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J D Stewart

Publications and source records attributed to J D Stewart.

At least 55 records · Page 3Linked to original sources
PubMed1995

Detection of a catalytic antibody species acylated at the active site by electrospray mass spectrometry.

The chemical interactions between a catalytic antibody Fv fragment and ester substrates were examined using pneumatically assisted electrospray (ion spray) mass spectrometry. Upon addition of the p-nitrophenyl ester substrate to the antibody fragment, an antibody fragment species that represents approximately 8% of the total Fv concentration is clearly observed in the electrospray spectrum. The observed increase in molecular weight of the Fv fragment corresponds to the mass of the acyl group of the substrate. Formation of the acyl-Fv species is blocked by preincubation of the antibody fragment with hapten inhibitor, suggesting that the acyl linkage involves a residue in the active site of the antibody. The acyl-Fv species is not observed when the corresponding p-chlorophenyl ester substrate is used, indicating that the level of this species is dependent on the leaving group of the substrate. The acylated species is not observed for a site-directed mutant lacking catalytic activity, His L91 Gln. The present results are consistent with modeling studies of the structure of the Fv fragment and provide strong confirmatory evidence for the multistep kinetic mechanism previously proposed for this antibody.

Acylation↗
PubMed1995

New insight into the catalytic mechanism of chorismate mutases from structural studies.

Chorismate mutase catalyzes the rearrangement of chorismic acid to prephenic acid, which is the first committed step in the biosynthesis of aromatic amino acids. Its catalytic mechanism has been much studied, but is poorly understood. Recent structural information on enzymes from two species, and on an antibody that catalyzes the same reaction, has shed new light on this topic.

Animals↗
PubMed1995

Stomatal and mesophyll limitations of photosynthesis in black spruce seedlings during multiple cycles of drought.

Container-grown black spruce (Picea mariana (Mill.) B.S.P.) seedlings were planted in trays containing a sand and peat mixture, and placed in a climate-controlled greenhouse. One group of seedlings was kept well-watered, and another group was subjected to three cycles of drought. Gas exchange analysis showed that mesophyll photosynthetic function was largely unimpaired by drought. In contrast, stomatal conductance was sensitive to drought, although it became less sensitive with each drought cycle. Both stomatal and mesophyll conductances increased with time in control and drought-stressed seedlings, but mesophyll conductance increased with time more rapidly than did stomatal conductance. Limitation of photosynthetic rate was dominated by the mesophyll. In control seedlings, relative stomatal limitation increased from 6 to 16% by the end of the experiment. In drought-stressed seedlings, relative stomatal limitation of photosynthesis reached 40% during the first drought, but decreased to near control values immediately after rewatering. Because the third, most severe drought had only a minor effect on stomatal conductance, relative stomatal limitation of photosynthesis was similar to that in control seedlings by the end of the experiment. Inhibition of ontogenetic change during drought stress may be responsible for the apparent acclimation of mesophyll photosynthetic processes. We conclude that it would be more effective to select for high photosynthetic capacity than for reduced stomatal sensitivity when breeding for increased drought resistance in black spruce seedlings.

Journal Article↗
PubMed1994

Dissection of an antibody-catalyzed reaction.

Antibody 43C9 accelerates the hydrolysis of a p-nitroanilide by a factor of 2.5 x 10(5) over the background rate in addition to catalyzing the hydrolysis of a series of aromatic esters. Since this represents one of the largest rate accelerations achieved with an antibody, we have undertaken a series of studies aimed at uncovering the catalytic mechanism of 43C9. The immunogen, a phosphonamidate, was designed to mimic the geometric and electronic characteristics of the tetrahedral intermediate that forms upon nucleophilic attack by hydroxide on the amide substrate. Further studies, however, revealed that the catalytic mechanism is more complex and involves the fortuitous formation of a covalent acyl-antibody intermediate as a consequence of complementary side chain residues at the antibody-binding site. Several lines of evidence indicate that the catalytic mechanism involves two key residues: His-L91, which acts as a nucleophile to form the acyl-antibody intermediate, and Arg-L96, which stabilizes the anionic tetrahedral moieties. Support for this mechanism derives from the results of site-directed mutagenesis experiments and solvent deuterium isotope effects as well as direct detection of the acyl-antibody by electrospray mass spectrometry. Despite its partial recapitulation of the course of action of enzymic counterparts, the reactivity of 43C9, like other antibodies, is apparently limited by its affinity for the inducing immunogen. To go beyond this level, one must introduce additional catalytic functionality, particularly general acid-base catalysis, through either improvements in transition-state analog design or site-specific mutagenesis.

