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At least 991 records · Page 55Linked to original sources
PubMed1991

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.

Amides↗
PubMed1996

Depopulation of highly excited singlet states of DNA model compounds: quantum yields of 193 and 245 nm photoproducts of pyrimidine monomers and dinucleoside monophosphates.

Formation of uracil and orotic acid photodimers, uridine and 5'-UMP photohydrates, TpT photodimers and (6-4)photoproducts, dCpT photohydrates and (6-4)photoproducts and UpU, CpC and CpU photohydrates were studied in neutral deoxygenated aqueous solution at room temperature upon irradiation at either 193 or 254 nm. The photoproducts were identified and quantified and the contribution from photoionization to substrate decomposition, using lambda irr = 193 nm, was separated. The ratio of the quantum yields of respective stable products, eta = phi 193/phi 254, is indicative of the yield of internal conversion from the second to the first excited singlet state, S2-->S1. For the observed photodimers eta decreases from 0.94 for uracil to 0.7 for TpT and further to 0.55 for orotic acid. For the (6-4)photoproducts of TpT and dCpT eta = 0.5-0.8 and for the photohydrates in the cases of UpU, CpC, CpU and dCpT eta ranges from 0.55 to 1.

DNA↗
PubMed1977

Nitrogen retention in rats fed on diets enriched with arginine and glycine. 1. Improved N retention after trauma.

1. Nitrogen retention was measured in adult rats (250-350 g) subjected to the trauma of hind-leg fracture and given diets with or without arginine plus glycine supplementation. Observations were also recorded on excretion of creatine, creatinine, allantoin, and orotic acid. Liver and skeletal muscle transaminase activities were also determined. 2. When traumatized rats weighing approximately 250 g were given a diet with 200 g casein/kg, supplemented with 20 g arginine and 10 g glycine/kg (EC diet) or a casein diet made isonitrogenous with the EC diet by addition of aspartic acid (C diet), a 60-70% increase in N retention was observed for the first 5 d post-injury for animals consuming the EC diet. A soya-bean (S) diet, isonitrogenous to the diet containing 20% casein, supplemented with arginine and glycine was as effective as the EC diet in promoting significantly better N retention of traumatized rats (350 g) in comparison to rats given the C diet. 3. When the dietary casein content was reduced to 100 g/kg, supplements of 10 g arginine and 5 g glycine or 20 g arginine and 10 g glycine/kg did not improve N retention. It is suggested that both protein quality and protein quantity are important following injury. 4. An increased excretion of creatine was observed in traumatized rats given the high-protein diets supplemented with arginine and glycine. No consistent changes were noted for urine creatinine. 5. 5. Urine allantoin levels remained stable after leg-fracture in rats consuming either the C or EC diets. Differences in the levels of urine orotic acid were found during both the pre- and post-injury periods in rats given the C, EC or S diets. 6. The mechanisms responsible for the improved N retention of traumatized rats consuming the high-protein diets with supplements of arginine and glycine may be related to the role of arginine both as a constituent of muscle tissue and as an intermediate in the urea cycle. 7. In traumatized rats fed the C or EC diets, liver transaminase activity increased whereas the transaminase activity in skeletal muscle decreased. These results support the recent concept that the increased excretion of N following injury arises from diminished reutilization of amino acids by muscle tissue without an acute increase in the rate of muscle catabolism.

Alanine Transaminase↗
PubMed1973

The distribution of ribosomes between different functional states in livers of fed and starved mice.

1. To investigate the role of ribosome function in regulating protein synthesis, the activity, distribution and functional states of ribosomal particles were investigated in livers of mice fed ad libitum or starved overnight. 2. The distribution of protein-synthesizing activity between polyribosomes of different sizes was analysed after incorporation of radioactive leucine, and the quantitative distribution of ribosomes as native subunits, monomers and polyribosomes was analysed after incorporation of orotic acid. Precursors labelled with (3)H or (14)C were given separately to fed and starved mice, so that livers from the two groups of animals were processed together. 3. The former experiments showed that starvation has little effect on the distribution of protein-synthesizing activity across polyribosome sedimentation patterns, though the latter experiments showed that the proportion of ribosomes existing as monomers increased from 9.5% to 15.2%, whereas the proportion existing as polyribosomes decreased from 81.4% to 75.6%. Starvation had a negligible effect on the proportion of native subunits, which accounted for 9.1% and 9.2% of the ribosomes in fed and starved mice respectively. 4. The monomeric ribosome fraction was isolated and subjected to ionic conditions which selectively dissociate single ribosomes. Starvation increased the proportion of monomers that dissociated from 59% to 72%, so the monomers that accumulate in livers of starved animals are single ribosomes and not monoribosomes resulting from degradation of polyribosomes. 5. The fate of newly formed ribosomal particles was studied by measuring the specific radioactivity of native subunits, monomers and polyribosomes at different times after injection of radioactively labelled orotic acid. Starvation did not appear to affect equilibration between newly formed particles and polyribosomes, and the radioactivity of polyribosomes in both groups of mice reached about 90% of that in native subunits after 4h. The radioactive labelling of monomers proceeded at a slower rate, especially after starvation. At 4h, the radioactivity of monomers was 64% and 55% that of native subunits in fed and starved mice respectively.

