Influence of D-galactosamine on the synthesis of sugar nucleotides and glycoconjugates in rat hepatocytes

Rat hepatocytes were incubated in the presence of a high concentrationof the hepatopathogenic agent D-galactosamine (GalN), and theeffect on the cellular concentrations of pyrimidine nucleotidesand nucleotide sugars was determined. The UTP pool became depleted.The pools of UMP and CMP in RNA decreased to 72%, indicativefor an inhibition of RNA synthesis. UDP-HexNAc (where HexNAcis GlcNAc + GalNAc) and UDP-HexN (where HexN is GlcN + GalN)levels increased, and those of UDP-hexose and UDP-GlcA (whereGlcA is glucuronic acid) decreased. The cellular concentrationof CTP did not change, whereas that of CMP-NeuAc (where NeuAcis N-acetylneuraminic add) showed a 2-fold increase. Labellingwith [¹⁴C]orotic acid and [³H]cytidine showed that the metabolicflow via the de novo pathway was not changed. The depletionof the so-called overflow pool of UTP [Pels Rijcken et al, Biochem.J., 293, 207–213, 1993] caused a release of the feedbackinhibition by UTP and thus an increased flow through the salvagepathway. Finally, it appeared that GalN, when added to hepatocytes,gives rise to a pool of UDP-GlcNAc (where GlcNAc is N-acetylglueosamine)that is separate from the pool of UDP-GlcNAc that is derivedfrom GlcN. D-galactosamine glycosylation sugar nucleotide biosynthesis     

RNA Synthesis in Phaseolus Chloroplasts: I. RIBONUCLEIC ACID SYNTHESIS IN CHLOROPLAST PREPARATIONS FROM PHASEOLUS VULGARIS L. LEAVES AND SOLUBILIZATION OF THE RNA POLYMERASE

Chloroplast preparations from the young primary leaves of Phaseolusvulgaris L. cv. Canadian Wonder carry out the DNA-dependentincorporation of UTP into RNA at rates between 8 and 14 pmolUTP µg^–1 chlorophyll h^–1. It is estimatedthat 90% of the activity was localized in the chloroplasts.The incorporation proceeded for between 20 and 30 min at 35°C. The maximum rates of RNA synthesis were attained atpH 8.3, in the presence of 15 mM MgCl₂. Chloroplasts were alsoactive, to a lesser extent, with 1.5 mM MnCl₂. The simultaneouspresence of MnCl₂ and MgCl₂ resulted in inhibition of activity.Nuclear material prepared from young P. vulgaris leaves incorporatedUTP at a rate of about 12 pmol UTP µg^–1 DNA h^–1.On a chloroplast (Tritonsoluble) DNA basis chloroplast activitywas over 40-fold that of nuclei. Methods of solubilizing chloroplastRNA polymerase were explored. Yields of over 75% were achieved,but methods suitable for one species were not always successfulwhen applied to another. The highest yields of the P. vulgarisenzyme were obtained using EDTA and KCl. All methods resultedin solubilization of DNA. RNA synthesis by the soluble P. vulgarisenzyme proceeded for more than 40 min at 35 °C.     

Chloroplast Development in 4-Thiouridine-Cultured Radish Seedlings XI. Inhibition of RNA Synthesis by 4-Thiouridine Nucleotides during Germination of Radish Seeds

The effects of 4-thiouridine and its metabolites on RNA synthesisin radish (Raphanus sativus) cotyledons were investigated. The4-thiouridine nucleotides, 4-thioUTP and 4-thioUDP, were foundto inhibit DNA-dependent RNA polymerase activities of isolatednuclei and of chloroplasts from radish cotyledons. These inhibitorsappeared to compete with UTP for its binding to RNA polymerase.Neither 4-thiouridine nor 4-thioUMP inhibited RNA polymeraseactivities. Reduced RNA accumulation accompanied by the inhibition of protochlorophyllideaccumulation was observed in cotyledons of dark-grown radishseedlings germinated and grown with 4-thiouridine. On the otherhand, 4-thiouridine had no effect on chloroplast developmentin the control seedlings germinated and grown without 4-thiouridine.These results suggest that the inhibition of chloroplast developmentby 4-thiouridine may in part be due to the inhibition of RNAsynthesis by 4-thiouridine nucleotide during germination, resultingin inhibition of etioplast development. (Received December 9, 1985; Accepted June 26, 1986)     