Aniline Compounds↗
PubMed1994

Site-directed mutagenesis of a catalytic antibody: an arginine and a histidine residue play key roles.

Individual residues important for ligand binding and catalytic activity were identified by computer modeling and investigated by site-directed mutagenesis for catalytic antibody 43C9, which accelerates amide hydrolysis by a factor of 10(6). On the basis of a computer model, Tyr L32, His L91, Arg L96, His H35, and Tyr H95 were chosen for replacement by site-directed mutagenesis. To facilitate these studies, an expression system was developed in which properly folded 43C9 single-chain antibody was secreted from an engineered Escherichia coli host. Substitution of His L91 by Gln produced a mutant with no catalytic activity, but whose affinities for ligands were nearly the same as those of the wild-type, identifying His L91 as the nucleophile that forms the acyl intermediate implicated by previous kinetic studies. Arg L96 is also critical for catalytic activity and appears to function as a oxyanion hole for the tetrahedral transition states. Two substitutions for His H35 resulted in mutant proteins with no catalytic activity as well as altered affinities for ligands, indicating an important structural role for this residue. Substitutions for Tyr L32 and Tyr H95 were made in an attempt to improve the catalytic efficiency of 43C9. The results of these mutations allow us to propose a mechanism for 43C9-catalyzed hydrolysis: Substrate binding to 43C9 orients the scissile carbonyl group adjacent to both the His L91 and Arg L96 side chains. The imidazole of His L91 acts as a nucleophile, forming an acyl-antibody intermediate that breaks down by hydroxide attack to afford the products and regenerate the catalyst.

Antibodies, Catalytic↗
PubMed1994

Quantitative sweat testing using acetylcholine for direct and axon reflex mediated stimulation with silicone mold recording; controls versus neuropathic diabetics.

Sweat glands were stimulated by iontophoresed acetylcholine (ACh). Using a two-chamber capsule, one group of sweat glands was stimulated directly by ACh, and another stimulated indirectly via axon reflexes. Sweat droplets were recorded with a silicone mold and counted by a dissecting microscope and a computerized scanner. Responses from 32 diabetics with generalized somatic peripheral neuropathy were compared with 32 controls. Counting sweat droplets by computerized scanning was more accurate than by microscope. The numbers of droplets, their total areas, and mean areas were all larger in the direct response. Counts of droplet numbers was more diagnostically useful than measuring the total area and the mean areas of the droplets. There was a high correlation between droplet numbers of the direct and the indirect sweat responses. Droplet number counts of the direct response had a slightly higher diagnostic yield than those of the technically more complicated indirect response: for a specificity of 95%, the direct response had a sensitivity of 55%, the indirect being 50%.

Acetylcholine↗
PubMed1994

Induction of in vivo DNA adducts by 4 industrial by-products in the rat-lung-cell system.

Benz[a]anthracene (BA), dibenz[a,h]anthracene (DBA), dibenzo[a,i]pyrene (DBP), and dibenz[a,h]acridine (DBAC) are by-products found in many industrial wastes and emissions. Workers in the related occupational settings are potentially exposed to these substances through inhalation. In the present study, induction of DNA adducts in vivo by these chemicals was investigated using 32P-postlabeling analysis in the rat-lung-cell system. The potency of DNA-adduct inducing activity was also compared to that of two cytogenetic endpoints i.e., sister-chromatid exchange (SCE) and micronucleus formation. Via intratracheal instillation, male CD rats (6/group) were dosed 3 times with BA, DBA, DBP or DBAC in a 24-h interval. Lung cells were enzymatically separated and used to determine the frequency of DNA adducts, SCE and micronuclei. Results show that all 4 test compounds induced DNA adducts, SCEs, and micronuclei in the rat-lung cell in vivo and that the postlabeling DNA adduct assay detected genotoxic activity at lower dose levels than the two cytogenetic assays. These findings suggest that BA, DBA, DBP or DBAC are rat pulmonary genotoxicants and the DNA-adduct assay is more sensitive than SCE or micronucleus assays for detecting the pulmonary genotoxicity of these industrial PAHs in the in vivo rat-lung-cell system.

Acridines↗
PubMed1993

An idealized dynamical model of simple diffusional interactions between macromolecules and between macromolecules and surfaces.

A model for hard-sphere, nonelectrostatic diffusional interactions between macromolecules and between macromolecules and surfaces is presented. Quantities such as average free path and impact frequency are derived, being validated by full three-dimensional simulations of large numbers of random flights. A method for obtaining the total collision rate is developed based on absorption boundary conditions applied to the time-independent diffusion equation, multiplied by the probable number of subsequent encounters. A distribution function is obtained that describes both the spatial and temporal impact profiles of diffusing molecules with surfaces as well as the detailed nature of individual encounters. Conclusions are that the interaction of diffusing macromolecules with surfaces is an extremely efficient process; trajectories are typified by clusters of relatively closely spaced encounters separated by relatively long excursions prior to reimpact. Results should be applicable to the development of kinetic models for describing such complex phenomena as ligand binding to receptors on cell surfaces.

