was the clear enhancement in the enzymatic hydrolysis rates of the P34HB/OA blends compared to that of neat P34HB. The synergetic improvement in these properties may be of significant importance for the wider practical application of biosourced P34HB.

Purine and pyrimidine metabolism during the partial drying treatment of white spruce (Picea glauca) somatic embryos

The imposition of a partial drying treatment (PDT) on mature white spruce somatic embryos is a necessary step for successful germination and embryo conversion into plantlets. Purine and pyrimidine metabolism was investigated during the PDT of white spruce somatic embryos by following the metabolic fate of 14C‐labeled adenine, adenosine, and inosine, as purine intermediates, and orotic acid, uridine, and uracil, as pyrimidine intermediates, as well as examining the activities of key enzymes. Both the salvage and the degradation pathways of purines were operative in partially dried embryos. Adenine and adenosine were extensively salvaged by the enzymes adenine phosphoribosyltransferase and adenosine kinase, respectively. The activity of the former enzyme increased during the PDT. In both mature and partially dried embryos, a large proportion of inosine was recovered as degradation products. The de novo pathway of pyrimidine nucleotide biosynthesis, estimated by the incorporation of orotic acid into the nucleotides and nucleic acids, was high at the end of the maturation period and declined during the PDT. Uridine was the main substrate for the pyrimidine salvage pathway, since a large proportion of uracil was recovered as degradation products, i.e. CO2 and β‐ureidopropionic acid in both mature and partially dried embryos. Uridine was mainly salvaged by uridine kinase, whose activity was found to increase during the PDT. Taken together these results indicate that the PDT might be required for increasing the activity of adenine and uridine salvage enzymes, which could contribute to the enlargement of the nucleotide pool required at the onset of germination.

Alteration in de novo pyrimidine biosynthesis during uridine reversal of pyrazofurin‐inhibited dna synthesis

Pyrazofurin, a pyrimidine nucleoside analogue with antineoplastic activity, inhibits cell proliferation and DNA synthesis in cells by inhibiting uridine 5'-phosphate (UMP) synthase. It has been previously shown in concanavalin A (con A)-stimulated guinea pig lymphocytes (23) that pyrazofurin-inhibited DNA synthesis could be selectively reversed by exogenous uridine (Urd). In this report, we have examined possible mechanisms for the Urd reversal with experiments that determine the ability of exogenous Urd to (a) interfere with either the intracellular transport of pyrazofurin, or the conversion of pyrazofurin to its intracellularly active form, pyrazofurin-5'-phosphate; (b) reverse the pyrazofurin block of [14C]orotic acid incorporation into DNA; and (c) alter the pattern of exogenous [3H]Urd incorporation into DNA-thymine (DNA-Thy) and DNA-cytosine (DNA-Cyt) during pyrazofurin inhibition of pyrimidine de novo biosynthesis. The results of these experiments showed that Urd reversal does not occur through altered pyrazofurin transport or intracellular conversion to pyrazofurin-5'-phosphate, nor does it alter the distribution of [3H]Urd in DNA-Thy and DNA-Cyt. Instead, these findings indicate that the primary mechanism for exogenous Urd reversal of pyrazofurin inhibition of DNA synthesis involves the reversal of pyrazofurin inhibition of UMP synthase, thus restoring orotic acid incorporation into lymphocyte DNA through the pyrimidine de novo pathway.

Effects of ciprofloxacin on eucaryotic pyrimidine nucleotide biosynthesis and cell growth.

Several of the new 4-quinolones significantly increase the incorporation of [3H]thymidine into the DNA of mitogen-stimulated human lymphocytes. This study suggests that ciprofloxacin inhibits de novo pyrimidine biosynthesis, thereby resulting in a compensatory increase in the uptake of pyrimidine precursors through salvage pathways, and that additional effects may affect eucaryotic cell growth. Incorporation of deoxyuridine, uridine, and orotic acid as well as thymidine was increased in the presence of ciprofloxacin, one of the antibacterially most active of the new 4-quinolones. In contrast, the uptake was decreased in very high concentrations of the drug. Culture in HAT (hypoxanthine, aminopterine, thymidine) medium, which blocks de novo thymidylate synthesis, abrogated the increase in [3H]thymidine incorporation induced by ciprofloxacin. Ciprofloxacin also failed to increase the uptake of [14C]hypoxanthine or leucine, indicating a selective effect on pyrimidine and not on purine nucleotide biosynthesis. N-(Phosphonacetyl)-L-aspartate, an inhibitor of pyrimidine nucleotide biosynthesis, also increased [3H]thymidine incorporation in phytohemagglutinin-stimulated lymphocytes in a fashion similar to ciprofloxacin. The growth of several cell lines was partially inhibited by ciprofloxacin at 20 micrograms/ml and completely inhibited at 80 to 160 micrograms/ml. Growth inhibition by ciprofloxacin could not be restored by the addition of uridine to the medium. Chromosome breaks, gene amplification, or other genetic alterations could not be detected in human lymphocytes incubated with up to 25 micrograms of ciprofloxacin per ml.