Journal Article↗
PubMed1996

Hypoxanthine: a low molecular weight factor essential for growth of erythrocytic Plasmodium falciparum in a serum-free medium.

A low molecular weight factor in a basal medium essential for erythrocytic Plasmodium falciparum development in a serum-free medium using a cell growth-promoting factor derived from adult bovine serum was detected. The factor was hypoxanthine. The optimal hypoxanthine concentration for parasite growth was between 15 and 120 microM. The contribution of hypoxanthine to increased parasite growth was clearly evident in cultures on day 4. Among various low molecular weight supplements tested, adenine, adenosine, AMP, ATP, cyclic AMP, guanine, guanosine, inosine, inosine monophosphate, xanthine, NAD, NADH, NADP, NADPH and deoxyguanosine triphosphate showed a similar effect to that of hypoxanthine in the serum-free culture system. On the other hand, the addition of uric acid, FAD, thymidine, uridine, orotic acid, deoxythymidine triphosphate, deoxycytidine triphosphate, deoxyadenosine triphosphate, ribose-1-phosphate, or ethanolamine was not beneficial to the parasite growth. The results presented here will not only be of practical value, but will provide important information about the developmental requirements of the parasite.

Animals↗
PubMed1991

[Total protein and RNA biosynthesis in the liver cells of the long-tailed suslik Citellus undulatus in different functional states].

The synthesis of proteins and RNA have been studied by means of labeled precursors of 14C-amino acids and 14C-orotic acid in liver cells of hibernating, arousing and active ground squirrels (Citellus undulatus). The investigations conducted testify to the possibility of CHI-inhibited synthesis of proteins in liver cells of hibernating ground squirrels, which occurs, apparently, on preexisting polyribosomes. The low level of protein biosynthesis in this state is stipulated not only by the Arrhenius dependence, but by the dissociation of polyribosomes into monosomes as well. In the course of arousal, an intensive assembling of polyribosomes was detected even under the inhibition of RNA synthesis by actinomycin D, testifying to the presence of a pool of mRNA [correction of iRNA] in liver cells, which are necessary for the observed activation of translation. The intensity of protein synthesis on arousal is also stimulated by the activation of transcription.

Animals↗
PubMed1983

Increased levels of UMP synthase protein and mRNA in pyrazofurin-resistant rat hepatoma cells.

Rat hepatoma cells that have undergone stepwise selection in increasing concentrations of pyrazofurin have coordinately increased levels of both orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine-5'-phosphate decarboxylase (EC 4.1.1.23) activity. These two activities catalyze the conversion of orotic acid to UMP in de novo pyrimidine biosynthesis. Cells selected in 50 microM pyrazofurin have over 40 times the wild type level for both activities. A single polypeptide of approximately 55,000 daltons is increased in the resistant cells in amounts corresponding to the increase in the two activities. Resistant cell lines that are grown for extended periods in the absence of pyrazofurin are unstable, losing their elevated levels of both enzyme activities and the increased specific protein. Antibody prepared against a purified protein containing both enzyme activities binds specifically to this increased protein. These results corroborate other evidence indicating the two enzyme activities are contained within a single polypeptide called UMP synthase. Poly(A+) mRNA isolated from wild type and resistant lines was analyzed by in vitro translation for production of UMP synthase protein. Immunoprecipitation of the translation products shows the resistant cells have a 17-fold increase in translatable mRNA activity coding for UMP synthase. The synthase accounts for 0.24% of the total in vitro translation products synthesized with poly(A+) mRNA from the pyrazofurin-resistant cells as opposed to 0.014% with wild type mRNA. A cloned UMP synthase cDNA sequence hybridizes strongly to a 1.8-kilobase mRNA in the resistant cells. This mRNA is only barely detectable in equivalent preparations from wild type cells. Quantitation of the mRNA by dot hybridization techniques indicates a 16-fold increase in UMP synthase mRNA in the resistant cells. Although this increase in mRNA for UMP synthase could explain most of the increased protein, it is not sufficient to totally account for the 40-fold increase in UMP synthase.