Cell Membrane↗
PubMed1993

Cerebral vein thrombosis due to hereditary antithrombin III deficiency.

Cerebral vein thrombosis, also called superior sagittal sinus thrombosis, is a well recognized clinical and radiologic entity associated with a variety of medical disorders. We report a patient with fatal cerebral vein thrombosis following myelography, in whom the cause was familial antithrombin III (AT3) deficiency. Unsuspected AT3 deficiency should be considered in cases of unexplained cerebral venous thromboses.

Adult↗
PubMed1992

Distal small fiber neuropathy: results of tests of sweating and autonomic cardiovascular reflexes.

The purpose of this study was to evaluate sweating and cardiovascular autonomic function in patients with distal small fiber neuropathy (DSFN). Sweat testing by the quantitative sudomotor axon reflex test was abnormal in 32 of 40 (80%) patients. The thermoregulatory sweat test was abnormal in 18 of 25 (72%) patients; one or both tests were abnormal in 36 of 40 (90%). Minor heart rate abnormalities were present in 11 of 40 (28%) patients. We conclude that, in patients with DSFN: (a) the sympathetic sudomotor fibers are frequently affected, and that evaluation of sweating is a useful diagnostic test; (b) that the autonomic nerves controlling heart rate are less affected.

Autonomic Nervous System Diseases↗
PubMed1992

Comparison of DNA adduct detection between two enhancement methods of the 32P-postlabelling assay in rat lung cells.

32P-Postlabeling analysis is a useful assay system for detecting the covalent binding of mutagens and/or carcinogens to DNA. The detection ability of this system has been tremendously enhanced by the incorporation of butanol extraction or nuclease P1 treatment into the experimental protocol. In this study, the sensitivity of adduct detection between these two enhancement methods was compared in vivo or in vitro with 2-aminoanthracene (2AA), 2,4,7-trinitro-9-fluorenone (TNF), and nitrosated coal dust extract (NCDE) using the lung cells of rats. For the in vivo assay, male CD rats were dosed 3 times via intratracheal instillation, whereas for the in vitro study, rat lungs cut into small pieces were treated with test substances for 16 h without exogenous activation. Although, under the conditions tested, both the butanol and the nuclease P1 methods detected DNA adducts caused by all 3 test agents in rat lung cells in vivo or in vitro, a higher adduct detecting ability was found with the butanol enhancement for 2AA and TNF, and with the nuclease P1 enhancement for NCDE. The results suggest that overall the butanol enhancement method is a more sensitive protocol. However, for detecting unknown adduct-forming chemicals, especially when they are present in complex mixtures, both enhancement methods may have to be used.

Animals↗
PubMed1992

Evaluation of the HCFA model for the analysis of mortality following hospitalization.

From 1987 through 1990, the Health Care Financing Administration (HCFA) evaluated variations in the mortality rates experienced by patients admitted to hospitals participating in the Medicare program. This study was conducted to evaluate the adequacy of the model used for that purpose. Detailed clinical data were gathered on 42,773 patients admitted to 84 statistically selected hospitals. The effect of risk adjustment using the HCFA model, which is based on claims data, was compared to a risk-adjustment model based on physiologic and clinical data. Models that include claims data were markedly superior to those containing only demographic characteristics in predicting the probability of patient death, and the addition of clinical data resulted in further improvement. The correlation of ranks of hospitals based on a model that uses only the claims data and on one that uses, in addition, clinical data, was .91. As a screen for the identification of "high (mortality) outlier" hospitals, the claims model had moderate sensitivity (81 percent) and specificity (79 percent), a high negative predictive value (90 percent), and a low positive predictive value (64 percent) when compared to the clinical model. The two mortality models gave similar results when used to determine which structural characteristics of hospitals were related to mortality rates: hospitals with a higher proportion of registered nurses or board-certified physician specialists, or with a greater level of access to high-technology equipment had lower risk-adjusted mortality rates. These data suggest that the current claims-based risk-adjustment procedure may satisfactorily be used to characterize variations in mortality rates associated with hospitalization. The procedure could also be used as a basis for further epidemiological analyses of factors that affect the probability of patient death. However, it does not positively identify outlier hospitals as providers of problematic care.

Centers for Medicare and Medicaid Services, U.S.↗