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J A Harrington

Publications and source records attributed to J A Harrington.

At least 19 recordsLinked to original sources
PubMed2001

Art or science? Understanding medicine and the common law.

Those of you who have either had or seen the process of a magnetic resonance imaging (MRI) scan cannot fail to be impressed with the scientific or technologic marvel of this huge diagnostic machinery. After a relatively noisy series of manoeuvres, the machine turns out a beautiful set of images on film. That is the science. What happens next, though, is that the films are given to a radiologist who gazes at them, puzzles for awhile and then gives an opinion on what they might mean. That is the art.

Art↗
PubMed1995

Enzymatic elimination of fluoride from alpha-fluoro-beta-alanine.

Rat liver homogenates catalyzed the elimination of fluoride from (R,S)-alpha-fluoro-beta-alanine. The substrate specificity and physical properties of the defluorinating enzyme were similar to those of mitochondrial L-alanine-glyoxylate aminotransferase II (EC 2.6.1.44, AlaAT-II). Furthermore, AlaAT-II activity, measured with L-alanine and glyoxylate as substrates, copurified with the alpha-fluoro-beta-alanine-defluorinating enzyme. The NH2-terminal sequence (18 residues) of the enzyme did not show significant sequence similarity with any of the proteins currently listed in GenBank. The purified enzyme catalyzed the transamination of L-alanine (Ala) and glyoxylate (glyx) at pH 8.5 by a ping-pong mechanism with kinetic parameters of kcat = 17 sec-1, KL-Ala = 3.2 mM, and Kglyx = 0.3 mM, respectively. The kinetic parameters for the defluorination of (R)-alpha-fluoro-beta-alanine and (S)-alpha-fluoro-beta-alanine were kcat = 6.2 and 2.6 min-1, respectively, and Km = 2.7 and 0.88 mM, respectively. L-Alanine potently inhibited the defluorination reaction with an apparent Ki of 0.024 mM. (R,S)-alpha-Fluoro-beta-alanine converted the optical spectrum of the enzyme-bound cofactor from the pyridoxal form to the pyridoxamino form, which indicated that this cofactor may participate in the defluorination reaction. The product of the enzymatic reaction, malonic semialdehyde, reacted nonenzymatically with (R,S)-alpha-fluoro-beta-alanine to form an adduct that was detected spectrally. AlaAT-II was not inactivated during dehalogenation of (R,S)-alpha-fluoro-beta-alanine but was inactivated completely during dehalogenation of beta-chloro-L-alanine.

Alanine↗
PubMed1995

Purification and characterization of a rat liver enzyme that hydrolyzes valaciclovir, the L-valyl ester prodrug of acyclovir.

Valaciclovir is an oral prodrug of the antiherpetic agent acyclovir. An enzyme that hydrolyzes valaciclovir to acyclovir, valaciclovir hydrolase (VACVase), was purified from rat liver and characterized. VACVase was a basic (pI 9.4) protein associated with mitochondria. It was monomeric and had a molecular mass of 29 kDa. Amino acid sequences of six VACVase peptides, including its NH2 terminus (13 amino acids) and accounting for approximately 20% of its complete sequence, were not found in the SwissProt protein data base. VACVase hydrolyzed other amino acid esters of acyclovir in addition to valaciclovir (kcat/Km = 58 mM-1 s-1), with a preference for the L-alanyl (kcat/Km = 226 mM-1 s-1) and L-methionyl (kcat/Km = 200 mM-1 s-1) esters. It did not hydrolyze other types of esters or numerous di- and tripeptides and aminoacyl-beta-naphthylamides. Hydrolysis of valaciclovir by VACVase was not inhibited by amastatin, antipain, aprotinin, bestatin, chymostatin, E-64, EDTA, ebelactone A, ebelactone B, elastatinal, leupeptin, pepstatin, or phosphoramidon. It was neither inhibited nor activated by Ca2+, Co2+, Mg2+, Mn2+, or Zn2+. Therefore, this enzyme is not a typical esterase or peptidase and, to our knowledge, it has not been described previously. Its physiological function is not known; however, it may play a significant role in the biotransformation of valaciclovir to acyclovir.

