Biomedical subjects

D M Grant

Publications and source records attributed to D M Grant.

At least 73 records · Page 4Linked to original sources

PubMed1997

Post-1980 black population redistribution trends in the United States.

"Using data from the 1990 Public Use Microdata Samples (PUMS) and other U.S. census documents, this paper demonstrates how three rather dramatic shifts in the migration behavior of blacks, which took shape during the 1970s, continued to contribute to the geographical redistribution of the black population down the urban hierarchy during the 1980s. Analyses of black migration flows into six metropolitan areas suggest that liberal welfare benefits play, at best, a minor role in contemporary black population redistribution trends, kinship ties (i.e., location-specific capital), the search for affordable housing, and employment in the hospitality services industry appear to be the dominant forces influencing black migration into the case-study communities."

Black or African American↗

PubMed1996

One dimensional chemical shift modulated correlation spectroscopy of single-crystals.

A simple 1D modulation method is presented for determining the spatial correlation of chemical shifts in two different single-crystal orientations. This proposed chemical shift modulated correlation (CSMC) spectroscopy utilizes a Jeener pulse sequence to give 1D spectra containing peaks that are amplitude modulated by the chemical shifts associated with the single-crystal's evolution orientation. Conversely, the spectral frequencies in these 1D CSMC spectra designate the shifts in the crystal's orientation during the detection period. By observing the amplitude modulation of these spectral frequencies, the spatial correlation between the two chemical environments can be established for a specific nucleus from a single 1D spectrum. The CSMC procedure finishes by acquiring a normal 1D static spectrum for the crystal oriented in the evolution direction, thereby providing very accurate evolution frequencies approximated by the amplitude modulation in the CSMC spectra. The CSMC method improves the time efficiency over both the traditional goniometer method and the 2D chemical shift-chemical shift correlation method, and therefore is beneficial for the study of samples with longer T1 values.

Algorithms↗

PubMed1996

The use of differential transverse relaxation to detect mobile species in solids.

Delayed acquisition of the proton NMR in selected organic molecular solids (L-alanine, durene, ethyl fumarate, and p-hydroxybenzoic acid) is shown to allow the observation of mobile species in the presence of relatively rigid bulk molecules. The mobility is found to be thermally activated. The combination of the thermally activated motion and magic-angle spinning leads to a fraction of these species moving nearly isotropically on the time scale of the inverse of the homonuclear dipolar splitting. In the case of ethyl fumarate and alanine, there exist populations with differing values of T1 and T1 row. This indicates the co-existence of relatively rigid and relatively mobile molecules in the same sample. The intensities under delayed acquisition cannot always be trusted to yield quantitative information. Comparison of spectra taken under delayed acquisition and under the CRAMPS (B.C. Gerstein, R.G. Pembleton, R.C. Wilson and L.M. Ryan, J. Chem. Phys., 66 (1977)361) technique is made.

Alanine↗

PubMed1996

Covalent binding of sulfamethoxazole reactive metabolites to human and rat liver subcellular fractions assessed by immunochemical detection.

Potentially serious idiosyncratic reactions associated with sulfamethoxazole (SMX) include systemic hypersensitivity reactions and hepatotoxicity. Covalent binding of SMX to proteins subsequent to its N-hydroxylation to form N4-hydroxysulfamethoxazole (SMX-HA) is thought to be involved in the pathogenesis of these reactions. A polyclonal antibody was elicited in rabbits against a SMX--keyhole limpet hemocyanin conjugate that recognized covalent protein adducts of SMX in microsomal protein and was used to characterize the covalent binding of SMX and its putative reactive metabolites to hepatic protein in vivo and in vitro. In vitro covalent binding of SMX to rat and human liver microsomal protein was NADPH-dependent, while binding of SMX-HA was not dependent on NADPH. SMX and SMX-HA produced similar patterns of covalent binding, with major protein targets in the region of 150, 100 (two bands), 70 (two bands), and 45-55 kDa. The pattern of covalent binding to human and rat liver microsomal protein was similar. Binding of SMX-HA was completely eliminated by GSH or by addition of cytosolic fractions and acetylcoenzyme A. The acetoxy metabolite of SMX also led to covalent binding, but it was primarily attributable to the formation of SMX-HA from acetoxySMX. In vivo exposure of rats to SMX did not result in detectable covalent binding by the methods employed. When rat liver slices were incubated with 2 mM SMX or 500 microM SMX-HA, no toxicity was observed and yet covalent binding of SMX-HA to 130, 100, 70, and 55 kDa proteins could be detected. These results confirm that covalent binding of SMX occurs via the formation of SMX-HA and that covalent binding of SMX-HA in vitro results from its conversion to the more reactive nitroso metabolite. Acetylation of SMX-HA protected against its covalent binding. Further studies are required to determine how this in vitro covalent binding relates to in vivo covalent binding in humans and to either direct or immune-mediated cytotoxicity in SMX idiosyncratic drug reactions.

