Metabolism of Uridine 5′-Diphosphate-Glucose in Golgi Vesicles from Pea Stems

Uridine 5′-diphosphate-glucose (UDP-Glc) is transported into the lumen of the Golgi cisternae, where is used for polysaccharide biosynthesis. When Golgi vesicles were incubated with UDP-[³H]Glc, [³H]Glc was rapidly transferred to endogenous acceptors and UDP-Glc was undetectable in Golgi vesicles. This result indicated that a uridine-containing nucleotide was rapidly formed in the Golgi vesicles. Since little is known about the fate of the nucleotide derived from UDP-Glc, we analyzed the metabolism of the nucleotide moiety of UDP-Glc by incubating Golgi vesicles with [α-³²P]UDP-Glc, [β-³²P]UDP-Glc, and [³H]UDP-Glc and identifying the resulting products. After incubation of Golgi vesicles with these radiolabeled substrates we could detect only uridine 5′-monophosphate (UMP) and inorganic phosphate (Pi). UDP could not be detected, suggesting a rapid hydrolysis of UDP by the Golgi UDPase. The by-products of UDP hydrolysis, UMP and Pi, did not accumulate in the lumen, indicating that they were able to exit the Golgi lumen. The exit of UMP was stimulated by UDP-Glc, suggesting the presence of a putative UDP-Glc/UMP antiporter in the Golgi membrane. However, the exit of Pi was not stimulated by UDP-Glc, suggesting that the exit of Pi occurs via an independent membrane transporter.     

Uridine 5′-diphosphate glucose 4-epimerase from ehrlich ascites carcinoma cells

Uridine 5′-diphosphate glucose 4-epimerase (EC 5.1.3.2) from Ehrlich ascites carcinoma cells was purified to apparent homogeneity using conventional procedures and NAD-hexane-agarose affinity chromatography. The protein had a molecular weight of 96,000. The ascites enzyme had an absolute requirement for exogenously added NAD (10 ΜM) for stability. This appears to be a unique feature of ascites epimerase since epimerase from other mammalian sources did not exhibit such a dependence. Exogenously added NAD was also needed for catalysis with an apparentK _m value of 2.5 ΜM. NADH was a very potent competitive inhibitor (K _i = 0.11 ΜM with respect to NAD) of the enzyme activity at pH values close to intracellular pH. The dependence of the enzyme on NAD for stability and its inhibition by NADH may have some potential significance in tumor metabolism     

Stereoselective Synthesis of 1′-C-Branched Uracil Nucleosides from Uridine

Abstract

Face-selective electrophilic addition (bromo-pivaloyloxylation) to 1-[3,5-bis-O-(tert-butyldimethylsilyl)-2-deoxy-D-erythro-pent-1-enofurano-syl]uracil (1), when combined with nucleophilic substitution using organosilicon or organoaluminum reagents, provides a new and highly divergent C-C bond forming method at the anomeric position.     

Uridine 5′-diphosphate-xylose: anthocyanidin 3-O-glucose-xylosyltransferase from petals of Matthiola incana R.Br.

Petals of genetically defined lines of Matthiola incana R.Br. contain a glycosyltransferase which catalyzes the transfer of the xylosyl moiety of uridine 5-diphosphate-xylose to the glucose of cyanidin 3-glucoside. The enzyme also uses 3-glucosides of pelargonidin and delphinidin, cyanidin 3-(p-coumaroyl)-glucoside and 3-(caffeoyl)-glucoside as substrates. The xylosyltransferase exhibits a pH optimum of 6.5. The enzyme activity depends on the stage of bud and flower development. Accumulation of cyanidin 3-glucoside during flower development is correlated with xylosyltransferase activity.Abbreviations HPLC high-performance liquid chromatography - UDP uridine 5-diphosphate     

Synthesis of Uridylyl (3′-5′) Uridine Derivatives Containing 5-(Methylamino-Methyl) Uridine as A Modified Nucleoside Found from E. COLI Minor tRNAArg

Abstract

5-(Methylaminomethyl)uridine-containing uridylyl (3′-5′) uridine derivatives (14, 26, and 29), which were the original and modified sequences corresponding to the first letter (position 34) and the 5′-upper ribonucleoside (position 33) in the anticodon loop of minor tRNA^Arg, have been synthesized via 5-(methylaminomethyl)uridine derivatives (4 and 24).     