Acyclovir↗
PubMed1995

Attenuation of the antitumor activity of 5-fluorouracil by (R)-5-fluoro-5,6-dihydrouracil.

5-Ethynyluracil (5-EU; 776C85) is a potent mechanism-based inactivator of dihydropyrimidine dehydrogenase that improves the antitumor activity of 5-fluorouracil (5-FU) to a greater extent than can be accounted for by the improved 5-FU pharmacokinetics that result from preventing the catabolism of 5-FU. We therefore tested the effects of (R)-5-fluoro-5,6-dihydrouracil (5-FUH2), the 5-FU catabolite extensively formed in the absence of 5-EU, on the antitumor activity and toxicity of 5-FU in 5-EU-treated rats bearing large s.c. tumors. Rats were dosed once weekly for 3 weeks with the following regimens: 100 mg/kg 5-FU (maximum tolerated dose), 10 mg/kg 5-FU 1 h after 1 mg/kg 5-EU, or 10 mg/kg 5-FU plus 90 mg/kg 5-FUH2 1 h after 1 mg/kg 5-EU. The latter regimen was designed to approximate the exposure produced from 5-FU in the absence of 5-EU, where > 80% of the dose is catabolized. 5-FU produced complete and sustained tumor regressions in 94% of the animals pretreated with 5-EU. In contrast, 5-FU in combination with 5-FUH2 produced complete regression in only 38% of the 5-EU-treated rats, which was similar to the antitumor activity of 5-FU in the absence of 5-EU. All treatments resulted in 7-11% transient weight loss. 5-FU produced no other notable toxicity in 5-EU-treated rats. However, 5-FUH2 added to this regimen caused transient diarrhea and stomatitis in 13% of the animals, which was similar to the toxicity produced by 5-FU in the absence of 5-EU. Thus, 5-FUH2, or other downstream catabolites of 5-FU, impaired the antitumor activity and slightly increased the toxicity of 5-FU. Accordingly, 5-EU approved to improve the efficacy of 5-FU by preventing the formation of 5-FU catabolites.

Animals↗
PubMed1994

(R)-5-fluoro-5,6-dihydrouracil: kinetics of oxidation by dihydropyrimidine dehydrogenase and hydrolysis by dihydropyrimidine aminohydrolase.

The biologically active isomer of 5-fluoro-5,6-dihydrouracil [(R)-5-fluoro-5,6-dihydrouracil, R-FUH2] was synthesized to study the kinetics of its enzymatic oxidation and hydrolysis by homogeneous dihydropyrimidine dehydrogenase (DPDase) and dihydropyrimidine aminohydrolase (DPHase), respectively. DPDase catalyzed the slow oxidation of R-FUH2 at pH 8 and 37 degrees with a Km of 210 microM and a kcat of 0.026 sec-1 at a saturating concentration of NADP+. The catalytic efficiency (kcat/Km) of DPDase for R-FUH2 was 1/14th of that for 5,6-dihydrouracil (UH2). In the opposite direction, DPDase catalyzed the reduction of 5-fluorouracil (FU) with a Km of 0.70 microM and a kcat of 3 sec-1 at a saturating concentration of NADPH. Thus, DPDase catalyzed the reduction of FU 30,000-fold more efficiently than the oxidation of R-FUH2. In contrast to the slow oxidation of R-FUH2 by DPDase, R-FUH2 was hydrolyzed very efficiently by DPHase with a Km of 130 microM and a kcat of 126 sec-1. The catalytic efficiency of DPHase for the hydrolysis of R-FUH2 was approximately twice that for the hydrolysis of UH2. Because R-FUH2 is hydrolysis of R-FUH2 was approximately twice that for the hydrolysis of UH2. Because R-FUH2 is hydrolyzed considerably more efficiently than it is oxidized and because the activity of DPHase was 250- to 500-fold greater than that of DPDase in bovine and rat liver, the hydrolytic pathway should predominate in vivo.

Amidohydrolases↗
PubMed1994

5-ethynyl-2(1H)-pyrimidinone: aldehyde oxidase-activation to 5-ethynyluracil, a mechanism-based inactivator of dihydropyrimidine dehydrogenase.

