Biomedical subjects

S Naylor

Publications and source records attributed to S Naylor.

At least 19 recordsLinked to original sources

PubMed1995

Expression of Desulfovibrio gigas desulforedoxin in Escherichia coli. Purification and characterization of mixed metal isoforms.

The dsr gene from Desulfovibrio gigas encoding the nonheme iron protein desulforedoxin was cloned using the polymerase chain reaction, expressed in Escherichia coli, and purified to homogeneity. The physical and spectroscopic properties of the recombinant protein resemble those observed for the native protein isolated from D. gigas. These include an alpha 2 tertiary structure, the presence of bound iron, and absorbance maxima at 370 and 506 nm in the UV/visible spectrum due to ligand-to-iron charge transfer bands. Low temperature electron paramagnetic resonance studies confirm the presence of a high-spin ferric ion with g values of 7.7, 5.7, 4.1, and 1.8. Interestingly, E. coli produced two forms of desulforedoxin containing iron. One form was identified as a dimer with the metal-binding sites of both subunits occupied by iron while the second form contained equivalent amounts of iron and zinc and represents a dimer with one subunit occupied by iron and the second with zinc.

Amino Acid Sequence↗

PubMed1995

Preliminary investigations of preconcentration-capillary electrophoresis-mass spectrometry.

Analyte preconcentration on-line with capillary electrophoresis-mass spectrometry (PC-CE-MS) is described. Preconcentration cartridges were fabricated from PTFE tubing filled with ca. 1-2 mm bed of reversed-phase C18 HPLC packing or polymeric reversed-phase beads. The particle size of the stationary phase was of larger dimension than the internal diameter of the CE capillary. Therefore, PC-CE capillaries were assembled without frit material and held together by friction. The wide applicability of on-line PC-CE-MS is demonstrated by the analysis of solutions containing peptides, proteins, and synthetic analogues of putative metabolites of the neuroleptic agent haloperidol.

Amino Acid Sequence↗

PubMed1995

Simultaneous structure-activity determination of disulfiram photolysis products by on-line continuous-flow liquid secondary ion mass spectrometry and enzyme inhibition assay.

Disulfiram (DSF) is used in the treatment of recovering alcoholics and exerts its effect by inhibiting the enzyme aldehyde dehydrogenase (ALDH). We analyzed a mixture of products derived photochemically from DSF with on-line microbore HPLC-continuous-flow liquid secondary ion mass spectrometry (HPLC-CF-LSI-MS). By utilizing the post-HPLC column split of solvent flow, a small proportion (ca. 5%) was sent directly into the mass spectrometer, and the remainder was collected. Simultaneous MS analysis and enzyme inhibition studies on ALDH were then possible. Furthermore, using HPLC-CF-LSI-MS-MS, we were able to structurally characterize an interesting sulfine compound that inhibited ALDH.

Aldehyde Dehydrogenase↗

PubMed1995

Characterization of 2-amino-1-benzylbenzimidazole and its metabolites using tandem mass spectrometry.

We have investigated the in vitro hamster hepatic microsomal metabolism of the amino-azaheterocycle, 2-amino-1-benzylbenzimidazole (ABB). Three major metabolites were isolated and structurally characterized, using a combination of off-line HPLC, in conjunction with both electron ionization and fast atom bombardment ionization tandem mass spectrometry. ABB was shown to be debenzylated to afford 2-aminobenzimidazole (AB), as well as N- and C-oxidized to give 1-benzyl-N2-hydroxyaminobenzimidazole (BHB) and 2-amino-1-benzyl-hydroxybenzimidazole, respectively. The possible reasons for formation of the exocyclic hydroxylamine BHB are discussed. Furthermore, ABB is proposed as a suitable model compound for investigating parameters that control formation of toxic hydroxylamines derived from amino-azaheterocycles.

Animals↗

PubMed1995

Microcell mediated chromosome transfer maps the Fanconi anaemia group D gene to chromosome 3p.