Chemical Conversion of Ribonucleoside 5′-S-Methyl Phosphorothiolates into Ribonucleotide Anhydrides Including Adenosine 5′-Triphosphate and Uridine Diphosphate Glucose

Ribonucleotide anhydrides have been prepared from corresponding ribonucleoside 5′-S-methyl phosphorothiolates by demethylthiolation with iodine in dry pyridine at room temperature in the presence of appropriate phosphates such as inorganic orthophosphate, inorganic pyrophosphate or glucose 1-phosphate. Thus synthesis of ribonucleotide anhydrides have been achieved and three ribonucleoside 5′-triphosphates (ATP, CTP and UTP), three ribonucleoside 5′-diphosphates (ADP, CDP and UDP) and a pyrophosphate coenzyme (UDPG) have been synthesized and isolated as lithium salts by charcoal treatment followed by ion exchange chromatography.     

Synthesis and Properties of 2′4′- and 3′-5′-Linked Ribodinucleoside Monophosphates Containing 2-Aminoadenosine and Uridine

Abstract

Self complementary diribonucleoside monophosphates containing 2-aminoadenosine (n²A) and uridine (U) residues, (2′-5′) n²ApU (1), (3′-5′) n²ApU (2), (2′-5′) Upn²A (3) and (3′-5′) Upn²A (4), were synthesized by condensation of suitably protected nucleoside and nucleotide units using dicyclohexylcarbodiimide (DCC). The dimers, (3) and (41, were also obtained from uridine 2′,3′-cyclic phosphate and unprotected 2-aminoadenosine using 2,4,6-triisopropylbenzenesulfonyl chloride (TPS-Cl) as the condensing agent. The conformational properties of these dimers were examined by UV, CD and NMR spectroscopy. The results reveal that the 2′-5′ isomers take a stacked conformation, which contains a larger base-base overlap and is more stable against thermal perturbation with respect to the 3′-5′ isomers. The n²ApU isomers have more stacked structure than the Upn²A isomers.     

5′-Nucleotidase from Yeast,Having Special Affinity for 5′-AMP

Three kinds of diketopiperazines which have retarditive activity for the growth of plant seedlings and plant roots at concentrations ranging from 1 : 2,500 to 1 : 100,000, were isolated from the neutral fraction by extracting the cultured broth of Rosellinia necatrix. These three diketopiperazines have been proved to be l-prolyl-l-leucine anhydride, l-prolyl-l-valine anhydride and l-prolyl-l-phenylalanine anhydride respectively, and the last one seems to be a new diketo-piperazine.

Furthermore, a crystalline wax having m.p. 52°C, a physiologically inactive substance, was also isolated from the same neutral fraction and presumed to be the saturated hydrocarbon of n-pentacosane C₂₅H₅₂.     

4-Substituted Uridine 5′-Triphosphates as Agonists of the P2Y2 Purinergic Receptor

Abstract

Uridine 5′-O-triphosphate (UTP) is a potent agonist of the purinergic receptor designated P_2Y2. UTP is rapidly metabolized in human tissue. To find a compound with similar activity that may be more slowly metabolized, a series of 4-substituted uridine 5′-triphosphates were prepared and evaluated in a P_2Y2 receptor second messenger assay.     

4′-hydroxy-3,5,6,7,3′,5′-hexamethoxyflavone from Murraya paniculata

4′-Hydroxy-3,5,6,7,3′,5′-hexamethoxyflavone has been isolated from the leaves of Sri Lankan Murraya paniculata.     

Adenosine 5′-triphosphate sulphurylase from Saccharomyces cerevisiae

1. ATP sulphurylase from Saccharomyces cerevisiae was purified 140-fold by using heat treatment, DEAE-cellulose chromatography and Sepharose 6B gel filtration. 2. The enzyme was stable at -15 degrees C, optimum reaction velocity was between pH7.0 and 9.0, and the activation energy was 62kJ/mol (14.7kcal/mol). 3. The substrate was shown to be the MgATP(2-) complex, free ATP being inhibitory. 4. Double-reciprocal plots from initial-velocity studies were intersecting and the K(m) of each substrate was determined at infinite concentration of the other (K(m) MgATP(2-), 0.07mm; MoO(4) (2-), 0.17mm). 5. Radio-isotopic exchange between the substrate pairs, adenosine 5'-[(35)S]sulphatophosphate and SO(4) (2-), (35)SO(4) (2-) and adenosine 5'-sulphatophosphate, occurred only in the presence of either MgATP(2-) or PP(i). This suggests, along with the initial-velocity data, a sequential reaction mechanism in which both substrates bind before any product is released. 6. The enzyme reaction was specific for ATP and was not inhibited by l-cysteine, l-methionine, SO(3) (2-), S(2)O(3) (2-) (all 2mm) nor by p-chloromercuribenzoate (1mm). 7. Competitive inhibition of the enzyme with respect to MoO(4) (2-) was produced by SO(4) (2-) (K(i)=2.0mm) and non-competitive inhibition by sulphide (K(i)=3.4mm). 8. Adenosine 5'-sulphatophosphate inhibited strongly and concentrations as low as 0.02mm altered the normal hyperbolic velocity-substrate curves with both MgATP(2-) and MoO(4) (2-) to sigmoidal forms.     