5-Ethynyluracil is a potent mechanism-based inactivator of dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2) in vitro (Porter et al., J Biol Chem 267: 5236-5242, 1992) and in vivo (Spector et al., Biochem Pharmacol, 46: 2243-2248, 1993. 5-Ethynyl-2(1H)-pyrimidinone was rapidly oxidized to 5-ethynyluracil by aldehyde oxidase. The substrate efficiency (kcat/Km) was 60-fold greater than that for N-methylnicotinamide. In contrast, xanthine oxidase oxidized 5-ethynyl-2(1H)-pyrimidinone to 5-ethynyluracil with a substrate efficiency that was only 0.02% that of xanthine. Because 5-ethynyl-2(1H)-pyrimidinone did not itself inactivate purified DPD in vitro and aldehyde oxidase is predominately found in liver, we hypothesized that 5-ethynyl-2(1H)-pyrimidinone could be a liver-specific inactivator of DPD. We found that 5-ethynyl-2(1H)-pyrimidinone administered orally to rats at 2 micrograms/kg inactivated DPD in all tissues studied. Although 5-ethynyl-2(1H)-pyrimidinone produced slightly less inactivation than 5-ethynyluracil, the two compounds showed fairly similar patterns of inactivation of DPD in these tissues. At doses of 20 micrograms/kg, however, 5-ethynyl-2-pyrimidinone and 5-ethynyluracil produced equivalent inactivation of DPD. Thus, 5-ethynyl-2(1H)-pyrimidinone appeared to be an efficient, but not highly liver-selective prodrug of 5-ethynyluracil.

Aldehyde Oxidase↗
PubMed1993

5-Ethynyluracil (776C85): inactivation of dihydropyrimidine dehydrogenase in vivo.

5-Ethynyluracil (776C85), a potent, mechanism-based, irreversible inactivator (Porter et al., J Biol Chem 267:5236-5242, 1992) of purified dihydropyrimidine dehydrogenase (DPD, uracil reductase, EC 1.3.1.2), readily inactivated DPD in vivo. DPD was assayed in tissue extracts by measuring the release of 14CO2 from [2-14C]uracil with an improved method. Specific activities from 0.1 to > 1000 U/mg protein were reproducibly measured. After rats were orally dosed with 20 micrograms/kg 5-ethynyluracil, liver, intestinal mucosa, lung, and spleen DPD were inactivated by 83-94%. The dose required to inactivate rat liver, rat brain, and mouse liver DPD by 50% was 1.8, 11, and 8.9 micrograms/kg, respectively. Rat liver DPD was inactivated completely within 25 min after an oral dose of 500 micrograms/kg 5-ethynyluracil. New DPD was synthesized with a half-time of 63 hr. We also developed an assay based on stoichiometric inactivation of DPD by 5-ethynyluracil to measure 5-ethynyluracil in plasma samples. Samples containing 5-ethynyluracil were incubated with rat liver extract for 24 hr at 12 degrees and then assayed for DPD. DPD activity decreased linearly with the concentration of 5-ethynyluracil (between 0 and 20 nM 5-ethynyluracil). The assay could detect 5-ethynyluracil at concentrations as low as 6 nM in human plasma and was not affected by high concentrations of uracil.

Animals↗
PubMed1993

3'-azido-3'-deoxythymidine (AZT) monophosphate: an inhibitor of exonucleolytic repair of AZT-terminated DNA.

A 3'-exonuclease(s) that excised 3'-azido-3'-deoxythymidine (AZT) monophosphate (AZTMP) from the 3' terminus of DNA was partially purified from two human cell lines. AZTMP inhibited the hydrolysis of AZTMP-terminated single-stranded and double-stranded DNA substrates. Thus, high levels of AZTMP might inhibit the exonuclease and trigger the toxicity of AZT by impairing the repair of AZTMP-terminated DNA.

Cell Line↗
PubMed1992

2-Acetylpyridine thiocarbonohydrazones. Potent inactivators of herpes simplex virus ribonucleotide reductase.