Fanconi anaemia (FA) is an autosomal recessive disorder characterized by progressive pancytopenia, short stature, radial ray defects, skin hyperpigmentation and a predisposition to cancer. Cells from FA patients are hypersensitive to cell killing and chromosome breakage induced by DNA cross-linking agents such as mitomycin C (MMC) and diepoxybutane (DEB). Consequently, the defect in FA is thought to be in DNA crosslink repair. Additional cellular phenotypes of FA include oxygen sensitivity, poor cell growth and a G2 cell cycle delay. At least 5 complementation groups for Fanconi anaemia exist, termed A through E. One of the five FA genes, FA(C), has been identified by cDNA complementation, but no other FA genes have been mapped or cloned until now. The strategy of cDNA complementation, which was successful for identifying the FA(C) gene has not yet been successful for cloning additional FA genes. The alternative approach of linkage analysis, followed by positional cloning, is hindered in FA by genetic heterogeneity and the lack of a simple assay for determining complementation groups. In contrast to genetic linkage studies, microcell mediated chromosome transfer utilizes functional complementation to identify the disease bearing chromosome. Here we report the successful use of this technique to map the gene for the rare FA complementation group D (FA(D)).

Cell Line↗

PubMed1992

Identification of metabolites of 4,4'-diaminodiphenylmethane (methylene dianiline) using liquid chromatographic and mass spectrometric techniques.

The in vitro metabolism of 4,4'-diaminodiphenylmethane (methylene dianiline, MDA) was investigated using rabbit liver microsomes. Minimal clean-up of the microsomal incubations was carried out using zinc sulphate precipitation followed by solid-phase extraction on Sep-Pak C18 cartridges. Three metabolites were detected in hepatic microsomal incubations, namely the azodiphenylmethane (azo) azoxydiphenylmethane (azoxy) and 4-nitroso-4'-aminodiphenylmethane (nitroso) compounds. The azo and azoxy metabolites were produced enzymatically whereas the nitroso compound may have been formed via a non-enzymatic process. Reversed-phase high-performance liquid chromatography-plasma spray mass spectrometry was used to initially detect these metabolites. Fast atom bombardment mass spectrometry and fast atom bombardment tandem mass spectrometry were utilized to further structurally characterise these compounds. Comparison of mass spectral data obtained from synthesised standards with data obtained on the putative metabolites substantiated the characterisation of these compounds.

Aniline Compounds↗

PubMed1992

Rapid and efficient purification of cimetropium bromide and mifentidine drug metabolite mixtures derived from microsomal incubates for analysis by mass spectrometry.

A comparative study of the use of organic solvent extraction versus Sep-Pak C18 cartridges in the recovery and analysis of phase I (unconjugated) drug metabolites using mass spectrometry is presented. Standard mixtures of putative metabolites of the anticholinergic drug cimetropium bromide and the H2-antagonist mifentidine were purified from inactivated liver microsomal preparations using both methods, and subsequently the recovery of each compound was quantitated. In general, the percentage recovery and degree of purification were greater when using Sep-Pak C18 cartridges compared with organic solvent extraction. Even more efficient recovery was achieved when zinc sulphate precipitation of proteins in the liver microsomal mixtures was carried out prior to analysis. Also, the HPLC-grade solvents used in this study contained a variety of ultraviolet-inactive, hydrophobic components. This leads to problems of suppression in fast atom bombardment mass spectrometric analysis. Using Sep-Pak C18 cartridges directly prior to analysis by fast atom bombardment with single or tandem mass spectrometry leads to far superior mass spectral results compared with organic solvent extraction.

Androgen Antagonists↗

PubMed1992

Bioanalytical applications of tandem mass spectrometry in the in vitro metabolism of the anticholinergic drug cimetropium bromide to detect differences in species metabolism.