Further studies on the diurnal rhythm of uridine incorporation into RNA in the long-day duckweed, Lemna gibba G3

Using the long-day duckweed Lemna gibba G3, the changes in theactivities of RNA synthesis in isolated nuclei and chloroplastsand of the reaction prerequisite for the incorporation of exogenousuridine into RNA were examined. When the duckweed was exposedto either a light-dark cycle or to continuous light, the activityof RNA synthesis in the nuclear and chloroplast fractions changeddiurnally and reached its highest levels during the night phase.The changes coincided with uridine incorporation into RNA invivo. However, the amount of radioactive uridine taken up intothe acid-soluble fraction remained unchanged during the wholeday. The proportion of radioactivity incorporated into phosphorylateduridine compounds as well as UTP+UDP to the radioactivity inthis fraction remained constant. Thus, the diurnal rhythm ofuridine incorporation into RNA was related to the diurnal rhythmof RNA synthesis in isolated nuclei and chloroplasts. The loweractivity of uridine incorporation into RNA under continuousdarkness may be determined by the activity of RNA synthesisin nuclei and chloroplasts as well as the uptake rate of uridineinto the duckweed cells, not by the activity of its phosphorylation. (Received August 30, 1977; )     

UTP inhibits Na+ absorption in wild-type and Delta F508 CFTR-expressing human bronchial epithelia

Ca²⁺-mediated agonists,including UTP, are being developed for therapeutic use in cysticfibrosis (CF) based on their ability to modulate alternativeCl conductances. As CF isalso characterized by hyperabsorption ofNa⁺, we determined the effect ofmucosal UTP on transepithelial Na⁺transport in primary cultures of human bronchial epithelia (HBE). Insymmetrical NaCl, UTP induced an initial increase in short-circuit current (I_sc)followed by a sustained inhibition. To differentiate between effects onNa⁺ absorption andCl secretion,I_sc was measuredin the absence of mucosal and serosal Cl(I_Na). Again,mucosal UTP induced an initial increase and then a sustained decreasethat reduced amiloride-sensitiveI_Na by 73%. TheCa²⁺-dependent agonists histamine,bradykinin, serosal UTP, and thapsigargin similarly induced sustainedinhibition (62-84%) ofI_Na. Mucosal UTPinduced similar sustained inhibition (half-maximal inhibitory concentration 296 nM) ofI_Na in primarycultures of human CF airway homozygous for the F508 mutation.BAPTA-AM blunted UTP-dependent inhibition ofI_Na, butinhibitors of protein kinase C (PKC) and phospholipaseA₂ had no effect. Indeed, directactivation of PKC by phorbol 12-myristate 13-acetate failed to inhibitNa⁺ absorption. Apyrase, a tri-and diphosphatase, did not reverse inhibitory effects of UTP onI_Na, suggesting along-term inhibitory effect of UTP that is independent of receptoroccupancy. After establishment of a mucosa-to-serosaK⁺ concentration gradient andpermeabilization of the mucosal membrane with nystatin, mucosal UTPinduced an initial increase in K⁺current followed by a sustained inhibition. We conclude that increasingcellular Ca²⁺ induces a long-terminhibition of transepithelial Na⁺transport across normal and CF HBE at least partly due todownregulation of a basolateral membraneK⁺ conductance. Thus UTP may havea dual therapeutic effect in CF airway:1) stimulation of aCl secretory response and2) inhibition ofNa⁺ transport.     