Animals↗

PubMed1996

Genotyping of the polymorphic N-acetyltransferase (NAT2*) gene locus in two native African populations.

The hepatic N-acetyltransferase enzyme encoded by the NAT2* gene locus is responsible for the human polymorphic acetylation of numerous arylamine or hydrazine-containing drugs and xenobiotics including AIDS-related therapeutic agents such as isoniazid and sulphonamides. The genetic basis underlying the human acetylation polymorphism has been extensively studied in several populations but native African populations were poorly documented. In the present study, 117 unrelated black Africans, namely Dogons from Mali and Gabonese, were investigated for NAT2* allelic variability and genotype distribution. Thirteen NAT2* alleles were unambiguously identified by combined use of allele-specific reamplifications and restriction endonuclease digestions. Our results confirm the African origin of G191->A substitution in the NAT2* coding region which was previously associated with slow acetylation in African-Americans. The finding of high allelic diversity in the studied populations is consistent with the hypothesis of a single African origin for NAT2*-associated polymorphism. Finally, no excess of the slow acetylator phenotype is predicted in these populations, implying no need for fitting NAT2* polymorphism-sensitive therapies to black Africans, compared to Caucasians.

Acetylation↗

PubMed1995

Genetic analysis of microsomal epoxide hydrolase in patients with carbamazepine hypersensitivity.

Carbamazepine therapy is occasionally complicated by hypersensitivity reactions, the mechanism of which is poorly understood. It has been suggested that affected individuals may have a genetically-determined defect of microsomal epoxide hydrolase. The aim of this study was to determine whether a single genetic mutation or pattern of mutations could be used to predict individual susceptibility to carbamazepine-hypersensitivity. DNA was isolated from 10 carbamazepine-hypersensitive patients and 10 healthy volunteers. The patients had developed various forms of toxicity with carbamazepine, including toxic epidermal necrolysis, Stevens-Johnson syndrome, hepatitis and pneumonitis. The technique of polymerase chain reaction single-strand conformation polymorphism analysis (PCR-SSCP) was used to screen for mutations in all nine exons of the microsomal epoxide hydrolase gene. Any new mutations detected by this method were characterised by direct sequencing of the DNA. In addition, in the most severely affected patient, we sequenced all nine exons of the gene. There was a higher frequency of mutations in the hypersensitive group when compared with the controls, but there was no consistent mutation (or pattern of mutations) in the microsomal epoxide hydrolase gene which was common to the hypersensitive group. DNA sequencing of all nine exons of the microsomal epoxide hydrolase gene from the most severely affected patient showed the sequence to be "wild-type," when compared to the previously published sequences. The results of this study suggest that a single mutation within the coding region of the microsomal epoxide hydrolase gene cannot be the sole determinant of the predisposition to carbamazepine hypersensitivity.

Adolescent↗

PubMed1995

Relationship of 13C NMR chemical shift tensors to diffraction structures.

13C chemical shift tensor measurements on single crystals provide a powerful method to study changes in the electron environment of nuclei with changes in molecular structure. Thus, diffraction structures are critical to an understanding of chemical shift tensors. This work explores the general reliability of using structural data to predict components of the symmetrical chemical shift tensor. Imprecision in the hydrogen positions introduces considerable scatter in the simulated 13C shift tensors, and optimized C-H bond distances in methyl-beta-D-glucopyranoside used with the X-ray positions of the heavier C and O atoms greatly improve the simulated chemical shifts. Acenaphthene, with two crystallographically different molecules per unit cell, offers an excellent example for comparing and contrasting structural differences in the two molecules. A recently improved X-ray structure of naphthalene obtained at low temperature provides chemical shift simulations which are comparable to those from neutron diffraction methods and appear to reflect breaks in the D2h symmetry measured in the NMR chemical shift tensors. These data illustrate the close relationship between NMR and diffraction structures.

Carbohydrate Conformation↗

PubMed1995

13C-coupled relaxation studies of a leucine zipper peptide using polarization-transfer pulse sequences.