1. In vitro metabolism of the anticholinergic drug, cimetropium bromide, was investigated using four different animal hepatic microsomal incubates derived from rat, hamster, guinea pig, and mouse livers. 2. Constant neutral loss (CNL) tandem mass spectrometry was used to detect the presence of the N-methylenecyclopropyl-scopine functionality by monitoring loss of 54 daltons (corresponding to loss of methylenecyclopropane) in microsomal incubates. 3. A CNL loss of 46 daltons was used to screen for the presence of ester hydrolysis products. 4. A comparison of the daughter ion spectra obtained on ions detected by CNL scanning, with daughter ion spectra of synthetic standards, determined the presence of ten metabolites of cimetropium bromide. 5. Hydroxylation of the aromatic ring in the ester side-chain was found to be the major metabolic pathway, and ester bond hydrolysis was a minor metabolic pathway. 6. N-Demethylation of the bridgehead nitrogen was observed only in rat and hamster incubates. 7. Using the method of CNL scanning it was possible to screen different animal microsomal incubates without resorting to any major purification procedures such as h.p.l.c. 8. This scanning method revealed differences between species in the metabolic pathways of cimetropium bromide.

Animals↗

PubMed1991

Isolation of two N-monosubstituted protoporphyrins, bearing either the whole drug or a methyl group on the pyrrole nitrogen atom, from liver of mice given griseofulvin.

1. A hepatic green pigment with inhibitory properties towards the enzyme ferrochelatase has been isolated from the liver of mice treated with griseofulvin and identified as N-methylprotoporphyrin. 2. All four structural isomers of N-methylprotoporphyrin have been demonstrated to be present, NA, where ring A of protoporphyrin IX is N-methylated, being the predominant isomer. 3. In addition to N-methylprotoporphyrin, a second green pigment, present in far greater amounts, was also isolated from the liver of griseofulvin-treated mice. This second green pigment is also an N-monosubstituted protoporphyrin, but in this case the substituent on the pyrrole nitrogen atom appears to be intact griseofulvin rather than a methyl group. 4. The fragmentation of this adduct in tandem m.s. studies suggests that griseofulvin is bound to the pyrrole nitrogen through one of its carbon atoms and further suggests that N-methylprotoporphyrin may arise as a secondary product from the major griseofulvin pigment.

Animals↗

PubMed1991

Gas chromatographic-mass spectrometric analysis of diols and tetrols from reactions of polycyclic aromatic hydrocarbon epoxides with hemoglobin.

We have evaluated both electron ionization (EI) and negative-ion chemical ionization (NICI) methods for the analysis of trimethylsilyl derivatives of a series of polycyclic aromatic hydrocarbon (PAH) alcohols including styrene diol, benzo[e]pyrene diol and tetrols, cyclopenta[c,d]pyrene diols, benzo[a]pyrene-4,5-diols, chrysene tetrols, benz[a]anthracene tetrols I and II, and syn- and anti-benzo[a]pyrene tetrols. NICI is the more sensitive method for all compounds except styrene diol. Detection limits are compound-dependent and range from 1 fmol for cyclopenta[c,d]pyrene diol to 1 pmol for benzo[e]pyrene diol. The EI detection limit for styrene diol is 60 fmol. PAH alcohols related to the compounds listed above were observed following hydrolysis of hemoglobin which had been reacted with PAH epoxides in vitro. Benzo[a]pyrene tetrols and a chrysene tetrol were observed following hydrolysis of hemoglobin isolated from human smokers' blood. Hydrolysis of styrene oxide treated hemoglobin in 18O-labeled water revealed at least two mechanisms of ester hydrolysis, including the BAL 1 pathway.

Alcohols↗

PubMed1991

Mutation in the TP53 gene in colorectal carcinoma detected by polymerase chain reaction.

The human TP53 gene is a possible tumor suppressor since TP53 gene mutations are observed in greater than 70% of sporadic colorectal carcinoma DNAs. In genomic DNAs from seven colon cancer cell samples, a 405 base pair DNA fragment containing exon 5, intron 5, and exon 6 of the TP53 gene was amplified by polymerase chain reaction and analyzed for mutations. One sample [human colon cancer (HCC) 278] was found to have a TP53 mutation altering the amino acid glutamine 167 in exon 5. A deletion of 2 bases changed glutamine 167 (CAG) to alanine (GCA) and the resulting frame-shift produced an in-frame stop codon at amino acid 179. While the normal TP53 gene gives rise to a 53 kD protein, the estimated size of this mutant TP53 protein if expressed would be approximately 20 kD.