Relationships between the effect of O2 on energy metabolism and RNA synthesis in Rhodotorula gracilis

In the aerobic yeast Rhodotorula gracilis, a decrease in O₂availability, from saturating conditions (pO₂ = 100 mm Hg) topO₂ = 1 mm Hg, causes a decrease in the growth rate accompaniedby a fall in the accumulation rate of RNA and, to a lesser extent,of proteins. At pO₂ = 1, no relevant changes in the levels oftriphosphate nucleotides (ATP, GTP, CTP and UTP) are observedwith respect to the controls, while the ADP and AMP levels markedlyincrease. The uptake of labelled precursors and qualitative analysis ofRNA suggest that the decrease in the RNA accumulation rate atpO₂ = 1 is due to a decreased RNA synthesis rate. This paper examines the mechanism by which low availabilityof O₂ causes a decrease in the RNA synthesis rate even thoughtotal triphosphate nucleotide levels remain high. (Received December 28, 1977; )     

Substrate properties of C'-methylnucleoside triphosphates in a reaction of RNA synthesis catalyzed by Escherichia coli RNA-polymerase

2 theta-C-methyl substituted and phosphonate analogs of UTP were prepared and together with the synthesized earlier 3'-C-methyl-UTP were investigated in the RNA synthesis reaction catalysed by Escherichia coli RNA-polymerase. Substrate properties of UTP analogs were studied in the presence of all natural triphosphates, in the absence of UTP and under conditions of soil substrate reaction. It was shown that UTP(3'CH3) is incorporated into the RNA chain and terminates further RNA elongation. Another analog UTP (2'CH3) may substitute natural UTP in RNA synthesis, but the effectivity of its incorporation is diminished. Phosphonate analog UTP(5'CH2) is a pseudoterminator of RNA synthesis. The conformational analysis of 2'- and 3'C-methylnucleosides by force-field method of calculation was carried out in order to find energetically forbidden conformations of these analogs due to the collision of bulky methyl group and a heterocyclic base. An attempt was made to fix the conformation of the substrate during its enzymatic transformation.     

Possible in vitro repair of viral RNA by ligase-like enzyme(s) in poliovirus-infected cells.

A soluble polymerase-template complex prepared from poliovirus-infected cells was found to incorporate radioactive UTP into trichloroacetic acid-insoluble RNA linearly for 8 h in the presence of ATP and Mg2+. Radioactive CTP or GTP was not incorporated under identical conditions. Nearest-neighbor analysis of the in vitro product demonstrated that ATP was added to the viral RNA in the form of polyadenylic acid; UTP was added internally to the 3'-OH group of all four nucelotides. The data can best be explained by the addition of the UTP to the 3'-OH groups of single-stranded breaks in the double-stranded viral RNA and ligation to the adjacent 5'-phosphate groups. The enzymatic activity was also found in encephalomyocarditis virus- and rhinovirus type 1A-infected cells but not in uninfected cells.     

The relationship between the turnover of UTP and RNA synthesis in cultured cells treated with aflatoxin B1.

Methods have been adapted to measure the specific activity of UTP in cells in monolayer culture. In HeLa cells labelled with [3H]uridine and treated with aflatoxin B1 there was reduced radioactivity in crude acid extracts, but the toxin did not affect the radioactive incorporation into UTP. Using cells in which the UTP was pre-labelled, the subsequent addition of aflatoxin B1 inhibited UTP incorporation into RNA. Accordingly aflatoxin B1 did not inhibit the uptake of uridine or the latter's conversion to UTP but inhibited the incorporation of UTP into RNA.     

Incorporation of exogenous precursors into uridine and ribonucleic acid. Nucleotide compartmentation in the renal cortex in vivo