A series of 2-acetylpyridine thiocarbonohydrazones was synthesized for evaluation as potential antiherpetic agents. The compounds were prepared by the condensation of 2-acetylpyridine with thiocarbonohydrazide followed by treatment with isocyanates or isothiocyanates. Many were found that were potent inactivators of ribonucleotide reductase encoded by HSV-1 and weaker inactivators of human enzyme. Several thiocarbonohydrazones (e.g. 38 and 39) inactivated HSV-1 ribonucleotide reductase at rate constants as much as seven times that of lead compound 2. In general, those substituted with weak electron-attracting groups offered the best combination of potency and apparent selective activity against the HSV-1 enzyme. Seven new thiocarbonohydrazones (21, 25, 31, 36, 38, 39, and 40) were apparently greater than 50-fold more selective than 2 against HSV-1 ribonucleotide reductase versus human enzyme. The results indicated new compounds worthy of further study as potentiators of acyclovir in combination topical treatment of herpes virus infections.

Antiviral Agents↗
PubMed1992

Ab-interno neodymium:YAG versus erbium:YAG laser sclerostomies in a rabbit model.

This study was undertaken to determine whether thermally-induced tissue necrosis was a factor in ab-interno contact-laser sclerostomy failure. A rabbit model was used to compare the continuous-wave Neodymium (Nd):YAG with the pulsed Erbium (Er):YAG laser with respect to such failure. Laser energy was focused into a fused-silica fiber optic (400 microns) for the Nd:YAG laser (12 W; 3 to 5 seconds), and into a single-crystal, uncladded sapphire fiber optic (250 microns) for the Er:YAG laser (7 to 8 mJ; 250 microseconds; 6 to 8 pulses). The Nd:YAG and Er:YAG lasers required from 21 to 35 J and from 42 to 64 mJ, respectively, to create the sclerostomies. Filtering blebs and intraocular pressure reduction lasted longer (log-rank test; P less than .03) and surgical complications were fewer in the Er:YAG group than in the Nd:YAG group. By creating sclerostomies with minimal thermal damage, the Er:YAG laser may offer significant clinical advantages over lasers producing larger thermal effects.

Animals↗
PubMed1991

Human ribonucleotide reductase. Activation and inhibition by analogs of ATP.

Sixteen ATP analogs were studied as activators of CDP reduction catalyzed by human ribonucleotide reductase. Activation constants were determined. Three analogs, 3-deazaATP, 5'-adenylimidodiphosphate, and 3'-dATP, activated approximately as efficiently as ATP. Four analogs were partial activators. These seven activators were also accessory activators of GDP reduction. Furthermore, two other analogs, adenosine-5'-O-(1-thiotriphosphate) and 8-bromoATP, selectively stimulated GDP reduction. Ten analogs, at equal molar concentrations with ATP, inhibited ATP-dependent activation of CDP reduction and/or accessory activation of GDP reduction by greater than 45%. No analog inhibited as potently as 2'-dATP, which had an IC50 of 30-50 microM versus the stimulation of CDP and GDP reduction by 2.0 mM ATP.

Adenosine Triphosphate↗
PubMed1991

Herpes and human ribonucleotide reductases. Inhibition by 2-acetylpyridine 5-[(2-chloroanilino)-thiocarbonyl]-thiocarbonohydrazone (348U87).

The mode of inactivation of herpes simplex virus type 1 and human ribonucleotide reductases by 2-acetylpyridine 5-[(2-chloroanilino)-thiocarbonyl]-thiocarbonohydrazone++ + (348U87) was determined and compared to that described previously [Porter et al. Biochem Pharmacol 39: 639-646, 1990] for 2-acetylpyridine 5-[(dimethylamino)thiocarbonyl]-thiocarbonohydrazone (A1110U). 348U87 inactivated herpes ribonucleotide reductase faster than did A1110U. Moreover, iron-complexed 348U87 was a considerably more potent inactivator than iron-complexed A1110U. It appeared to efficiently form an initial complex with the viral enzyme prior to rapid enzyme inactivation. The combination of 348U87 and iron-complexed 348U87 inactivated with a rate constant that was slightly greater than the sum of their individual rate constants of inactivation. The corresponding combination of A1110U species inactivated with a rate constant that was much greater than the sum of the individual rate constants of inactivation. Herpes ribonucleotide reductase that had been inactivated by either species of 348U87 was reactivated by diluting the enzyme and inactivators into assay medium containing excess iron. 348U87 was also an effective inactivator of herpes simplex virus type 2 and varicella zoster virus ribonucleotide reductases. The iron-complexed forms of 348U87 and A1110U exhibited very different modes of inactivation of human ribonucleotide reductase. Iron-complexed 348U87 was a tight-binding inactivator, whereas iron-complexed A1110U was only a weak, non-inactivating, inhibitor. Furthermore, the inactivation by iron-complexed 348U87 was not stimulated by either 348U87 or A1110U, whereas the weak inhibition by iron-complexed A1110U was converted to rapid inactivation by A1110U. Excess iron prevented the inactivation by iron-complexed 348U87. Uncomplexed 348U87 was similar to uncomplexed A1110U in that it was not an inhibitor of the human enzyme.