Adenocarcinoma↗

PubMed1991

Takayasu's arteritis in a pregnant woman. A case report.

Takayasu's arteritis in a pregnant white patient is described. This case highlights the fact that, irrespective of race, any patient who presents for the first time in pregnancy with pulseless hypertensive disease or other features suggestive of Takayasu's arteritis, should have their management in labour determined by the number of complications that are present. These are retinopathy, arterial aneurysms, hypertension and aortic regurgitation. These prognostic criteria will result in a classification of patients that will lead to appropriate management.

Adult↗

PubMed1990

Molecular dosimetry of polycyclic aromatic hydrocarbon epoxides and diol epoxides via hemoglobin adducts.

Ten reactive metabolites of five polycyclic aromatic hydrocarbons and styrene were investigated to determine the generality of ester adduct formation with human hemoglobin in the form of RBC and hydrolysis to the corresponding tetrahydrotetrols or dihydrodiols. No exceptions were noted among the compounds tested, which included the anti-diol epoxides of benzo[a]pyrene (BaP), chrysene, and benz[a]anthracene; the syn-diol epoxide of BaP; a mixture of syn- and anti-diol epoxides of benzo[e]pyrene; and epoxides of styrene, benzo[e]pyrene, BaP, and cyclopenta[c,d]pyrene. A test of the propensity of the simplest benzylic epoxide, styrene oxide, to form esters that hydrolyze via a BAL1 mechanism was performed. Hydrolysis of styrene oxide-adducted hemoglobin in H2(18)O at neutral pH yielded 18O incorporation results that suggest this mechanism of hydrolysis is operant to a minor degree in styrene oxide-hemoglobin ester adducts. A method was developed for the isolation and quantification of the polycyclic aromatic alcohols, which consists of enzymatic proteolysis, immunoaffinity chromatography, and gas chromatography-mass spectrometry or fluorimetry. The method allows for routine analysis of hemoglobin from individual samples as small as 1 ml of whole blood. Analysis of blood from different human populations revealed that hemoglobin adducts of the anti-diol epoxide of BaP dominated the spectrum of adducts formed by the selected metabolites.

Chromatography, Affinity↗

PubMed1990

Effect of different target gases on low-energy collision-activated dissociation of peptides.

Experimental variables affecting the daughter-ion spectra of a series of protonated peptides [MH]+, produced by fast-atom bombardment ionization, using a low energy (0-450 eV) quadrupole collision cell are investigated. The parameters studied include target gas pressure, collision energy, cross-sectional area and acidity of the target gas. The results show that low-mass immonium ions are preferentially formed both at high collision energies (greater than 200 eV) and at target gas pressures greater than 10(-6) mBar (where multiple collisions occur in the gas cell). Positive fragment ion abundance is maximized when acidic gases are used as the target gases, and this is rationalized on the basis of a proton-transfer reaction from the target gas to the amide nitrogen of the peptide bond promoting fragmentation.

Enkephalin, Leucine↗

PubMed1990

Collision-activated dissociation studies of alkylamines formed from copper-induced dealkylation of N-alkylporphyrins.

A low-energy (5-450 eV) collision-activated dissociation (CAD) study of a series of aliphatic amines revealed that at collision energies above 200 eV, charge-site-initiated fragmentation occurs. The resulting fragment ions can be utilized in the characterization of alkyl substituents of di- and trisubstituted aliphatic amines. In the presence of Cu2+ and a suitable nucleophile, such as n-dodecylamine, N-alkyl protoporphyrins dealkylate to afford copper protoporphyrin and an alkyl-dodecylamine adduct. A CAD study of a number of alkyl-dodecylamine adducts derived from the copper-induced dealkylation of synthetic N-alkyl protoporphyrins, using charge-site-initiated fragment ions, showed that the alkyl group was trapped by the nucleophilic amine present. Subsequently this method was used to identify the alkyl group of a biologically derived N-alkyl protoporphyrin.