The possibility of compartmentation of UTP in vivo was investigated in the renal cortex of unanaesthetized rats. In addition, liver and spleen were studied in order to compare tissues with different utilization of precursors for pyrimidine nucleotide synthesis. After continuous 2h infusions of [(3)H]uridine or [(3)H]orotate, their incorporation into UTP, UDP-sugars and RNA was quantified. Rates of RNA synthesis were calculated by dividing the incorporation of precursor into RNA by the average specific radioactivity of the UTP pool. Although similar RNA-synthesis rates might have been expected with the two precursors, higher rates were found with uridine than with orotate. The relative incorporation into UDP-sugars of these precursors was also different. Similar results were obtained in the liver. In the spleen, equal amounts of both precursors were incorporated into UTP, but [(3)H]orotate incorporation did not lead to labelling of RNA. To evaluate the heterogeneity of cells with respect to the metabolism of pyrimidines, precursor incorporation was studied in isolated glomeruli and by radioautography. Incorporation into glomeruli was qualitatively similar to but quantitatively different from results in the renal cortex. Although there is obvious tissue heterogeneity, compartmentation of UTP pools is the most credible explanation for the results obtained with the renal cortex and liver. Consequently RNA and UDP-sugars may originate from two different UTP pools. Tissue heterogeneity is the likely explanation for the results obtained in the spleen. Studies of synthesis of pyrimidine and RNA, particularly in relation to growth and regeneration, must take into consideration the precursor used, the apparent existence of UTP compartmentation and the degree of cellular heterogeneity.     

Utilizations of various uridine 5'-triphosphate analogues by DNA-dependent RNA polymerases I and II purified from liver nuclei of the cherry salmon (Oncorhynchus masou)

Various 5-substituted UTPs (methyl, ethyl, n-propyl, n-butyl, fluoro, chloro, bromo, and iodo) and sulfur-containing UTP analogues (4-thio-, 2-thio-, 5-methyl-2-thio-, and 5-methyl-4-thio-) were synthesized chemically and their utilization by DNA-dependent-RNA polymerases I and II of the cherry salmon (Oncorhynchus masou) were studied in substitution experiments under the condition of limited RNA synthesis in vitro. RNA polymerase I utilized the 5-methyl-, chloro, bromo, and iodo derivatives of UTP more efficiently than unmodified UTP, but RNA polymerase II utilized UTP most efficiently. 5-Methyl-4-thiouridine 5'-triphosphate (4-thio TTP) was utilized more efficiently than UTP by RNA polymerase I. On the other hand, it was found that 4-thio TTP was a selective substrate for RNA polymerase I and that its incorporation by RNA polymerase II was very slow. Thus recognition of UTP analogues as substrates by RNA polymerase I and II was different. These observation were attributed from kinetic analyses to differences in catalytic activity (Vmax).     

Separate pyrimidine-nucleotide pools for messenger-RNA and ribosomal-RNA synthesis in HeLa S3 cells.

Kinetic analyses of mRNA and 28-S RNA labeling [3H]uridine revealed distinctly different steady-state specific radioactivities finally reached for uridine in mRNA and 28-S RNA when exogenous [3H]uridine was kept constant for several cell doubling times. While the steady-state label of (total) UTP and of uridine in mRNA responded to the same extent to a suppression of pyrimidine synthesis de novo by high uridine concentrations in the culture medium, uridine in 28-S RNA was scarcely influenced. Similar findings were obtained with respect to labeling of cytidine in the various RNA species due to an equilibration of UTP with CTP [5-3H]Uridine is also incorporated into deoxycytidine of DNA, presumably via dCTP. The specific radioactivity of this nucleosidase attained the same steady-state value as UTP, uridine in mRNA and cytidine in mRNA. The data indicate the existence of two pyrimidine nucleotide pools. One is a large, general UTP pool comprising the bulk of the cellular UTP and serving nucleoplasmic nucleic acid formation (uridine and cytidine in mRNA, deoxycytidine in DNA). Its replenishment by de novo synthesis can be suppressed completely by exogenous uridine above 100 muM concentrations. A second, very small UTP (and CTP) pool with a high turnover provides most of the precursors for nucleolar RNA formation (rRNA). This pool is not subject to feedback inhibition by extracellular uridine to an appreciable extent. Determinations of (total) UTP turnover also show that the bulk of cellular RNA (rRNA) cannot be derived from the large UTP pool.     