Adenosine-5′-phosphosulfate kinase from Thermobifida fusca

Sulfur, as a macronutrient, is essential for all kinds of organisms. Sulfate, the primary available source of sulfur, is firstly activated by adenylation catalyzed by ATP sulfurylase (ATPS) to form adenosine 5′-phosphosulfate (APS), which will be further phosphorylated into 3′-phosphoadenosine 5′-phosphosulfate (PAPS) by APS kinase (APSK). In some organisms, sulfate activating related enzymes are assembled to form sulfate-activating complex (SAC) to facilitate APS synthesis, the thermodynamically unfavorable reaction. In genome of a moderate thermophilic bacterium, Thermobifida fusca, there are presumably GTPasecoupled ATPS and one putative bifunctional ATPS/APSK type SAC. In this study, this putative SAC of T. fusca was prokaryotically expressed, purified and characterized. Activity assays showed that it contained APSK activity, while lacked ATPS activity. SAC of T. fusca was further used as a coupling enzyme to assay APS formation catalyzed by yeast ATPS. Based on the sequence alignment and modeled structure, we infer that the divergences of two conserved motifs and the missing of a loop and a helix-turn-helix motifs may contribute to the deficiency of ATPS activity.     

Excretion of 5′-Nucleotides from Bacterial Cells

Incubating the dried cells of Brevibacterium sojae No. 425-40 in alkaline buffer, the excretion of 5′-nucleotides accompanying with the decrease of intracellular RNA was observed. Then the determination of the optimum condition of the excretion and the investigation on the enzyme responsible for the degradation of endogenous RNA were carried out.

In the experiments using sonicate and disrupted cells, it appeared that orthophos-phate and Mg⁺⁺ might be accelerative or essential for the degradation of endogenous RNA and, in addition to four 5′-nucleotides (AMP, GMP, UMP and CMP), each nucleoside 5′-diphosphate was also contained in its degraded products. Nucleoside 2′- or 3′-monophos-phates were not detected. Although it was not clear whether phosphodiesterase concerned with the degradation of intracellular RNA or not, it was suggested that polynucleotide phos-phorylase acted mainly on the degradation.

The maximal excretion of 5′-nucleotides from dried cells was obtained by suspending 1 to 2% of dried cells in 0.05 M carbonate-bicarbonate buffer (pH 10) and incubating it at 60°C for two to three hours. Orthophosphate and Mg⁺⁺ were not required for the excretion.     

Cobalt-requiring 5′-deoxy-5′-methylthioadenosine nucleosidase from soybean (Glycine max) cotyledon

5??-Deoxy-5??-methylthioadenosine nucleosidase (MTA nucleosidase, EC 3.2.2.9) was purified from soybean (Glycine max) cotyledon. The nucleosidase was a trimer consisting of three identical subunits with a molecular mass of 59.5?kDa. The nucleosidase was a cobalt-requiring enzyme for its catalytic function. The enzymatic activity increased in a dose-dependent manner in the presence of cobalt. Cobalt was bound to the nucleosidase with a stoichiometry of 1 equivalent of cobalt/subunit. A thiol group-specific reagent reduced the enzymatic activity. Four cysteinyl residues of each subunit are considered to play an important role in binding cobalt.     

Continuous production of 5′-ribonucleotides from yeast RNA by hydrolysis with immobilized 5′-phosphodiesterase and 5′-adenylate deaminase

The production of 5-IMP and 5-GMP by enzymatic conversion from RNA using a continuous two packed-bed reactor was investigated. 5-Phosphodiesterase (5PD) and 5-adenylate deaminase (5AD) were immobilized in an acrylic resin to produce derivatives with about 15 U/g of support. The kinetic properties of the enzymes were described by Michaelis-Menten models: no significant differences were found in the K _m value of the free and immobilized 5AD (60 and 20 m, respectively), whereas for 5PD the K _m value was one order of magnitude higher for the immobilized enzyme (4.85 mg RNA/ml), probably due to diffusional limitations. Both enzymes remained stable after 8 h of use in a continuous packed-bed reactor whereas the half lives of the free enzymes were 193 min and 240 min at 40°C and 70°C for 5AD and 5PD, respectively. A procedure is proposed for the design of a continuous two packed-bed column process.F. Olmedo and F. Iturbe are with the Depto. de Alimentos y Biotecnologia, Facultad de Química, UNAM, México 04510, D.F., Mexico. J. Gomez-Hernández is with the Depto. de Biotecnología, UAM-1, Apdo. Postal 55-535, México 09340, D.F., Mexico. A. López-Munguía is with the Instituto de Biotecnología, Apartado Postal 510-3, Cuernavaca, Mor. 62271, Mexico     

