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T P Bradshaw

Publications and source records attributed to T P Bradshaw.

14 recordsLinked to original sources
PubMed2000

Hydration changes implicated in the remarkable temperature-dependent membrane permeation of cyclosporin A.

Cyclosporin A is a cyclic peptide believed to exist as multiple conformers in aqueous solution. Two major conformations, distinguished by a single cis-trans isomerization and the presence of four either intramolecular or intermolecular hydrogen bonds, have been confirmed depending on whether CsA is characterized in organic solvents or bound in aqueous complex with cyclophilin. The relationship between CsA conformation and its ability to penetrate biological membranes is currently unknown. Using Caco-2 cell monolayers, we documented a remarkable increase (more than 2 orders of magnitude) in the membrane permeation of the peptide as temperature was increased from 5 to 37 degrees C. The solubility of CsA was 72 microM at 5 degrees C, but decreased by more than an order of magnitude at 37 degrees C. Moreover, CsA partitioned into non-hydrogen bond donating solvents linearly as a function of increasing temperature, suggestive of a significant conformational change. However, while NMR spectra of CsA confirmed the previously predicted presence of multiple conformers in aqueous solution, the equilibrium between the two major species was not affected by changes in temperature. These NMR data indicated that the observed temperature-dependent changes in the membrane permeability of CsA do not originate from changes in the peptide backbone conformation. Sedimentation equilibrium analysis revealed that CsA behaves in a highly nonideal manner over the temperature range tested. We interpret this behavior as a change in the hydration state with a smaller (or weaker) hydration shell surrounding the peptide at higher temperatures. Such a change would result in lower peptide desolvation energy, thereby promoting partitioning into cellular membranes. We contend that changes in membrane penetration result from alterations in the hydration state of CsA and are not related to the interconversion of the defined conformations.

Caco-2 Cells↗
PubMed1997

Formation of free radicals and protein mixed disulfides in rat red cells exposed to dapsone hydroxylamine.

The hemolytic activity of dapsone is well known to reside in its N-hydroxylamine metabolites. Addition of dapsone hydroxylamine (DDS-NOH) to red cell suspensions causes damage such that when reintroduced into the circulation of isologous rats, the injured cells are rapidly removed by the spleen. Hemolytic activity is associated with the extensive formation of disulfide-linked hemoglobin adducts on red cell membrane skeletal proteins. To determine if free radicals could be involved in this process, rat red cells were incubated with DDS-NOH in the presence of the spin trap, 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) and subjected to EPR analysis. Addition of DDS-NOH (25-50 microM) to a red cell suspension gave rise to a four-line (1:2:2:1) EPR spectrum with coupling constants identical to those of a DMPO-hydroxyl radical adduct (DMPO-OH) standard. No other radicals were detected; however, preincubation of red cells with cysteamine caused the DDS-NOH-generated DMPO-OH signal to be replaced by a cysteamine thiyl radical adduct signal. DDS-NOH-treated red cells were also found to contain ferrylhemoglobin, indicating the presence of hydrogen peroxide. Furthermore, DDS-NOH was found to stimulate salicylate hydroxylation in red cell suspensions, confirming the presence of oxygen radicals. These data support the hypothesis that oxygen radicals are involved in the mechanism underlying dapsone-induced hemolytic anemia.

Animals↗
PubMed1996

CYP3A-like cytochrome P450-mediated metabolism and polarized efflux of cyclosporin A in Caco-2 cells.

Transport of cyclosporin A (CsA) across Caco-2 cells is modulated by its directional efflux, mediated by a p-glycoprotein-like pump (Augustijns et al., Biochem. Biophys. Res. Comm. 197:360-365, 1994). In addition to this unidirectional flux, oxidative metabolism of CsA by cytochrome P450 is likely to influence the absorption of this cyclic peptide across intestinal mucosa. Thus, metabolism of CsA in the in vitro Caco-2 cell culture system was investigated. Formation of several metabolites was observed during the course of CsA transport across Caco-2 cell monolayers. Results from LC/MS/MS experiments revealed that the major metabolite was 1eta-hydroxy CsA (M-17), one of the three major metabolites produced by CYP3A4 present in both the liver and small intestine in humans. Preincubation of Caco-2 cell monolayers with troleandomycin, a specific inhibitor for the microsomal CYP3A protein, reduced the formation of the metabolite M-17, suggesting that an enzyme that functionally resembles CYP3A is responsible for the formation of this metabolite. However, formation of only the M-17 metabolite suggests that the isozyme present in the Caco-2 cells is distinct from CYP3A4, which also catalyzes the formation of significant quantities of the metabolites 9gamma-hydroxy cyclosporin A (M-1) and 4N-desmethyl cyclosporin A (M-21) from CsA. Interestingly, the amount of M-17 accumulating on the apical (AP) side was much greater than that on the basolateral (BL) side during the AP --> BL transport of CsA across Caco-2 cell monolayers. This is consistent with p-glycoprotein pump-mediated efflux of the metabolite to the apical side. Furthermore, formation of the M-17 metabolite on the AP side of cell monolayers during the AP --> BL transport of CsA was much greater than that during the BL --> AP transport. This result suggests that the p-glycoprotein efflux pump causes an increase in the metabolism of CsA during the course of its AP --> BL transport by effectively slowing down the transport of CsA molecules across Caco-2 cells. Thus, Caco-2 cells serve as an excellent model to dissect the relative roles played by p-glycoprotein-mediated efflux and CYP3A-catalyzed oxidation in modulating the overall absorption of CsA and other such compounds.

