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At least 73 records · Page 4Linked to original sources
PubMed1997

Site-directed mutagenesis and characterization of uracil-DNA glycosylase inhibitor protein. Role of specific carboxylic amino acids in complex formation with Escherichia coli uracil-DNA glycosylase.

Bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) protein inactivates uracil-DNA glycosylase (Ung) by acting as a DNA mimic to bind Ung in an irreversible complex. Seven mutant Ugi proteins (E20I, E27A, E28L, E30L, E31L, D61G, and E78V) were created to assess the role of various negatively charged residues in the binding mechanism. Each mutant Ugi protein was purified and characterized with respect to inhibitor activity and Ung binding properties relative to the wild type Ugi. Analysis of the Ugi protein solution structures by nuclear magnetic resonance indicated that the mutant Ugi proteins were folded into the same general conformation as wild type Ugi. All seven of the Ugi proteins were capable of forming a Ung.Ugi complex but varied considerably in their individual ability to inhibit Ung activity. Like the wild type Ugi, five of the mutants formed an irreversible complex with Ung; however, the binding of Ugi E20I and E28L to Ung was shown to be reversible. The tertiary structure of [13C,15N]Ugi in complex with Ung was determined by solution state multi-dimensional nuclear magnetic resonance and compared with the unbound Ugi structure. Structural and functional analysis of these proteins have elucidated the two-step mechanism involved in Ung.Ugi association and irreversible complex formation.

Bacillus Phages↗
PubMed2003

Complexes of the uracil-DNA glycosylase inhibitor protein, Ugi, with Mycobacterium smegmatis and Mycobacterium tuberculosis uracil-DNA glycosylases.

Uracil, a promutagenic base, appears in DNA either by deamination of cytosine or by incorporation of dUMP by DNA polymerases. This unconventional base in DNA is removed by uracil-DNA glycosylase (UDG). Interestingly, a bacteriophage-encoded short polypeptide, UDG inhibitor (Ugi), specifically inhibits UDGs by forming a tight complex. Three-dimensional structures of the complexes of Ugi with UDGs from Escherichia coli, human and herpes simplex virus have shown that two of the structural elements in Ugi, the hydrophobic pocket and the beta1-edge, establish key interactions with UDGs. In this report the characterization of complexes of Ugi with UDGs from Mycobacterium tuberculosis, a pathogenic bacterium, and Mycobacterium smegmatis, a widely used model organism for the former, is described. Unlike the E. coli (Eco) UDG-Ugi complex, which is stable to treatment with 8 M urea, the mycobacterial UDG-Ugi complexes dissociate in 5-6 M urea. Furthermore, the Ugi from the complexes of mycobacterial UDGs can be exchanged by the DNA substrate. Interestingly, while EcoUDG sequestered Ugi into the EcoUDG-Ugi complex when incubated with mycobacterial UDG-Ugi complexes, even a large excess of mycobacterial UDGs failed to sequester Ugi from the EcoUDG-Ugi complex. However, the M. tuberculosis (Mtu) UDG-Ugi complex was seen when MtuUDG was incubated with M. smegmatis (Msm) UDG-Ugi or EcoUDG(L191G)-Ugi complexes. The reversible nature of the complexes of Ugi with mycobacterial UDGs (which naturally lack some of the structural elements important for interaction with the beta1-edge of Ugi) and with mutants of EcoUDG (which are deficient in interaction with the hydrophobic pocket of Ugi) highlights the significance of both classes of interaction in formation of UDG-Ugi complexes. Furthermore, it is shown that even though mycobacterial UDG-Ugi complexes dissociate in 5-6 M urea, Ugi is still a potent inhibitor of UDG activity in mycobacteria.

Amino Acid Sequence↗
PubMed2000

Solid-state transformation of 4, 2'-anhydro-5-(beta-D-arabinofuranosyl)uracil dihydrate to 4, 2'-anhydro-5-(beta-D-arabinofuranosyl)uracil monohydrate.

Crystals of 4,2'-anhydro-5-(beta-D-arabinofuranosyl)uracil, (I), obtained from an aqueous solution, were characterized as the dihydrate, C(9)H(10)N(2)O(5).2H(2)O, (Ia). In air, these crystals slowly transform to the monohydrate, C(9)H(10)N(2)O(5).H(2)O, (Ib), but remain crystalline. The solid-state transformation proceeds with the loss of one water molecule and a rearrangement of hydrogen-bonded layers of molecules. The furanose ring in (I) has an approximate C4'-exo,O4'-endo twist conformation. The central five-membered ring is slightly puckered. The uracil group is planar within experimental uncertainty.

Arabinofuranosyluracil↗
PubMed1969

Uracil photoproducts from uracil irradiated in ice.

Two new products were isolated from uracil irradiated with ultraviolet light in frozen aqueous solution. As judged by mass, nuclear magnetic resonance, and ultraviolet and infrared spectra, one is a photopolymer, U(3) and the structure of the other is probably 6-4'-[ pyrimidin-2'-one]-uracil. Formations of these products between pyrimidine bases are apparently common photoreactions, and may be important to the study of the photochemistry and photobiology of nucleic acids.