Effect of Chlorpromazine on Protein, Phospholipid and RNA Synthesis or Degradation in Rat Liver

Chlorpromazine (CPZ) administration to rats (25 mg/kg ip) significantly stimulated (14C)-leucine incorporation to liver microsomal proteins, (14C)-orotic acid incorporation to liver RNA and (32P) incorporation to liver microsomal lipids. CPZ administration did not modify the decay of radioactivity of liver microsomal lipids prelabeled with (32P) or that of microsomal proteins prelabeled with [(14C)-guanidino]-arginine. Results suggest that CPZ administration stimulates protein, phospholipids and RNA synthesis but does not affect their degradation. The relation of these findings with CPZ preventive effects on CCl4-induced liver injury is discussed.

Triiodothyronine-stimulated RNA synthesis in primary cultures of adult rat hepatocytes

We have studied the effect of triiodothyronine (T 3) on RNA synthesis by primary cultures of rat hepatocytes in order to ascertain whether hepatocyte transcriptional activity is directly stimulated by this hormone. The results demonstrate that T 3 stimulates RNA synthesis as measured by [ 3H]orotic acid incorporation into RNA and by RNA polymerase activity. The responsiveness of cultured hepatocytes to T 3 becomes evident only after a fairly long latency period required for the recovery of T 3 nuclear binding sites. The response of RNA synthesis to T 3 was absent unless the hepatocytes were simultaneously exposed to insulin and dexamethasone, indicating a permissive role of these factors in the action of T 3 on RNA synthesis.

Hepatic artery administration of degradable starch microspheres

The effects of hepatic artery administration of degradable starch microspheres on liver energy charge and nucleic acid anabolism were studied in rats. Liver energy charge was evaluated 20 and 60 min after the injection of degradable starch microspheres. As compared to controls the microspheres had no effect on liver energy charge. The incorporation of orotic acid, uracil, and thymidine into liver RNA or DNA was studied 1 h after hepatic artery injection of precursor alone or together with degradable starch microspheres. Orotic acid and uracil incorporation into RNA was studied in normal rats and the DNA incorporation of thymidine in animals with regenerating livers. Orotic acid and thymidine were given in trace amounts. Uracil was given in amounts corresponding to a therapeutic dose of 5-fluorouracil. The addition of microspheres had no effects on the incorporation of the nucleic acid precursors into RNA or DNA. Thus, in the normal liver degradable starch microspheres administered by the hepatic artery had no influence on liver energy charge or RNA anabolism in the liver. Also the microspheres had no negative effects on the DNA anabolism in proliferating liver cells.

Effect of amines on fibrinogen synthesis

l-Epinephrine, serotonin, and isoproterenol stimulate the incorporation of [ 14C ]leucine into thrombin-induced clottable protein; this stimulation was abolished by actinomycin D. The incorporation of 32P into total RNA of rat liver, the site of fibrinogen synthesis, was stimulated by epinephrine and was highest at 2 h after 32P administration. [ 14C ]Orotic acid incorporation into polysomal RNA of liver was also increased significantly by epinephrine and serotonin. The immunoprecipitation of newly synthesized protein by monospecific antibody raised against pure rat fibrinogen clearly demonstrates that l-epinephrine increased fibrinogen formation in vivo under the experimental condition. Translation of poly (A)-containing RNA from total polysomal RNA clearly indicates that l-epinephrine increased mRNA specific for fibrinogen.

RNA synthesis in regenerating mouse liver evaluated by incorporation of [methyl-14C]methionine and by determination of RNA polymerase activity.