A 2′-Deoxy-2′-Fluoro-2′-C-Methyl Uridine Cyclopentyl Carbocyclic Analog and Its Phosphoramidate Prodrug as Inhibitors of HCV NS5B Polymerase

The 2 ′-deoxy-2 ′-fluoro-2 ′-C-methyluridine nucleotide prodrug, PSI-7851 and its single diastereomer PSI-7977 have displayed potent antiviral activity against hepatitis C virus in clinical trials, and PSI-7977 is currently in Phase III studies. As part of our SAR study of the 2 ′-deoxy-2 ′-fluoro-2 ′- C-methyl class of nucleosides, we prepared the cyclopentyl carbocyclic uridine analog 11 and its phosphoramidate prodrug 15. Both 11 and 15 were shown not to inhibit HCV replication. This lack of activity might be attributed to the inability of the monophosphate to be converted to the corresponding diphosphate or triphosphate or the inactivity of triphosphate of 11 as an inhibitor of the polymerase.     

Overexpression of Inosine 5′-Monophosphate Dehydrogenase Type II Mediates Chemoresistance to Human Osteosarcoma Cells

Background

Chemoresistance is the principal reason for poor survival and disease recurrence in osteosarcoma patients. Inosine 5′-monophosphate dehydrogenase type II (IMPDH2) encodes the rate-limiting enzyme in the de novo guanine nucleotide biosynthesis and has been linked to cell growth, differentiation, and malignant transformation. In a previous study we identified IMPDH2 as an independent prognostic factor and observed frequent IMPDH2 overexpression in osteosarcoma patients with poor response to chemotherapy. The aim of this study was to provide evidence for direct involvement of IMPDH2 in the development of chemoresistance.

Methodology/Principal Findings

Stable cell lines overexpressing IMPDH2 and IMPDH2 knock-down cells were generated using the osteosarcoma cell line Saos-2 as parental cell line. Chemosensitivity, proliferation, and the expression of apoptosis-related proteins were analyzed by flow cytometry, WST-1-assay, and western blot analysis. Overexpression of IMPDH2 in Saos-2 cells induced strong chemoresistance against cisplatin and methotrexate. The observed chemoresistance was mediated at least in part by increased expression of the anti-apoptotic proteins Bcl-2, Mcl-1, and XIAP, reduced activation of caspase-9, and, consequently, reduced cleavage of the caspase substrate PARP. Pharmacological inhibition of IMPDH induced a moderate reduction of cell viability and a strong decrease of cell proliferation, but no increase in chemosensitivity. However, chemoresistant IMPDH2-overexpressing cells could be resensitized by RNA interference-mediated downregulation of IMPDH2.

Conclusions

IMPDH2 is directly involved in the development of chemoresistance in osteosarcoma cells, suggesting that targeting of IMPDH2 by RNAi or more effective pharmacological inhibitors in combination with chemotherapy might be a promising means of overcoming chemoresistance in osteosarcomas with high IMPDH2 expression.     

The 5′-Nor Aristeromycin Analogues of 5′-Deoxy-5′-Methylthioadenosine and 5′-Deoxy-5′-Thiophenyladenosine

To extend the potential of 5′-noraristeromycin (and its enantiomer) as potential antiviral candidates, the enantiomers of the carbocyclic 5′-nor derivatives of 5′-methylthio-5′-deoxyadenosine and 5′-phenylthio-5′-deoxyadenosine have been synthesized and evaluated. None of the compounds showed meaningful antiviral activity.     

Use of A Uridine Nucleotide as an Affinity Handle and 5′ Phosphorylating Agent for Synthetic DNA

Abstract

The 5′-(O-cyanoethyl N, N-diisopropyl phosphoramidite) of 2′,3′-O-bis(4,4′-dimethoxytrityl)uridine can be used to attach a uridine residue through a 5′-5′ phosphodiester linkage to a synthetic oligodeoxyribonucleotide. This 5′-terminal structure allows the oligomer to be selectively retarded on a chromatographic support containing dihydroxyboryl substituents, and to be converted upon periodate oxidation and p-elimination to the form possessing a 5′ phosphate group.