Three new pulse sequences are described for perturbing magnetization modes in 13C-coupled relaxation experiments via polarization-transfer techniques. Relative to non-polarization-transfer pulse sequences, these new pulse sequences result in carbon NMR spectra with significantly improved signal-to-noise ratios, a condition required for coupled relaxation studies of larger biomolecules. These pulse sequences are used to study molecular tumbling of a 13C-labeled leucine zipper peptide, GCN4-p1, in aqueous solution. Experimental data obtained for the AX2 spin system associated with the 13CH2 moiety of the peptide are fitted to the Favro diffusion model via nonlinear least-squares minimization. The least-squares fits provide values for rotational diffusion coefficients. Diffusion coefficients from relaxation studies performed at different temperatures yield enthalpies for the diffusional motion. Deficiencies in the fits of the relaxation data suggest the need for expanded relaxation models that account for proximate protons in the vicinity of the 13CH2 moiety.

Amino Acid Sequence↗

PubMed1995

Use of relaxation agent doping to shorten very long spin-lattice relaxation times in a magic-angle turning experiment.

A practical method is described for measuring the principal values of the chemical shift tensors in compounds with very long proton spin-lattice relaxation times (T1). This technique involves shortening the effective proton T1. by mixing a compound of interest with another compound having a much shorter T1 value. The doped mixture, partly consisting of a monophasic glass, allows efficient intermolecular spin diffusion between the two compounds. Using a slow magic-angle turning (MAT) experiment, we have successfully used such mixtures to measure the principal values of the chemical shift tensors of all the carbons in dibenzofuran in just four days. Without using this technique the experimental time required for the pure compound would have been several months.

Benzofurans↗

PubMed1995

Oleanane triterpene saponins from the Chinese medicinal herb Clinopodium chinensis.

Four oleanne triterpene saponins, 3beta,16beta, 23-trihydroxy-keton-13beta,28-epoxyolean-9(11)-en-3-yl -[beta-D-glucopranosyl(1-->2)]-[beta-D-glucopyranosyl(1-->3)]-beta -D-fucopyranoside [1] (clinopodiside D), 16beta-propionyl-3beta,23-dihydroxyoleana-11-21-dien-3 -yl-[beta-D-glucopyranosyl(1-->2)]-[beta-D-glucopranosyl(1-->3)]-b eta-D-fucopyranoside [2] (clinipodiside E), 3beta,16beta,21alpha,23,28-pentahydroxy-11-methoxyolean++ +-12-en-3-yl-[beta-D-glucopyranolyl(1-->2)]-[beta-D-glucopyranosyl (1-->3)]-beta-D-fucopyranoside [3] (clinopodiside F), and 3beta,16beta,21beta,23,28-pentahydroxyoleana-11,13(18) -dien-3-yl-[beta-D-glucopyranosyl(1-->2)]-[beta-D-glucopyranosyl(1 -->3)]-beta-D-fucopyranoside [4] (clinopodiside G), were isolated and purified from the plant Clinopodium chinensis. Their structures were elucidated on the basis of interpretation of nmr and ms data and from chemical evidence. The results are of potential interest for medicinal reasons.

Carbohydrate Sequence↗

PubMed1995

Triterpenoid saponins from Gypsophila oldhamiana.

Three new triterpenoid saponins were isolated from the roots of Gypsophila oldhamiana. Their structures were elucidated, using a combination of homonuclear and heteronuclear 2D nmr and fabms, as 3-0-beta-D-galactopyranosyl-(1-->2)-[beta-D-xylopyranolyl-(1-->3)] -beta-D-glucuronopyranosyl quillaic acid methyl ester [1], 3-0-beta-D-galactopyranosyl-(1-->2)-[beta-D-xylopyranosyl-(1-->3)]-beta- D-glucuronopyranosyl gypsogenin methyl ester [2], and 3-0-beta-D-galactopyranolsyl-(1-->2)-[beta-D-xylopyranosyl-(1-->3) ]-beta-D-glucuronopyranosyl quillaic acid 28-[0-beta-D-fucopyranosyl(1-->4)-beta-D-glucopyranosyl(1-->3)]-alpha-L- rhamnopyranosyl] ester.

Carbohydrate Sequence↗

PubMed1995

Nomenclature for N-acetyltransferases.

A consolidated classification system is described for prokaryotic and eukaryotic N-acetyltransferases in accordance with the international rules for gene nomenclature. The root symbol (NAT) specifically identifies the genes that code for the N-acetyltransferases, and NAT* loci encoding proteins with similar function are distinguished by Arabic numerals. Allele characters, denoted by Arabic numbers or by a combination of Arabic numbers and uppercase Latin letters, are separated from gene loci by an asterisk, and the entire gene-allele symbols are italicized. Alleles at the different NAT* loci have been numbered chronologically irrespective of the species of origin. For designation of genotypes at a single NAT* locus, a slash serves to separate the alleles; in phenotype designations, which are not italicized, alleles are separated by a comma.