The synthesis of RNA during mouse liver regeneration was studied by two different methods at 24 and 48 h after partial hepatectomy. Total chromatin-bound RNA polymerase activity showed an increase of 32% at 24 h after partial hepatectomy. At 48 h a slight increase in total activity was also observed in regenerating liver, but the difference was not significant. The increase in total RNA polymerase activity was due to a rise in RNA polymerase I plus III activity. This enzyme activity was increased at both 24 and 48 h. The increase was 57% at 24 h and 51% at 48 h. When [methyl-14C]methionine was used for labelling of methyl groups in rRNA, there was an increased specific radioactivity of this class of RNA at both 24 h and 48 h. The increases were 263 and 103% at 24 and 48 h respectively. Thus both methods revealed an increased synthesis of rRNA during mouse liver regeneration. The results are discussed in relation to previous results from this laboratory [Yngner, Carlberg, Lewan & Engelbrecht (1979) Hoppe-Seyler's Z. Physiol. Chem. 360, 1069-1074; Yngner, Engelbrecht, Lewan & Annerfeldt (1979) Biochem. J. 178, 1-8; Yngner, Bengtsson, Carlberg, Engelbrecht & Wieslander (1980) Exp. Cell. Biol. 48, 393-403], which have shown that the incorporation of orotic acid or uridine into RNA is not increased in mouse liver regenerating after partial hepatectomy.

Radioautographic visualization of differences in the pattern of [3H]uridine and [3H]orotic acid incorporation into the RNA of migrating columnar cells in the rat small intestine.

The epithelium of rat small intestine was radioautographed to examine whether RNA is synthesized by the salvage pathway as shown after [3H]uridine injection or by the de novo pathway as shown after [3H]orotic acid injection. The two modes of RNA synthesis were thus investigated during the migration of columnar cells from crypt base to villus top, and the rate of synthesis was assessed by counting silver grains over the nucleolus and nucleoplasm at six levels along the duodenal epithelium--that is, in the base, mid, and top regions of the crypts and in the base, mid, and top regions of the villi. Concomitant biochemical analyses established that, after injection of either [5-3H]uridine or [5-3H]orotic acid: (a) buffered glutaraldehyde fixative was as effective as perchloric acid or trichloracetic acid in insolubilizing the nucleic acids of rat small intestine; (b) a major fraction of the nucleic acid label was in RNA, that is, 91% after [3H]uridine and 72% after [3H]orotic acid, with the rest in DNA; and (c) a substantial fraction of the RNA label was in poly A+ RNA (presumed to be messenger RNA). In radioautographs of duodenum prepared after [3H] uridine injection, the count of silver grains was high over nucleolus and nucleoplasm in crypt base cells and gradually decreased at the upper levels up to the villus base. In the rest of the villus, the grain count over the nucleolus was negligible, while over the nucleoplasm it was low but significant. After [3H]-orotic acid injection, the number of silver grains over the nucleolus was negligible at all levels, whereas over the nucleoplasm the number was low in crypt cells, but high in villus cells with a peak in mid villus. The interpretation is that, except for a small amount of label incorporated into DNA from either precursor by crypt cells, the bulk of the label is incorporated into RNA as follows. In the crypts, cells make almost exclusive use of uridine, that is, of the salvage pathway, for the synthesis of ribosomal RNA in the nucleolus and of messenger and transfer RNA in the nucleoplasm. However, when cells pass from crypt to villus, they mainly utilize orotic acid--i.e., the de novo pathway--for the synthesis of messenger and transfer RNA within the nucleoplasm.

RNA synthesis in primary cultures of adult rat hepatocytes.

The ability of hepatocyte monolayers to synthesize RNA was investigated by measuring [3H]orotic acid incorporation into RNA and the total nuclear RNA polymerase activity as a function of the time in culture. The results demonstrate that primary cultures of hepatocytes maintained in a chemically defined serum- and hormone-free medium are able to synthesize RNA actively. This ability increases within the first 2 d of culture, despite the concomitant decrease in [3H]orotic acid uptake, and decreases only after 3 d. Factors such as serum, insulin, and dexamethasone, known to improve maintenance of functional hepatocytes, markedly stimulate the uptake of labeled precursor without apparently affecting the rate of RNA synthesis by cultured cells. It is suggested that the culture of adult rat hepatocytes provides a useful experimental model for the studies of hormonal regulation of transcription in liver.

Studies on the effect of indomethacin and sulfinpyrazone on the rates of synthesis of albumin and acute-phase α 1-acid glycoprotein by rat liver slices

The rates of synthesis of albumin and α 1-acid glycoprotein were determined in the presence of indomethacin and sulfinpyrazone. There were significant reductions in synthesis rates of both proteins in the presence of concentrations of 0.5 m m or greater in the case of both drugs tested. Incorporation studies showed that decreased synthesis rates were accompanied by inhibition of incorporation of radioactive precursors into albumin and α 1-acid glycoprotein and into total protein isolated from liver and incubation medium. Incorporation of [ 3H]orotic acid into liver RNA was also subject to inhibition by the drugs used in the present work as well as by salicylate and phenylbutazone, two drugs used in an earlier study. Synthesis of RNA followed by orotic acid incorporation was more sensitive to inhibition by the four drugs in question; substantial inhibition was observed in the presence of 0.25 m m or greater of indomethacin, sulfinpyrazone, and phenylbutazone, and in the presence of 2 m m or greater of salicylate.