Anti-Bacterial Agents↗
PubMed1995

Identification of free radicals produced in rat erythrocytes exposed to hemolytic concentrations of phenylhydroxylamine.

Previous studies have shown that incubation of rat red blood cells in vitro with phenylhydroxylamine (50-300 microM) induces rapid splenic sequestration of the red cells on reintroduction to isologous rats. EPR and the spin trapping agent, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), were utilized to determine if free radical species could be identified under these experimental conditions. Hemolytic concentrations of phenylhydroxylamine, in the presence of DMPO and 5-20% lysed or intact rat erythrocyte suspensions, gave rise to a four-line (1:2:2:1) EPR spectrum. No signal was obtained if phenylhydroxylamine, DMPO, or red cells was omitted. Comparison of the phenylhydroxylamine-induced signal with authentic hydroxyl radical- and GSH thiyl radical-DMPO standard adduct signals identified the phenylhydroxylamine-induced species as a GSH thiyl free radical. Removal of GSH from a red cell lysate abolished the GSH thiyl radical signal without the appearance of any other signal, while addition of exogenous GSH resulted in its return. When erythrocytes were exposed to concentrations of phenylhydroxylamine > or = 200 microM, a time-dependent transition of the GSH thiyl radical signal to a hemoglobin thiyl radical signal was observed. The data are consistent with the postulate that thiyl radical species, generated from the interaction of phenylhydroxylamine and oxyhemoglobin, play a key role in the development of hemolytic injury to the rat red cell.

Animals↗
PubMed1995

Dapsone-induced hemolytic anemia.

Dapsone, an old drug introduced and used almost exclusively for the treatment of leprosy, is now utilized in an increasing number of therapeutic situations. However, its hemotoxicity is potentially severe and is often dose limiting. Effective countermeasures, based on resolution of the mechanisms underlying dapsone-induced hemotoxicity, could significantly enhance the therapeutic value of the drug. In studies on rat red cells, we have established that the N-hydroxy metabolites of dapsone, DDS-NOH and MADDS-NOH, are direct-acting hemolytic agents, that they are formed in amounts sufficient to account for the hemotoxicity of the parent drug, and that the action of these toxic metabolites in the red cell induces premature sequestration by the spleen. Incubation of rat red cells with hemolytic concentrations of arylhydroxylamines leads to the generation of hydroxyl, glutathiyl, and hemoglobinthiyl radicals, and the formation of protein-glutathione mixed disulfides. Disulfide-linked adducts are also formed between membrane skeletal proteins and hemoglobin monomers, as well as between the monomeric hemoglobin units forming dimers, trimers, tetramers, and pentamers. Profound morphological changes are seen with change from normal discoidocity to an extreme nonspherocytic enchinocyte shape. Parallel studies with human red cells indicate that the response of human cells is qualitatively similar but that there are notable differences in regard to skeletal membrane effects. A working hypothesis for the mechanism underlying dapsone hemolytic activity is proposed.

Anemia, Hemolytic↗
PubMed1993

Evidence for a polarized efflux system in CACO-2 cells capable of modulating cyclosporin A transport.