Antiviral Agents↗
PubMed1990

Herpes simplex virus type 1 ribonucleotide reductase: selective and synergistic inactivation by A1110U and its iron complex.

2-Acetylpyridine-5-[(dimethylamino)thiocarbonyl]thiocarbonohydr azone (A1110U) inactivated herpes simplex virus Type 1 ribonucleotide reductase (EC 1.17.4.1) by a first-order process (kinact) which had a maximum value (Mkinact) of 8 hr-1 and a Kd that was less than 1 microM. The stable complex between iron and A1110U, (A1110U)2Fe+i, inactivated this enzyme with a Mkinact of 7 hr-1 and a Kd of 7 microM. Free A1110U and its iron-complex synergized as inactivators of the enzyme. For example, the kinact for the combination of 2 microM A1110U and 1 microM (A1110U)2Fe+i as independent inactivators was calculated to be about 9 hr-1, while the observed value was 32 hr-1. The bimolecular rate constant for inactivation of the viral enzyme by (A1110U)2Fe+i in the presence of a saturating concentration of A1110U was 2.5 10(7) M-1 hr-1 at 30 degrees. Human ribonucleotide reductase was less sensitive to the inhibitory effects of A1110U and its iron-complex. This enzyme was neither inhibited nor inactivated by A1110U and was weakly inhibited by (A1110U)2Fe+i. Furthermore, inactivation required the combination of A1110U and (A1110U)2Fe+i. The bimolecular rate constant for inactivation of human ribonucleotide reductase by (A1110U)2Fe+i in the presence of a saturating concentration of A1110U was considerably smaller (3.8 10(6) M-1 hr-1 at 37 degrees) than the analogous constant for the viral enzyme. Several iron-chelating reagents with unrelated structures substituted for free A1110U in its various roles with both enzymes. However, the iron complexes of these alternative chelators did not substitute for (A1110U)2Fe+i. The rates of inactivation of both enzymes were independent of the oxidation state of iron in (A1110U)2Fe+i and of CDP concentration. The inactivated enzymes were reactivated rapidly by FeSO4, but were not reactivated by CoCl2, CuSO4, or NiCl2. MnCl2 inhibited reactivation of the viral enzyme by FeSO4.

Adenosine Triphosphate↗
PubMed1989

Purine deoxynucleoside salvage in Giardia lamblia.

Giardia lamblia is dependent on the salvage of preformed purines and pyrimidines, including deoxythymidine. Dependence on deoxynucleoside salvage is extremely unusual among eucaryotic cells (Moore, E. C., and Hurlbert, R. B. (1985) Pharmacol & Ther. 27, 167-196). The present study investigates the possibility that giardia lacks ribonucleotide reductase and depends entirely on deoxynucleoside salvage. A ribonucleotide reductase inhibitor, hydroxyurea, at concentrations up to 2 mM had no effect on the growth of giardia. This is 15-20 times the ED50 of hydroxyurea for the protozoans Trypanosoma cruzi, Trypanosoma gambiense, and Leishmania donovani. A lysate of giardia had no detectable ribonucleotide reductase. Although radiolabeled adenine, adenosine, guanine, and guanosine were readily incorporated into RNA by cultured cells, no adenine or adenosine and only trace amounts of guanine and guanosine were detectable in DNA. This is in contrast to deoxynucleosides, where 58% of deoxyadenosine and 10% of deoxyguanosine incorporated into nucleic acid were found in DNA. Phosphorylation of both deoxyadenosine and deoxyguanosine was catalyzed by a cell lysate of giardia when nucleoside kinase co-substrates were included in the assay but not when phosphotransferase co-substrates were present. The absence of detectable ribonucleotide reductase, the failure to incorporate purine nucleobases and nucleosides into DNA to any significant extent, the ready incorporation of deoxynucleosides into DNA, and the demonstration of a purine deoxynucleoside kinase suggest that giardia are dependent on the salvage of exogenous deoxynucleosides.