Alleles↗

PubMed1995

Interindividual variability in the glucuronidation of (S) oxazepam contrasted with that of (R) oxazepam.

Although conjugation with glucuronic acid is a major process for converting many xenobiotics into hydrophilic, excretable metabolites, relatively little has been reported concerning interindividual variability of glucuronidation in human populations. Oxazepam, a therapeutically active metabolite of diazepam, is one of a number of C3-hydroxylated benzodiazepines for which glucuronide conjugation is the predominant pathway of biotransformation. The drug is normally formulated as a racemic mixture of inactive (R) and active (S) enantiomers. In the present study we have investigated the use of oxazepam as a potential probe drug for studying the variability of glucuronide conjugation, and for demonstrating the extent to which genetic factors may be responsible. In preliminary studies we determined oxazepam pharmacokinetics metabolite profiles after administration of racemic (R,S) oxazepam to eleven human volunteers. The (S) glucuronide was preferentially formed and excreted in nine of the eleven subjects. The ratios of (S) to (R) glucuronide metabolites (S/R ratios) were 3.87 +/- 0.79 (mean +/- SD) and 3.52 +/- 0.60 in urine and plasma, respectively. However, both ratios were significantly lower in two subjects (p < 0.01). In these two atypical subjects, the half-life of (R,S) oxazepam was also markedly longer (14.7 and 15.9 h) than in the other subjects (8.1 +/- 3.2 h). A good correlation (rs = 0.90) between the S/R-glucuronide ratio in urine and the plasma clearance of (R,S) oxazepam suggested that a low S/R ratio may be a marker of poor elimination of oxazepam. In further investigations, the drug was administered to 66 additional subjects. The S/R-glucuronide ratio in 8 h pooled urine was bimodally distributed, with 10% of all subjects possessing ratios below an apparent antimode of 1.9. A survey of the in vitro formation of oxazepam glucuronides by microsomes from 37 human livers also showed that 10% of the livers displayed an abnormally high apparent Michaelis constant (Km) for the formation of the (S) glucuronide, but not of the (R) glucuronide. These results suggest that the glucuronidation of the pharmacologically active (S) enantiomer of oxazepam is decreased in a significant percentage (10%) of Caucasian individuals. The observed in vitro differences in apparent kinetics of the S-glucuronidation reaction may reflect defects at the genetic level, leading to structural changes in the isozyme(s) of UDP-glucuronyltransferase that catalyse this reaction.

Adult↗

PubMed1995

In vitro formation, disposition and toxicity of N-acetoxy-sulfamethoxazole, a potential mediator of sulfamethoxazole toxicity.

Variation in the formation and disposition of the hydroxylamine of (SMX-HA) is thought to play an important role in the pathogenesis of sulfamethoxazole (SMX)-induced idiosyncratic adverse drug reactions. We hypothesized that, in analogy to carcinogenic arylamines, SMX-HA might be further converted to an electrophilic N-acetoxy metabolite which could play a role in mediating SMX toxicity. Accordingly, we chemically synthesized N-acetoxy-SMX, and examined the characteristics of its formation, metabolism, cytotoxicity and mutagenicity in human and bacterial test systems. The human arylamine N-acetyl-transferases, (NAT)1 and NAT2, were capable of converting SMX-HA to N-acetoxy-SMX. NAT1 and NAT2 possessed similar affinities for SMX-HA (apparent Km values of 650 and 520 microM, respectively), but the apparent maximal velocity of the NAT1-mediated acetylation was higher than that of NAT2. (1332 vs. 37 nmol/min/U of immunoreactive NAT protein). Human peripheral blood mononuclear cells 12,000 x g supernatant fractions converted N-acetoxy-SMX mainly back to SMX-HA, and also to a lesser extent to SMX, at clinically relevant concentrations. Similar pathways were observed in human hepatic cytosolic fractions. In a cytotoxicity assay, N-acetoxy-SMX was significantly more toxic to human peripheral blood mononuclear cells than SMX-HA (16.6 vs. 11.5% dead cells at a concentration of 300 microM). N-acetoxy-SMX was weakly mutagenic to the Salmonella typhimurium TA100 strain in the Ames test. These data suggest that the N-acetoxy metabolites of sulfonamides could potentially play a role in mediating sulfonamide idiosyncratic adverse drug reactions.