The characteristics of cyclosporin A (CsA) transport across Caco-2 monolayers were investigated. CsA (0.25-5.0 microM) was transported in a time and concentration dependent manner. The total amount of apical (AP) to basolateral (BL) transport was non-linearly related to CsA concentration from 0.25 to 1 microM and was linear from about 1 to 5 microM. Average permeability coefficient (Papp) values obtained in the AP to BL direction showed CsA concentration (0.5 and 5.0 microM) dependence, whereas those of the reverse (BL to AP) process did not. Papp values for the AP to BL direction were also markedly lower. When the P-glycoprotein pump inhibitors, chlorpromazine and progesterone, were included in the transport medium we observed a significant increase in CsA (0.5 and 5.0 microM) transport from the AP to BL direction; transport was decreased in the reverse direction. This study suggests that CsA is transported across Caco-2 cells by passive diffusion, but that a polarized efflux system (presumably a P-glycoprotein pump) located at the apical membrane can attenuate the net AP to BL transport.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Characterization of a novel form of thymidylate synthase: a heterodimer isolated after specific chemical modification of the immobilized native enzyme.

A novel approach, utilizing covalent chromatography and selective chemical modification, is described for application in studying subunit interactions involved in the catalytic and regulatory mechanisms of certain oligomeric proteins. The specific objective was to prepare and characterize heterodimeric form of thymidylate synthase which would serve as a model for an intermediate stage of the catalytic mechanism in which the active-site cysteine of one subunit would be engaged in covalent catalysis while that of the other subunit would exist in the free sulfhydryl or thiolate anion form. Dimeric Lactobacillus casei thymidylate synthase was subjected to covalent chromatography on thiopropyl Sepharose 6B resin under conditions in which a mixed disulfide linkage was formed with the catalytic sulfhydryl group of just one of the two subunits. Specific chemical modification of the remaining essential sulfhydryl group of the immobilized group enzyme with N-ethylmalemide, followed by cleavage and elution with buffer containing 2-mercaptoethanol, yielded the desired soluble heterodimeric form of the enzyme. The specific activity of this unique form of the enzyme (1.55 units/mg) was approximately 60% that of native protein (2.61 units/mg). Gel electrophoretic analysis of the heterodimeric enzyme, incubated in the presence of FdUMP and 5,10-methylenetetrahydrofolate (CH2H4folate), resulted in the appearance of a single protein band corresponding to the 1:1:1 enzyme-FdUMP-CH2H4folate complex, confirming the new species as dimeric thymidylate synthase containing a single functional active site.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1993

Characterization of the covalent chromatography of thymidylate synthase on thiopropyl-Sepharose 6B.

We have examined the covalent chromatography of Lactobacillus casei thymidylate synthase on thiopropyl-Sepharose 6B resin. This enzyme is a dimer of identical subunits, each of which contains one active site that features a catalytic sulfhydryl group (Cys-198). Reversible coupling was achieved via the attack of one of the enzyme's two catalytic cysteine residues, causing displacement of 2-thiopyridone, the reactive moiety of the resin. To establish the usefulness of this matrix for immobilization and the conditions required for chromatography, model studies were conducted with 2,2'-dithiodipyridine. The chemical modification of thymidylate synthase with 2,2'-dithiodipyridine was shown to be specific for the catalytic sulfhydryl groups of the native dimer, titrating 1.51 sulfhydryl groups, while 2.93 cysteines were modified in the GnHCl-denatured protein. The former reaction, which resulted in total loss of enzyme activity, was reversible with complete recovery of control activity within 30 min after addition of 100 mM 2-mercaptoethanol. Characterization of the protein pools generated in the covalent chromatography procedure indicated that the enzyme fraction washing through the column without attachment had substantially lower catalytic and ligand binding activities than the original protein stock; conversely, the enzyme fraction eluted from the column by 2-mercaptoethanol exhibited higher levels of these activities. Gel electrophoresis studies further illustrated that the unique application of the covalent chromatography technique described herein fractionated homogeneous thymidylate synthase protein into enzyme pools exhibiting distinct biochemical properties. As immobilization reaction times were increased beyond 6 h, the coupling of thymidylate synthase was demonstrated to occur through more than one enzymic sulfhydryl group. Interestingly, no covalent coupling was detected in attempts using activated thiol-Sepharose 4B, a result underlining the importance of the structure of the resin linker arm in enzyme immobilization.

2,2'-Dipyridyl↗
PubMed1992

Catalysis and ligand binding by thymidylate synthase immobilized on thiopropyl-sepharose 6B.