Amides↗
PubMedDate not supplied

Hepatic encephalopathy and orotic aciduria associated with hepatocellular carcinoma in a noncirrhotic liver.

A 40-yr-old man presented with encephalopathy and was found to have hepatocellular carcinoma without cirrhosis. A large vascular hepatic mass was defined by CT scan and angiography; laparoscopy with biopsy confirmed the absence of chronic liver disease. A definitive tissue diagnosis of hepatocellular carcinoma was made at laparotomy; the tumor was unresectable. Peripheral arterial and selective portal and hepatic venous ammonia levels were high, and this finding suggested that the encephalopathy was nitrogenous and hepatic in origin. The proposed mechanisms of the encephalopathy are generation of ammonia from tumor breakdown and portosystemic shunting, a result of partial tumor occlusion of the hepatic veins. An unusually high urinary excretion of orotic acid was found similar to that seen in hereditary orotic aciduria.

Adult↗
PubMed2002

Behavior of alginate gel beads containing chitosan salt prepared with water-soluble vitamins.

Alginate gel beads were prepared which contained weak acid salts of chitosan (Alg-CS) and water-soluble vitamins (e.g. ascorbic acid (AS)) and the behavior of the beads, uptake of bile acids was investigated in vitro. The Alg-CS beads rapidly took up bile acid and this phenomenon was observed for both hydrogel beads and dried beads. About 120 micromol of taurocholic acid was taken up into Alg-CS (1 g) prepared with orotic acid. Dried Alg-CS is the granule which can be made easily, and keeps the ability of CS salt, and all elements can be taken as a food. Therefore, Alg-CS could serve as a useful dietary agent for the prevention of hyperlipidemia.

Alginates↗
PubMed1988

Analysis of UMP synthase gene and mRNA structure in hereditary orotic aciduria fibroblasts.

Hereditary orotic aciduria is an autosomal recessive disease in which there is a severe deficiency in the activity of the de novo pyrimidine pathway enzyme uridine 5'-monophosphate (UMP) synthase. UMP synthase is a bifunctional enzyme containing the two activities orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase, which catalyze the two-step conversion of orotic acid to UMP. Cell lines from three orotic aciduria patients have been characterized for UMP synthase gene and mRNA content. Restriction-enzyme analysis of DNA from the deficient cells revealed no changes in the gene structure compared with normal cell DNA structure. The amount of UMP synthase mRNA was not decreased, nor was there a detectable difference in the size of the UMP synthase mRNA in the deficient cells. Analysis of the mRNA by hybridization with a nearly full-length UMP synthase cDNA followed by S1 nuclease digestion showed no alteration in the mRNA structure. The UMP synthase activity of the deficient cells ranges from 2% to 7% of the normal cell level. The activity can be significantly increased by growing the deficient cells in barbituric acid. Our data indicate that UMP synthase gene transcription in the orotic aciduria cells produces the expected amount of a stable, correctly processed mRNA. The mRNA appears to code for a mutant enzyme that has reduced stability or altered kinetic properties.

Carboxy-Lyases↗
PubMed1978

The cytochemical demonstration of GERL in rat hepatocytes during lipoprotein mobilization.

When a semisynthetic diet containing 1% orotic acid (OA) is fed to rats, the endoplasmic reticulum (ER) of hepatocytes vesiculates and lipoprotein (LP) droplets accumulate within the vesicles. When clofibrate (ethyl chlorophenoxyisobutyrate, CPIB) is added to the orotic acid-rich diet, the ER cisternae reform and the LP is mobilized through the reconstituted ER. A remarkable restoration of normal hepatocyte ultrastructure occurs except for a few organelles. From their morphological appearance it was suggested that cisternae which became dilated with small LP particles were part of GERL, abnormally enlarged. The present communication validates this interpretation through ultrastructural cytochemistry which can distinguish GERL from the adjacent Colgi apparatus. GERL shows acid phosphatase (AcPase) but not thiamine pyrophosphatase (TPPase) activity. In contrast, the adjacent Golgi element shows thiamine pyrophosphatase but not acid phosphatase activity. From such cytochemical studies we have recently proposed that GERL in normal rat hepatocytes may be involved in transforming LP particles, by enzymes like lipases that were presumed to be present in this hydrolase-rich portion of smooth ER. In the situation studied in this communication, the addition of ethyl chlorophenoxyisobutyrate to the diet causes the release from the ER of large amounts of LP to the Golgi apparatus and to GERL. Apparently the capacity of GERL to metabolize LP is exceeded and lipid accumulates in the residual bodies.