Differential inhibitory effects of 5-substituted 1-beta-D-xylofuranosyluracil 5'-triphosphates and related nucleotides on DNA-dependent RNA polymerases I and II from the cherry salmon (Oncorhynchus masou)

Various 5-substituted 1-beta-D-xylofuranosyluracil 5'-triphosphates (hydrogen, methyl-, ethyl-, n-propyl, n-butyl, fluoro-, chloro-, bromo-, and iodo derivatives) and some of the 3'-deoxyribofuranosyl nucleotides (3'-deoxy UTP and 3'-deoxy TTP) were synthesized chemically and their inhibitory effects on DNA-dependent RNA polymerases I and II of the cherry salmon (Oncorhynchus masou) were studied systematically. These 3'-modified UTP analogues could not be utilized as substrates in place of UTP, but they did inhibit the incorporation of UMP into RNA in vitro. In contrast, 2'-modified UTP analogues, such as 2'-dTTP and Ara TTP, were neither substrates nor inhibitors. Kinetic analysis showed that the inhibition by these compounds was essentially competitive with substrate UTP. The K1 values of RNA polymerase I for the analogues were smaller (2-6 microM) than the Km value for UTP (8 microM), but those for xylo-EtUTP, xylo-PrUTP, and xylo-BuUTP were larger (about 20 microM) than the Km for UTP. In contrast to these alkyl groups with steric and electron-donating effects, halogen groups have electron-withdrawing effects on the uracil nucleus. Therefore, it was concluded that the inhibitory activity of these analogues on RNA polymerase I was not affected by the inductive effects of substituent groups at the 5-position of uracil nucleus but by their steric effects. On the other hand, all of the K1 values of RNA polymerase II for UTP analogues were smaller (0.4-3 microM) than the Km value for UTP (4 microM). In this case, neither steric effect nor an inductive effect of substituents on UTP analogues influenced the inhibitory activity towards RNA polymerase II.     

Carbocyclic analogues of dTTP and UTP: properties in polymerase enzyme-catalyzed reactions

Racemic carbocyclic analogues of dTTP [(+/-)-C-dTTP] and its ribo counterpart, 5-methyl-UTP [(+/-)-C-m5UTP] were synthesized and examined, in comparison with dTTP and UTP (and m5UTP), as potential substrates of E. coli DNA and RNA polymerases, respectively. Unexpectedly, only a very low (terminal) incorporation of C-dTMP into DNAs of different structure was observed, C-dTTP did not serve as a substrate for chain elongation by the Klenow DNA polymerase. Inhibition of DNA replication was, however, observed in the presence of (+/-)-C-dTTP. The UTP analogue, (+/-)-C-m5UTP proved neither a substrate nor an inhibitor of the RNA polymerase enzyme.     

Synthesis of genomic and subgenomic RNA in mosquito cells infected with two Sindbis virus nsP4 mutants: influence of intracellular nucleoside triphosphate concentrations

Cells infected with Sindbis virus (SV) make two positive-strand RNAs, a genomic-length RNA (G) RNA and a subgenomic (SG) RNA. In cells infected with SVstd, and in general in cells infected with wt alphaviruses, more SG RNA is made than G RNA. How the balance between synthesis of G RNA and SG RNA is regulated is not known. SVpzf and SVcpc are nsP4 mutants of SV which, in mosquito cells, make more G RNA than SG RNA. When low concentrations of pyrazofurin (inhibits the synthesis of UTP and CTP) were added to SVpzf-infected cells, the yield of virus was increased, and the ratio of SG/G RNA was changed from <1 to >1. These effects were reversed by uridine. In SVcpc-infected cells, but not in SVstd-infected cells, synthesis of viral RNA was inhibited by the addition of either uridine or cytidine, and viral yields were lowered. Our findings suggest that the activities of the viral RNA-synthesizing complexes in cells infected with SVpzf or SVcpc, in contrast to those in SVstd-infected cells, are sensitive to high concentrations of UTP or CTP. Using a cell-free system that synthesizes both SG and G RNA, we measured viral RNA synthesis as a function of the UTP/CTP concentrations. The results indicated that the presence of the SVpzf mutations in nsP4 and the SG promoter produced a pattern quite different from that seen with the SVstd nsP4 and SG promoter. As the UTP/CTP concentrations were increased, the SVpzf system, in contrast to the SVstd system, made more G RNA than SG RNA, reflecting the situation in cells infected with SVpzf.     