Lactobacillus casei thymidylate synthase, which employs the thiol of cysteine-198 as a covalent catalyst, was reversibly coupled to thiopropyl-Sepharose 6B through the catalytic sulfhydryl group of one of its two subunits, yielding an immobilized heterodimeric form of the enzyme possessing one free active site and one covalently modified active site. Enzyme inactivated by treatment with N-ethylmaleimide, which selectively modifies the active site cysteines but not the remaining cysteines (cys-244), failed to react with the resin. Modified assay procedures were developed and utilized to characterize the activity and ligand binding properties of this unique enzymic species. The immobilized enzyme was found to catalyze thymidylate formation at its free active site but exhibited lowered specific activity (13-fold) and kcat (16-fold) values and an increased Km for dUMP (4-fold) when compared to the native, soluble enzyme. Immobilized enzyme also formed a covalent inhibitory ternary complex with 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate with a stoichiometry of 0.32 (mol FdUMP: mol enzyme), about half the predicted value of 0.6-0.7. The results of this initial study suggest that the active sites of the native enzyme dimer are asymmetrical in nature, and that the chemical status of the catalytic sulfhydryl groups may play a key role in directing communication between the subunits.

Enzymes, Immobilized↗
PubMed1991

Role of metabolites in propanil-induced hemolytic anemia.

Hemolytic anemia and methemoglobinemia induced by exposure to certain arylamines, such as aniline and dapsone, are known to be mediated by their N-hydroxylamine metabolites. The arylamide propanil (3,4-dichloropropionanilide), a herbicide used extensively in rice fields, is also thought to induce methemoglobinemia through the action of metabolites. However, the hemolytic potential of this compound has not previously been reported. The present studies were undertaken to determine the hemolytic potential of propanil, and, if positive, the role of metabolites in this hemotoxicity. The survival of previously administered 51Cr-labeled erythrocytes in rats was reduced in a dose-dependent manner by ip administration of both propanil and its deacylated metabolite, 3,4-dichloroaniline (ED50 for both ca. 1.8 mmol/kg). When labeled erythrocytes were exposed in vitro to propanil or 3,4-dichloroaniline and then readministered to rats, no decrease in erythrocyte survival was observed, which indicated that these compounds were not direct-acting hemolytic agents. In contrast, erythrocyte survival was markedly reduced by ip administration or in vitro exposure to N-hydroxy-3,4-dichloroaniline. In addition, N-hydroxy-3,4-dichloroaniline was detected in the blood of propanil-treated rats in amounts sufficient to account for the hemolytic activity of the parent compound. These data indicate that N-hydroxy-3,4-dichloroaniline mediates propanil-induced hemolytic anemia, and that occupational exposure to propanil may result in an increased risk of hemolytic episodes.

Anemia, Hemolytic↗
PubMed1988

The removal of exogenous thiols from proteins by centrifugal column chromatography.

Centrifugal column chromatography was shown to provide a rapid, efficient, and useful means of separation of various low molecular weight thiols from proteins. The single chromatographic step procedure employed standard 5 ml plastic syringes containing Sephadex G-25 as the bed matrix and required less than 5 min to produce average dilutions of 5000-, 980-, and 25-fold, respectively, from 5 to 200 mM initial concentrations of 2-mercaptoethanol, dithiothreitol, and reduced glutathione in the sample as measured by titration with 5,5'-dithiobis-(2-nitrobenzoic acid). Dihydrofolate reductase solutions of 0.07-0.08 mM were separated from 50 mM 2-mercaptoethanol, dithiothreitol, or reduced glutathione with a minimum 16,500-fold dilution of the thiol after centrifugal chromatography on two consecutive columns. Thymidylate synthase solutions of 0.06 mM were effectively separated from 50 mM 2-mercaptoethanol or dithiothreitol with a minimum average 5900-fold dilution of the thiol after consecutive column chromatography. There was no change in either the physical or chemical properties of the enzyme throughout the course of the experiments as determined by activity, active site sulfhydryl group titration, and binding assays. Recoveries of protein obtained in the load fraction were usually in excess of 70% of the protein loaded with virtually no dilution from the initial concentration. This method was developed in order to facilitate the study of the active site sulfhydryl groups in enzymes.

Centrifugation↗
PubMed1986

Crystallization and crystallographic data for new forms of thymidylate synthase from Lactobacillus casei.

Several new crystal forms of thymidylate synthase (5,10-methlenetetrahydrofolate:dUMP C-methyltransferase; EC 2.1.1.45) were obtained by controlled pH change. In the crystals the dimeric molecule has a 2-fold symmetry axis coinciding with crystallographic symmetry. The crystals scatter to at least 2.7 A resolution in the synchrotron X-ray beam and appear to be suitable for high-resolution X-ray diffraction analysis. The crystals were successfully derivatized and preliminary results are reported for the covalent inhibitory ternary complex of thymidylate synthase, 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate.

Crystallography↗