Ice↗
PubMed1986

Tumour uptake of radiolabelled pyrimidine bases and pyrimidine nucleosides in animal models. IX. Radiolabelled 1-(2'-fluoro-2'-deoxy-beta-D-ribofuranosyl)uracil and 1-(2'-chloro-2'-deoxy-beta-D-ribofuranosyl)uracil.

The biodistribution of radiolabelled 1-(2'-fluoro-2'-deoxy-beta-D-ribofuranosyl)uracil (2'-FUdR) and 1-(2'-chloro-2'-deoxy-beta-D-ribofuranosyl)uracil (2'-ClUdR) was evaluated using in-vivo and in-vitro tumour models. 6-[3H]-2'-FUdR exhibited a maximum tumour:blood (T:B) ratio (Walker 256 carcinosarcoma) of 1.0 at 15 min after injection whereas 2-[14C]-2'-FUdR and 2'-[36Cl]-2'-ClUdR exhibited maximum T:B ratios (Lewis lung carcinoma) of 3.0 and 4.1 respectively at 120 min. Clearance of blood radioactivity after injection of 6-[3H]-2'-FUdR, 2-[14C]-2'-FUdR and 2'-[36Cl]-2'-ClUdR was rapid and best described by a biexponential function. The clearance half-lives of the short-lived components were calculated as 1.6 min (95.5%), 2.3 min (94.7%) and 1.2 min (96.0%) respectively. The clearance half-lives of the long-lived components were 23 h (4.5%) for 6-[3H]-2'-FUdR, 89 min (5.3%) for 2-[14C]-2'-FUdR, and 49 min (4.0%) for 2'-[36Cl]-2'-ClUdR. Less than 36% of the 14C radioactivity present in the urine 2 h after injection of 2-[14C]-2'-FUdR was associated with 2'-FUdR, whereas greater than 91% of the 36Cl radioactivity in the urine 2 h after injection of 2'-[36Cl]-2'-ClUdR was associated with 2'-ClUdR. Both 6-[3H]-2'-FUdR (less than 3.1 pmol/10(6) cells) and 6-[3H]-2'-ClUdR (1.0 +/- 0.2 pmol/10(6) cells) were incorporated into cultured Raji tumour cells in vitro to a lesser extent than the natural nucleosides uridine (137.2 +/- 3.8 pmol/10(6) cells) and 2'-deoxyuridine (23.5 +/- 2.5 pmol/10(6) cells) after a 20 min incubation.

Animals↗
PubMed1993

Nucleoside analogues. 13. The effect on anti-tumour activity of varying the uracil 5-substituent and the point of attachment (N1 or N3) of the uracil moiety in seco-nucleoside nitrosoureas.

The high activity against colon tumours in the mouse of nitrosoureas linked to seco-nucleosides with 5-fluorouracil or uracil as base component led us to synthesize and test similar drugs carrying other 5-substituted uracils attached by either N1 or N3. Not only were some of the new drugs active against the solid MAC 13 and against mammary and lung tumours, but unlike earlier compounds were particularly effective (especially N3 and alkoxy drugs like B.4083) against the ascitic MAC 15A, causing some cures. Further, these agents are valuable tools in the study, using modern techniques, of bifunctional alkylation of biological macromolecules, a vital principle of experimental cancer chemotherapy about which our knowledge is still very incomplete.

Animals↗
PubMedDate not supplied

Chromatographic analysis (TLC) of uracil arabinoside, cytosine arabinoside, uracil and cytosine in human plasma.

Cytosine arabinoside, uracil arabinoside, cytosine and uracil after solid-phase extraction from plasma with minicolumns Adsorbex were separated by TLC on silica gel 60 GF254 by ascending and horizontal technique using suitable mobile phases. The substances were identified by UV irradiation at 254 nm and by spraying of 1% KMnO4 solution (up to the amount 500 ng analysed substances).

Arabinofuranosyluracil↗
PubMed1991

High-performance liquid chromatographic determination of 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil and its metabolite (E)-5-(2-bromovinyl)uracil in serum.

A high-performance liquid chromatographic method was developed to assay 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil and its metabolite (E)-5-(2-bromovinyl)uracil in serum. The chloro analogue of the parent drug is used as internal standard. Human serum samples were assayed to establish the pharmacokinetic parameters. Acetonitrile, used as a protein precipitant, was evaporated to dryness and the residue, containing the analytes and internal reference, was dissolved in mobile phase prior to chromatographic analysis. The minimum quantifiable level was 0.02 micrograms of each analyte per ml of serum.

Antiviral Agents↗
PubMed1994

The pyrimidine biosynthesis operon of the thermophile Bacillus caldolyticus includes genes for uracil phosphoribosyltransferase and uracil permease.