P2 purinoceptors regulate calcium-activated chloride and fluid transport in 31EG4 mammary epithelia

It has been reported thatsecretory mammary epithelial cells (MEC) release ATP, UTP, and UDP uponmechanical stimulation. Here we examined the physiological changescaused by ATP/UTP in nontransformed, clonal mouse mammary epithelia(31EG4 cells). In control conditions, transepithelial potential (apicalside negative) and resistance were 4.4 ± 1.3 mV (mean ± SD, n = 12) and 517.7 ± 39.4  · cm², respectively. The apicalmembrane potential was 43.9 ± 1.7 mV, and the ratio of apicalto basolateral membrane resistance (R_A/R_B) was 3.5 ± 0.2. Addition of ATP or UTP to the apical or basolateral membranescaused large voltage and resistance changes with an EC₅₀ of~24 µM (apical) and ~30 µM (basal). Apical ATP/UTP (100 µM)depolarized apical membrane potential by 17.6 ± 0.8 mV (n = 7) and decreasedR_A/R_B by a factor of3. The addition of adenosine to either side (100 µM) hadno effect on any of these parameters. The ATP/UTP responses werepartially inhibited by DIDS and suramin and mediated by a transientincrease in free intracellular Ca²⁺ concentration (427 ± 206 nM; 15-25 µM ATP, apical; n = 6). This Ca²⁺ increase was blocked by cyclopiazonic acid, by BAPTA,or by xestospongin C. 31EG4 MEC monolayers also secreted or absorbedfluid in the resting state, and ATP or UTP increased fluid secretion by5.6 ± 3 µl · cm² · h¹(n = 10). Pharmacology experiments indicate that 31EG4epithelia contain P2Y₂ purinoceptors on the apical andbasolateral membranes, which upon activation stimulate apicalCa²⁺-dependent Cl channels and cause fluid secretion acrossthe monolayer. This suggests that extracellular nucleotides could playa fundamental role in mammary gland paracrine signaling and theregulation of milk composition in vivo.

     

Poliovirus cis-acting replication element-dependent VPg Uridylylation lowers the Km of the initiating nucleoside triphosphate for viral RNA replication

Initiation of RNA synthesis by RNA-dependent RNA polymerases occurs when a phosphodiester bond is formed between the first two nucleotides in the 5′ terminus of product RNA. The concentration of initiating nucleoside triphosphates (NTPi) required for RNA synthesis is typically greater than the concentration of NTPs required for elongation. VPg, a small viral protein, is covalently attached to the 5′ end of picornavirus negative- and positive-strand RNAs. A cis-acting replication element (CRE) within picornavirus RNAs serves as a template for the uridylylation of VPg, resulting in the synthesis of VPgpUpU_OH. Mutations within the CRE RNA structure prevent VPg uridylylation. While the tyrosine hydroxyl of VPg can prime negative-strand RNA synthesis in a CRE- and VPgpUpU_OH-independent manner, CRE-dependent VPgpUpU_OH synthesis is absolutely required for positive-strand RNA synthesis. As reported herein, low concentrations of UTP did not support negative-strand RNA synthesis when CRE-disrupting mutations prevented VPg uridylylation, whereas correspondingly low concentrations of CTP or GTP had no negative effects on the magnitude of CRE-independent negative-strand RNA synthesis. The experimental data indicate that CRE-dependent VPg uridylylation lowers the K_m of UTP required for viral RNA replication and that CRE-dependent VPgpUpU_OH synthesis was required for efficient negative-strand RNA synthesis, especially when UTP concentrations were limiting. By lowering the concentration of UTP needed for the initiation of RNA replication, CRE-dependent VPg uridylylation provides a mechanism for a more robust initiation of RNA replication.