Amines↗

PubMed1990

Identification of phosphoramide mustard/DNA adducts using tandem mass spectrometry.

The reaction pathway of alkylating agents is often exploited in the design of bifunctional anti-cancer drugs. These drugs form mono-DNA adducts as well as inter- and intra-strand cross-linked adducts, notably by reaction at DNA bases, including the N-7-position of guanine (G). A positive-ion fast-atom bombardment (FAB) mass spectrum of an in vitro preparation of DNA alkylated with phosphoramide mustard (the active metabolite of the anti-cancer drug cyclophosphamide) indicated the presence of the two mono-DNA adducts N-(2-chloroethyl)-N-[2-(7-guaninyl)ethyl] amine, designated NOR-G, and N-(2-hydroxyethyl)-N-[2-(7-guaninyl)ethyl] amine, designated NOR-G-OH, (MH+ 257/259 and 239, respectively) but not the presence of the cross-linked adduct N,N-bis-[2-(7-guaninyl)ethyl] amine, designated G-NOR-G (MH+ 372). Using synthetic standards, daughter-ion spectra of NOR-G, NOR-G-OH and G-NOR-G were obtained (matrix 0.2 M p-toluene sulphonic acid in glycerol) by positive-ion FAB tandem mass spectrometry (FAB-MS/MS). The daughter-ion spectra of both mono-DNA adducts NOR-G and NOR-G-OH contained a fragment ion at m/z 152 [G + H]+, whereas the cross-linked adduct, G-NOR-G, showed an ion at m/z 221, [MH-G]+. Evidence for the presence of NOR-G, NOR-G-OH and G-NOR-G in the in vitro preparation was obtained by performing a double parent-ion scan on m/z 152 and 221. The presence of G-NOR-G was further supported by performing a single parent-ion scan on m/z 221.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA↗

PubMed1990

Deuterium exchange studies in the identification of alkylated DNA bases found in urine, by tandem mass spectrometry.

Humans are exposed to a large number of carcinogens which may react at various sites throughout the body, including the N-7-, N2-, and O6-positions of guanine. The effects of this are various but may result in depurination and eventual excretion of the modified base in the urine. Various alkylguanine derivatives with substituents at the N-7-, N2- and O6-positions were synthesized and daughter-ion spectra obtained. Apart from the methyl and dialkylguanines all other spectra exhibited an ion at m/z 151 using electron ionization (EI) and m/z 152 using fast-atom bombardment (FAB). The daughter-ion spectra of dialkylguanines contained an ion at m/z 150 (EI). Hence, scans of m/z 150 and 151 using EI, to detect all parent-ions from which they are formed (parent-ion scans) should indicate the presence of alkyl and dialkylguanine bases in a complex biological matrix such as human urine. Parent-ion scans of m/z 150 and 151 (EI) of a partially purified human urine sample exhibited numerous ions, including a prominent ion at m/z 179. A daughter-ion spectrum of m/z 179 revealed fragment ions that suggested the presence of N2-dimethylguanine and an ethylated guanine. Any confusion due to the presence of daughter ions from different alkylguanines in the same spectrum can be resolved by the fact that an ethylated guanine has four exchangeable protons, whereas N2-dimethylguanine has only three. By performing hydrogen/deuterium exchange it is possible to distinguish the N2-dimethylguanine from ethylated guanine isomers.(ABSTRACT TRUNCATED AT 250 WORDS)

Guanine↗

PubMed1989

A rapid method for isolation of human hemoglobin benzo[a]pyrene diol epoxide derived adducts using high performance liquid chromatography.

A method is described to isolate rapidly human hemoglobin-benzo[a]pyrene diol epoxide adducts. A combination of 300 A pore size C4 reversed phase HPLC to effect separation of adducted protein from native protein, and mu-bore C18 reversed phase HPLC to isolate and partially characterize proteolytic peptide adducts (by UV), was used.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