403 CEREBRAL RNA AND PROTEIN SYNTHESIS IN NEONATAL SHEEP BRAIN: EFFECTS OF FETAL THYROIDECTOMY

Cerebral structural and metabolic changes have been associated with reduced fetal and perinatal thyroid function in several species. In the present study several indices of neuronal function were assessed in lambs who were thyroidectomized at 64-67 days of gestational age. RNA synthesis measured by the rate of C14 orotic acid incorporation into nuclear RNA from six samples each of several brain regions did not differ for the cerebral cortex of hypothyroid (T4=4.1 ± 1.2 μg/ml TSH=37.5 ± 10.1 ng/ml) as compared with euthyroid (T4=9.2 ± 1.4; TSH=3.1 ± 1.2) lambs; mitochrondrial RNA synthesis was significantly reduced in the thalamus and hypothalamus (12%; p <.02) and in the cerebellar cortex (18%; p <.02) of hypothyroid animals. Protein synthesis, measured by incorporation of C14 leucine into synaptosomal protein, was significantly reduced in the hypothyroid lambs; in presynaptic nerve endings from the cerebral cortex (32%; p<.01), thalamus and hypothalamus (17%, p<.02) and cerebellar cortex (53%; p<.01). The cellular and synaptic changes attending perinatal hypothyroidism are reflected in regional changes in the synthesis of macromolecules relevant to effective cerebral functions.

The effect of short term incubation in vitro on the RNA content of regenerating mouse epidermis.

The effect of short term incubation in vitro on RNA content in regenerating and normal epidermis has been investigated. Regenerating mouse epidermis was incubated for three hours, either attached to its underlying dermis or by itself, in either buffered sucrose or Roswell Park Memorial Institute culture medium at 26 degrees C or at 37 degrees C. There is a significant loss of RNA when regenerating epidermis is incubated without being attached to its underlying dermis, at either 26 degrees C or 37 degrees C, although there was little loss of DNA, good incorporation of (3H) orotic acid into RNA, as well as good preservation of epidermal histological details. In contrast, when regenerating epidermis was incubated attached to its dermis, little loss of RNA occurred. Similarly, incubating normal epidermis attached to its dermis results in no loss of RNA. These conditions also result in no significant loss of DNA, good incorporation of (3H) orotic acid into RNA, and preservation of epidermal histological details.

Possible significance of changes in the energy metabolism for the release of liver lactate dehydrogenase and for the uptake and incorporation of [14C]-orotic acid into liver ribonucleic acid after partial hepatectomy.

The mouse liver revealed no increased incorporation of [14C]-orotic acid into either the total acid-soluble fraction, the uridine triphosphate or the RNA at 6 and 24 h after partial hepatectomy. In regenerating mouse and rat liver, the concentration of adenosine triphosphate was decreased 15-20% at 6 h, but was in the same range as that of the controls at 24 h. The adenosine monophosphate concentration of mouse liver increased 4-fold and 2-fold at these times after partial hepatectomy, respectively. The results indicate no direct relationship between the energy metabolism and the uptake and incorporation of orotic acid into RNA of regenerating liver. The activity of mouse plasma lactate dehydrogenase 5 (LDH 5) was increased 12-fold at 6 h and 5-fold at 24 h after partial hepatectomy. In rat, the LDH 5 activity was increased 2-fold at 6 h but was not different from that of the controls at 24 h. An increased leakage of LDH 5, possibly related to the decreased energy content of the liver, was thus revealed by the partially hepatectomized mice.

Ribonucleic acid metabolism during planarian regeneration.

A method for the extraction of total RNA of the planarian Polycelis tenuis is described. This technique has been applied to the study of RNA synthesis in the course of regeneration. Synthesis of RNA begins 7 hrs after sectioning and proceeds in two phases. The first phase, from 7 to 21 hr is characterized by an increased rate of (3H) orotic acid incorporation into RNA up to the 18th after sectioning followed by a decrease between 18 and 21 hrs. The rate of precursor incorporation then had risen again in the second phase to the 30th hr and finally decreased slowly until completion of regeneration. Studies of the pool of nucleotide precursors, show that the observed variations of orotic acid incorporation into RNA reflect variations in RNA synthesis. Electrophoretic analysis of RNA labelled for 2 hrs at various times during regeneration shows that the product formed during the first phase of regeneration is mainly rRNA and low molecular weight RNA. The second phase of the regeneration process, from the 24th hr, is characterized by the appearance of both ribosomal and polydisperse RNAs. These heterogeneous RNA species are detected up to the 46th hr of regeneration, after which time rRNA again becomes predominent. The significance of the two phases of RNA metabolism of which this is the first observation during planarian regeneration is discussed in connection with other biochemical events which have been described during regeneration processes in planarians and other animals.