Animals↗
PubMed1989

Rapid sampling of multiple enzyme reactions.

A simple method of initiating and sampling six simultaneous reactions was devised. A commercially available vial rack was fitted with a Plexiglas overlaying sheet to stabilize the vials for the addition and sampling procedures. Glass vials were routinely used because of their thermal conductivity advantages. Samples were added and removed, and the reactions were mixed with a multichannel pipet using every other channel. The data showing six simultaneous progress curves for the rapid inactivation of herpes simplex virus ribonucleotide reductase were presented and analyzed. In addition, the time course of 12 reactions catalyzed by varicella zoster virus thymidine kinase were assayed at one min intervals generating 96 data points within 8.5 min. A second experiment generated data points every 30 s for six simultaneous replicate thymidine kinase reactions. The ease of use and high reproducibility of the method are demonstrated by these data.

Enzyme Activation↗
PubMed1989

2-Acetylpyridine 5-[(dimethylamino)thiocarbonyl]-thiocarbonohydrazone (A1110U), a potent inactivator of ribonucleotide reductases of herpes simplex and varicella-zoster viruses and a potentiator of acyclovir.

2-Acetylpyridine 5-[(dimethylamino)thiocarbonyl]thiocarbonohydrazone (A1110U) was found to be a potent inactivator of the ribonucleotide reductases (EC 1.17.4.1) encoded by herpes simplex virus types 1 and 2 and by varicella-zoster virus and to be a weaker inactivator of human ribonucleotide reductase. It also markedly potentiated the antiherpetic activity of acyclovir against these viruses in tissue culture. A1110U both decreased the dGTP pool that builds up when infected cells are treated with acyclovir and induced a large increase in the pool of acyclovir triphosphate. The resultant 100-fold increase in the ratio of the concentrations of acyclovir triphosphate to dGTP should facilitate the binding of the fraudulent nucleotide to its target enzyme, herpes virus-encoded DNA polymerase, and could account for the synergy between A1110U and acyclovir. A similar change in the acyclovir triphosphate-to-dGTP ratio was previously reported to be induced by another ribonucleotide reductase inhibitor, 2-acetylpyridine 4-(2-morpholinoethyl)thiosemicarbazone (A723U). However, A1110U is considerably more potent and may have better clinical potential. Synergistic toxic interactions between A1110U and acyclovir were not detected in uninfected cells.

Acyclovir↗
PubMed1987

Effector studies of 3'-azidothymidine nucleotides with human ribonucleotide reductase.

The 5'-mono-, di- and triphosphate derivatives (N3dTMP, N3dTDP and N3dTTP respectively) of 3'-azidothymidine (N3dThd), a new drug for the treatment of the acquired immune deficiency syndrome (AIDS), were synthesized. The abilities of these analog nucleotides to mimic the effector properties of the corresponding thymidine nucleotides with human ribonucleotide reductase were studied. Surprisingly, the mode of inhibition of CDP reduction by dTTP and dTDP was found to be competitive versus CDP. The Ki values were 22 and 78 microM respectively. Inhibition by N3dTTP and N3dTDP was considerably weaker, with Ki values of 1200 and 550 microM. Neither dTMP nor N3dTMP produced significant inhibition at concentrations up to 500 microM. dTTP was an essential activator for GDP reduction. In the presence of the accessory activator, ATP, the activation constant for dTTP was 7.8 microM. N3dTTP was neither an activator of GDP reduction nor an inhibitor of the activation by dTTP. In view of the intracellular concentrations of these analog nucleotides reached after incubations with N3dThd [Furman et al., Proc. natn. Acad. Sci. U.S.A. 83, 8333 (1986)] and the weakness of their interactions with ribonucleotide reductase, it is unlikely that the antiviral or toxic effects of N3dThd can be attributed to direct effects on this enzyme. The possible indirect effects caused by alterations in the pools of the natural effectors are discussed.

Antiviral Agents↗