Acid Phosphatase↗
PubMed1972

The effect of aflatoxin B 1 on normal and cortisol-stimulated rat liver ribonucleic acid synthesis.

1. Aflatoxin B(1), administered in vivo, inhibits the incorporation of [(14)C]orotic acid in vivo into rat liver nuclei, and also inhibits both Mg(2+)- and Mn(2+)-dependent RNA polymerase activities in nuclei assayed in vitro. 2. Aflatoxin B(1) inhibits the cortisol-induced increase in incorporation of [(14)C]leucine in vivo, but does not affect the control value of this activity. 3. Aflatoxin B(1) administered in vivo inhibits the increase in nuclear Mg(2+)-dependent RNA polymerase activity, assayed in vitro, which results from the treatment with cortisol. 4. Adrenalectomy causes a decrease in Mg(2+)-dependent RNA polymerase activity. The effect on this enzymic activity of adrenalectomy plus treatment with aflatoxin B(1) is no greater than that of treatment with aflatoxin B(1) alone. 5. These results suggest that the inhibition of cortisol-stimulated biochemical pathways by aflatoxin B(1) is due to an inhibition of cortisol-stimulated RNA synthesis. 6. The cytoplasmic action of aflatoxin is thought to be due to a competition for receptor sites on the endoplasmic reticulum between steroid hormones and aflatoxin B(1). No evidence was obtained for a similar competition for nuclear receptor sites between [(3)H]cortisol and aflatoxin B(1). 7. No differences were observed between the activities of RNA polymerase preparations solubilized from control or aflatoxin-inhibited nuclei. 8. No differences in ;melting' profiles were observed between DNA and chromatin preparations isolated from control nuclei or from aflatoxin-inhibited nuclei. 9. It is suggested that aflatoxin B(1) exerts its effect on RNA polymerase by decreasing the template capacity of the chromatin and that the aflatoxin ;target' area of the chromatin includes that region which is stimulated by cortisol. This process, however, does not involve inhibiting the movement of cortisol from the outside of the hepatic cell to the nuclear chromatin.

Adrenalectomy↗
PubMed1966

Studies in rapidly labelled ribonucleic acid in cell fractions and in tumour tissues.

1. Twenty minutes after injection of [(3)H]orotic acid into rats the rapidly labelled RNA from the liver is mainly associated with the nuclear fraction and little with the ribosomal cytoplasmic fraction. 2. The thermal denaturation of RNA from the fractions was not as reversible as that of the RNA extracted from whole liver. 3. Rapidly labelled RNA is synthesized by cells from a transplantable hepatoma when incubated in the presence of [(3)H]uridine and, after extraction and centrifugation, the label is present in three main fractions: one which sediments to the bottom of a gradient and is associated with DNA, a second which sediments to the heavy side of the 28s RNA, and a third which has a peak of activity between 28s RNA and 18s RNA and is associated with DNA. 4. After labelling and extraction of the RNA from Ehrlich ascites cells the distribution of radioactive components is similar to that of the material from the hepatoma cells. 5. The difference between the tumour cells and liver is due to some extent to the method of homogenizing the tissues and the nature of the components is discussed.

Animals↗
PubMed1977

[Possible mechanism of action of oxythiamine on nucleic acid metabolism].

Hydoxythiamine injections (400 mg/kg for 72 hours) resulted in the inhibition of transketolase activity and of 14C-orotic acid incorporation into RNA in rat liver tissue. At the same time, the decrease of RNA and phosphoribosylpyrophosphate levels has been observed, which suggests a regulation effect (via pentospohosphates) of hydroxythiamine on nucleotide and nucleic acid metabolism.

Animals↗
PubMed1975

The mechanism of action of 3-deazauridine in tumor cells sensitive and resistant to arabinosylcytosine.