Acetyltransferases↗

PubMed1994

Structure-function studies of human arylamine N-acetyltransferases NAT1 and NAT2. Functional analysis of recombinant NAT1/NAT2 chimeras expressed in Escherichia coli.

The human arylamine N-acetyltransferases NAT1 and NAT2 catalyze the biotransformation of primary aromatic amine or hydrazine drugs and xenobiotics. These enzymes share 81% amino acid sequence identity, yet differ markedly with respect to their acceptor substrate selectivities and intrinsic in vitro stabilities. To define the contribution of large regions of NAT1 and NAT2 polypeptide structure to enzyme integrity and catalytic specificity, we used selected restriction endonuclease digestions and fragment religation into the tac promoter-based phagemid pKEN2 to construct a panel of 18 NAT1/NAT2 hybrid gene vectors for heterologous expression in Escherichia coli. Induction of hybrid gene expression in recombinant transformants of E. coli strain XA90 led to the production of soluble, catalytically active acetylating enzymes in all cases. Chimeric proteins produced in this fashion were then compared to wild-type NAT1 and NAT2 with respect to their enzyme kinetic constants (apparent Km, Vmax, and Vmax/Km) for the NAT1-selective and NAT2-selective substrates p-aminosalicylic acid and sulfamethazine, respectively, and for their in vitro stabilities at 37 degrees C. The ratio of the Vmax/Km for sulfamethazine to that for p-aminosalicylic acid allowed for the unambiguous classification of each enzyme as either NAT1 or NAT2 type, except for one novel chimera possessing a low Michaelis constant and a high maximal velocity for the acetylation of both substrates. A central region (amino acids 112-210) within the 290-residue polypeptide appeared to play a role in determining NAT1- or NAT2-type behavior. On the other hand, the region (residues 47-111) encompassing the putative active site cysteine (Cys68) was important in contributing to a low apparent Km for p-aminosalicylic acid but not for sulfamethazine, while amino acids 211-250 affected Km for sulfamethazine and 251-290 influenced Km for both substrates. Maximal velocities were highest for both substrates when the central 112-210 amino acid region was derived from NAT1. Finally, the region from amino acids 211-250 in NAT2 was important in determining its greater intrinsic enzyme stability than that exhibited by NAT1.

Acetyltransferases↗

PubMed1994

Characteristics of a white-footed mouse (Peromyscus leucopus) population inhabiting a polychlorinated biphenyls contaminated site.

Demographic characteristics of a white-footed mouse (Peromyscus leucopus) population inhabiting a woodland containing a low level of PCBs contamination were compared to an uncontaminated reference site. Although population density was higher on the PCBs-contaminated grid, the population exhibited greater temporal variability among years and had a higher number of transient animals. These population trends are consistent with those observed in structurally suboptimal habitats, indicating that the PCBs-contaminated grid is functioning as a suboptimal habitat. However, the data are not compelling enough to firmly conclude that the differences are due to contamination rather than to undetected environmental variation between sites. If demographic differences due to contamination are slight, they will be difficult to detect against a background of demographic variation resulting from subtle environmental differences. We conclude that animals at this site experienced a level of contamination that was below threshold for unequivocal detection of demographic effects.

Animals↗

PubMed1994

Measurement of 13C chemical shift tensor principal values with a magic-angle turning experiment.

The magic-angle turning (MAT) experiment introduced by Gan is developed into a powerful and routine method for measuring the principal values of 13C chemical shift tensors in powdered solids. A large-volume MAT probe with stable rotation frequencies down to 22 Hz is described. A triple-echo MAT pulse sequence is introduced to improve the quality of the two-dimensional baseplane. It is shown that measurements of the principal values of chemical shift tensors in complex compounds can be enhanced by using either short contact times or dipolar dephasing pulse sequences to isolate the powder patterns from protonated or non-protonated carbons, respectively. A model compound, 1,2,3-trimethoxybenzene, is used to demonstrate these techniques, and the 13C principal values in 2,3-dimethylnaphthalene and Pocahontas coal are reported as typical examples.

Anisoles↗

PubMed1994

Structure elucidation of three triterpenoid saponins from Alphitonia zizyphoides using 2D NMR techniques.

High-field nmr experiments have been used to determine the structures of three new saponins, zizyphoisides C, D, and E [1-3], isolated from Alphitonia zizyphoides, a medicinal plant found in the Samoan rain forest. The saponins consist of the aglycone, jujubogenin, to which are attached three sugar units and an unsaturated side-chain. The three compounds are similar except for the location of an acetyl group on one of the sugar units.

Carbohydrate Sequence↗