Non-specific incorporation of nucleic acid precursors in Blastomyces dermatitidis and Histoplasma capsulatum.

Incorporation of thymidine, thymidine monophosphate (TMP), thymidine triphosphate (TTP), uridine and orotic acid into DNA, RNA and protein in Blastomyces dermatitidis and Histoplasma capsulatum was studied utilizing a specific acid hydrolysis technique developed for these fungi. Thymidine was incorporated to the greatest extent (approximately 0.5% of added label) followed by uridine, orotic acid, TMP and TTP. In Blastomyces, uridine and orotic acid labeled primarily RNA. TMP and TTP labeled RNA, DNA and protein at nearly the same level. In Histoplasma RNA was labeled poorly by any of these precursors. TMP and TTP labeled DNA predominately and protein to a slightly lower level. Deoxyadenosine or uridine media supplements of 250 micrograms/ml did not enhance incorporation. All precursors tested were found to be nonspecific in that RNA, DNA and protein were labeled. All data indicate that neither RNA nor DNA synthesis can be specifically measured in whole cells or acid precipitates by any of these precursors. Specific radiometric monitoring with these isotopes therefore requires the separation of these macromolecules.

Effect of age on RNA synthesis by rat hepatocytes

The incorporation of ( 3H) orotic acid into RNA by hepatocytes from rats of various ages was studied. A 37% decrease in the incorporation of ( 3H) orotic acid into RNA was observed by hepatocytes isolated from 30-month-old rats compared to 12-month-old rats. There was no age-related difference in the incorporation of ( 3H) orotic acid into acid-insoluble material by hepatocytes. The incorporation of ( 3H) orotic acid into UTP by hepatocytes was determined by high pressure liquid chromatography. The ( 3H) orotic acid was rapidly converted to UTP by hepatocytes, and the specific activity of the UTP reached a steady state after 10 min of incubation. There was no significant difference in the specific activity of the UTP in hepatocytes isolated from rats ranging in age from 6 to 25 months. Therefore, the age-related decrease in the incorporation of ( 3H) orotic acid into RNA by hepatocytes is not due to an age-related difference in the uptake or conversion of ( 3H) orotic acid into UTP.

Effect of Malnutrition and Rehabilitation on the Metabolism of Polyamines in Rat Liver

In adult male rats the activity of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMD) and the concentration of putrescine and spermidine increased markedly 3 to 4 weeks after feeding the animals a 6% casein diet. Incorporation of [14C]orotic acid into nuclear RNA also increased during this period of time. In contrast, spermine concentration remained unchanged. During the fifth week of protein restriction enzyme activities, putrescine and spermidine concentrations and orotic acid incorporation returned to control levels. After the restricted rats were put on a control diet there was a rapid increase above control levels in ODC activity and a proportionally smaller increase in SAMD activity. Putrescine and spermidine concentration also increase above control levels. Both enzyme activities and polyamine concentrations were within normal levels 96 hours after refeeding. Spermine values were unmodified by refeeding. After a transient rise 12 hours after refeeding, incorporation of labeled orotic acid into RNA decreased significantly for the rest of the experiment.

Ribonucleic acid synthesis in Phoenix Dactylifera

AbstractBased on the fact that ribonucleic acid (RNA) synthesis occurs in vitro, a system in which the incorporation of radioactive orotic acid into the RNA of the nucleus of Iraqi dates was devised. Orotic acid‐6‐14C, and substrates necessary for the synthesis of nucleotides, stimulated the incorporation of orotic acid‐6‐14C into the RNA of the nucleus. In addition, a system was devised to test the hypothesis that each of the ratios of ATP/GTP and UTP/CTP, respectively, was a factor in stimulating RNA synthesis in different varieties.The best incorporation of UTP‐3H into nuclear RNA was observed, and varied according to the date used and depended on the ATP/GTP ratio, and the best incorporation of ATP‐6‐14C into nuclear RNA was observed and varied according to the date used and depended on the UTP/CTP ratio. This in vitro system provides a method for studying RNA synthesis in different Iraqi Dates.