Deazauridine inhibited growth of tumor cells in culture and in culture and in vivo; this agent was significantly more effective against L1210/AraC than against the parent sensitive line. Inhibition of growth of tumor cells in culture was prevented by uridine and cytidine and was partially alleviated by deoxycytidine, but not by deoxyuridine or thymidine. DeazaUR inhibited nucleic acid synthesis but not protein synthesis in tumor cells in culture; deoxycytidine alleviated inhibition of nucleic acid synthesis. The labeling of pyrimidine ribonucleotides by 6-14C-orotic acid was inhbited by deazaUR. DeazaUR treatment of tumor cells in culture resulted in increased uptake of cytidine-3H into RNA, whereas uridine-3H uptake into RNA was inhibited. Labelling of DNA by uridine-3H/ and cytidine-H was inhibited by deazaUR. Pools of CMP, CDP, and CTP decreased markedly during deazaUR treatment of L1210 cells in culture and in vivo. These observations in growing cells pointed to deazaUR inhibition of the synthesis of cytidylic acid. Deazauridine 5'-triphosphate was found to be an inhibitor of the synthesis of CTP from UTP catalyzed by enzyme preparations from L1210 cells. This observation is in agreement with those of McPartland et al.19 that deazaUTP inhibited CTP synthetase purified from calf liver. Deazauridine treatment of L1210 cells in culture stimulated the uptake of deoxycytidine-3H into DNA while inhibiting the uptake of 3H-labeled deoxyuridine, thymidine, deoxyadenosine, and deoxyguanosine. Intracellular pools of dCTP were decreased by deazauridine treatment in L1210 cells in culture and in vivo. Deazauridine 5'-diphosphate inhibited the enzymatic reduction of pyrimidine ribonucleoside 5'-diphosphates to the corresponding deoxyribonucleotides. These results are consistent with the view that deazauridine, after its uptake and intracellular phosphorylation, strongly inhibits the formation of CTP. This is considered to be the primary metabolic effect of the analog. A secondary effect appears to be an inhibition of dCTP formation.

3-Deazauridine↗
PubMed1988

DNA analysis of ornithine transcarbamylase deficiency.

By analysing the restriction fragment length polymorphism (RFLP) detected by an ornithinetranscarbamylase (OTC) gene specific DNA probe, we followed the segregation of the defective gene in two families with OTC deficiency (X-linked disease). We were able to exclude some female family members as carriers. In one case a doubtful result obtained in a biochemical carrier detection test (by examining the renal orotic acid excretion after a protein load) could be clarified by DNA analysis. In every family with OTC deficiency, carrier detection should be biochemical with additional DNA analysis. Previous results of the biochemical carrier test should be controlled by DNA analysis, especially when "normal" results were obtained.

Amino Acid Metabolism, Inborn Errors↗
PubMed1969

Studies on a nucleoprotein prepared from rat liver polysomes by digestion with T1 ribonuclease.

1. Treatment of rat liver polysomes in a buffer containing 2.5mm-magnesium chloride with T(1) ribonuclease at a concentration of 330units/ml. of reaction medium at 37 degrees for 2hr. leads to the production of an insoluble nucleoprotein. 2. On the bases of analysis for protein and RNA and of u.v.-absorption spectra the nucleoprotein appears to have lost approx. 60% of the structural RNA originally present in the ribosome. Degradation of (3)H-labelled polysomes (structural RNA labelled with orotic acid) with T(1) ribonuclease leads to nucleoprotein preparations retaining approx. 30% of the radioactivity originally present in the polysomes. By means of sucrose-density-gradient centrifugation it is shown that the nucleoprotein preparations are free of single 73s ribosomes and ribosomal subunits. No evidence for the presence of 28s and 18s structural RNA was obtained on examination of extracted nucleoprotein-particle RNA by means of sucrose-density-gradient centrifugation. 3. Digestion of washed polysomes carrying (14)C-labelled nascent peptide chains with T(1) ribonuclease gives a nucleoprotein particle that retains approx. 70% of the original labelled chains. Treatment of labelled nucleoprotein particles with 1mm-puromycin in the absence of transfer factors releases 20% of the labelled chains. Addition of GTP (0.48mumole) increases this release to 37%. 4. Treatment of nucleoprotein particles carrying (14)C-labelled peptide chains with either EDTA (50mm) or ammonium chloride (0.5m) brings about a small release of labelled material (approx. 15%). 5. Disruption of nucleoprotein particles carrying (14)C-labelled peptide chains with either sodium dodecyl sulphate or 2m-lithium chloride, followed by addition of transfer RNA as marker and chromatography on Sephadex G-200, show in both cases that considerable amounts of labelled peptide material move well ahead of the added transfer RNA marker. Further, if nucleoprotein particles carrying labelled peptide chains are treated with 0.3m-potassium hydroxide at 20 degrees for 24 hr., neutralized to pH7.6, and then chromatographed on Sephadex G-200, the labelled peptide material moves much closer to the added transfer RNA marker. These results suggest that a proportion of the nascent (14)C-labelled peptides on the nucleoprotein are attached to transfer RNA or large fragments of transfer RNA. 6. [(3)H]Polyuridylic acid binds to nucleoprotein particles in 1mm-magnesium chloride. The rate of binding is rapid when measured at 20 degrees .

Ammonium Chloride↗