A 3-kb DNA segment of the Bacillus caldolyticus genome including the 5' end end of the pyr cluster has been cloned and sequenced. The sequence revealed the presence of two open reading frames, pyrR and pyrP, located immediately upstream of the previously sequenced pyrB gene encoding the pyrimidine biosynthesis enzyme aspartate transcarbamoylase. The pyrR and pyrP genes encoded polypeptides with calculated molecular masses of 19.9 and 45.2 kDa, respectively. Expression of these ORFs was confirmed by analysis of plasmid-encoded polypeptides in minicells. Sequence alignment and complementation analyses identified the pyrR gene product as a uracil phosphoribosyltransferase and the pyrP gene product as a membrane-bound uracil permease. By using promoter expression vectors, a 650-bp EcoRI-HincII fragment, including the 5' end of pyrR and its upstream region, was found to contain the pyr operon promoter. The transcriptional start point was located by primer extension at a position 153 bp upstream of the pyrR translation initiation codon, 7 bp 3' of a sequence resembling a sigma A-dependent Bacillus subtilis promoter. This established the following organization of the ten cistrons within the pyr operon: promoter-pyrR-pyrP-pyrB-pyrC-pyrAa-pyrA b-orf2-pyrD-pyrF-pyrE. The nucleotide sequences of the region upstream of pyrR and of the pyrR-pyrP and pyrP-pyrB intercistronic regions indicated that the transcript may form two mutually exclusive secondary structures within each of these regions. One of these structures resembled a rho-independent transcriptional terminator. The possible implication of these structures for pyrimidine regulation of the operon is discussed.

Amino Acid Sequence↗
PubMed1984

Direct determination of uracil in [32P,uracil-3H]poly(dA.dT) and bisulfite-treated phage PM2 DNA.

A simple but effective technique for determining the presence of uracil existing as either A:U base pairs or G:U base pairs in DNA was developed. DNA is degraded to deoxynucleoside 3'-monophosphates by a combination of micrococcal nuclease and spleen phosphodiesterase. The monophosphates are converted to 5'-end-labeled 32P-labeled diphosphates in a reaction catalyzed by T4 polynucleotide kinase. The resultant product is then converted to 5'-end-labeled deoxynucleoside monophosphates by P1 nuclease digestion, which specifically removes 3'-phosphates. Successful separation of labeled dUMP from conventional bases in DNA is achieved by two-dimensional polyethyleneimine chromatography, with its detection determined by autoradiography and liquid scintillation counting. The sensitivity of the technique described can detect a minimum 1 X 10(-16) mol of dUMP in DNA. Additionally, the detection of 5-methylcytosine in placental DNA demonstrates the flexibility of the technique for the analysis of modified bases in DNA.

5-Methylcytosine↗
PubMed1990

Use of the PBS2 uracil-DNA glycosylase inhibitor to differentiate the uracil-DNA glycosylase activities encoded by herpes simplex virus types 1 and 2.

The bacteriophage PBS2 encoded uracil-DNA glycosylase (UNG) inhibitor was examined for its effect upon the nuclear UNG activities of KB, HeLa, and Vero cells infected with herpes simplex virus (HSV) type 1 or 2 and mock-infected cells. UNG activity from HSV-1 infected cells exhibited the greatest sensitivity to inhibition by the inhibitor, while UNG activity from cells infected with HSV-2 exhibited the greatest resistance. This differential effect was dependent upon the virus, cell line, and buffer system used in the reaction. Furthermore, the PBS2 UNG inhibitor's differential effect, provides a means of distinguishing the herpesvirus UNG activities from one another, and from the cellular UNG activity. Therefore, this method of identification should prove to be useful for the purification and characterization of the viral enzymes from infected cell nuclear extracts.

Cells, Cultured↗
PubMed1995

[Blood levels of 5-FU and uracil on additional use of uracil in UFT therapy in gastrectomized patients].

Our previous report made it clear that on UFT therapy in the gastrectomized patients reconstructed with Billroth II or various types of esophagojejunostomy, Futraful (FT) kept remained as the non-metabolized in the blood for a long time, but Uracil (Ura) or derived 5-FU merely increased initially. In order to further utilize the remaining FT, additional Ura equivalent to the original composition of UFT was given, once or fractionally at one or two hours after receiving UFT, and consecutive blood levels of FT, Ura and 5-FU were compared with each control without additional use of Ura. When Ura was given one hour after per os UFT, the high blood level of 5-FU lasted another hour, and given at two hours later the concentration curve of 5-FU with two peaks was obtained. The area under curve of 5-FU during the observation period in all the modes of addition reached approximately two-fold against controls. On the other hand, that of Ura increased to 4- to 6-fold compared with each control. Therefore, if Ura is given intermittently on UFT therapy for the gastrectomized patients mentioned above, it would be possible to utilize the remaining FT by inhibiting the degradation of 5-FU derived from it, and Ura might be given in a smaller dose on this application.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1995

Manual and automated determination of 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil and its metabolite (E)-5-(2-bromovinyl)uracil in urine.

This paper describes the determination of 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil in urine. The method involves sample clean-up by liquid-liquid extraction with ethyl acetate followed by high-performance liquid chromatographic (HPLC) analysis. The sample preparation may be performed either manually or automatically using a Zymark Py-robotic system. The chloro analog of the parent compound, CV-araU, is used as the internal standard. As low as 0.1 microgram of analyte per ml of urine can be measured. This sensitivity is adequate for pharmacokinetic studies but could be improved quite easily if necessary.

Antiviral Agents↗