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James L Thomas

Publications and source records attributed to James L Thomas.

13 recordsLinked to original sources
PubMed2003

Structure/function relationships responsible for coenzyme specificity and the isomerase activity of human type 1 3 beta-hydroxysteroid dehydrogenase/isomerase.

Human type 1 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD/isomerase) catalyzes the two sequential enzyme reactions on a single protein that converts dehydroepiandrosterone or pregnenolone to androstenedione or progesterone, respectively, in placenta, mammary gland, breast tumors, prostate, prostate tumors, and other peripheral tissues. Our earlier studies show that the two enzyme reactions are linked by the coenzyme product, NADH, of the 3 beta-HSD activity. NADH activates the isomerase activity by inducing a time-dependent conformational change in the enzyme protein. The current study tested the hypothesis that the 3 beta-HSD and isomerase activities shared a common coenzyme domain, and it characterized key amino acids that participated in coenzyme binding and the isomerase reaction. Homology modeling with UDP-galactose-4-epimerase predicts that Asp36 is responsible for the NAD(H) specificity of human 3 beta-HSD/isomerase and identifies the Rossmann-fold coenzyme domain at the amino terminus. The D36A/K37R mutant in the potential coenzyme domain and the D241N, D257L, D258L, and D265N mutants in the potential isomerase domain (previously identified by affinity labeling) were created, expressed, and purified. The D36A/K37R mutant shifts the cofactor preference of both 3 beta-HSD and isomerase from NAD(H) to NADP(H), which shows that the two activities utilize a common coenzyme domain. The D257L and D258L mutations eliminate isomerase activity, whereas the D241N and D265N mutants have nearly full isomerase activity. Kinetic analyses and pH dependence studies showed that either Asp257 or Asp258 plays a catalytic role in the isomerization reaction. These observations further characterize the structure/function relationships of human 3 beta-HSD/isomerase and bring us closer to the goal of selectively inhibiting the type 1 enzyme in placenta (to control the timing of labor) or in hormone-sensitive breast tumors (to slow their growth).

Amino Acid Sequence↗
PubMed2003

Stable adhesion of phospholipid vesicles to modified gold surfaces.

Phospholipid vesicles are well-studied biomembrane mimics that are of increasing interest in drug delivery, immunoassays, and sensor chips. In a number of biosensor applications it is desirable to be able to adhere vesicles to a surface in a manner which does not result in their rupture or fusion. Such behavior should, in principle, be achievable by controlling the vesicle-surface and vesicle-vesicle interactions. We have varied vesicle composition and charge (phosphatidylcholine, phosphatidylcholine-phosphatidic acid 18 mol%) and solution ionic strength, to study the adhesion of fluorescent vesicles to glass, gold, and gold modified with chemisorbed acetyl-cysteine. The extent of chemisorption was characterized with angle-resolved X-ray photoelectron spectroscopy (ARXPS), and vesicle integrity and behavior was studied using entrapped and lipophilic fluorescent markers, together and in separate measurements. Vesicle fusion (by energy transfer), adhesion of intact vesicles (with entrapped calcein) and diffusion coefficients (by photobleaching recovery) were monitored using confocal fluorescence microscopy. Acetyl-cysteine modified gold surfaces were shown to be appropriate substrates for adhesion of intact vesicles. Finally, as a 'proof of principle' for fluorescence amplification, release of a self-quenching entrapped reporter dye (calcein) by the detergent Triton X-100 was followed in real time.

Adsorption↗
PubMed2002

Structure/function relationships responsible for the kinetic differences between human type 1 and type 2 3beta-hydroxysteroid dehydrogenase and for the catalysis of the type 1 activity.

Two distinct genes encode the 93% homologous type 1 (placenta, peripheral tissues) and type 2 (adrenals, gonads) 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD/isomerase) in humans. Mutagenesis studies using the type 1 enzyme have produced the Y154F and K158Q mutant enzymes in the Y(154)-P-H(156)-S-K(158) motif as well as the Y269S and K273Q mutants from a second motif, Y(269)-T-L-S-K(273), both of which are present in the primary structure of the human type 1 3beta-HSD/isomerase. In addition, the H156Y mutant of the type 1 enzyme has created a chimera of the type 2 enzyme motif (Y(154)-P-Y(156)-S-K(158)) in the type 1 enzyme. The mutant and wild-type enzymes have been expressed and purified. The K(m) value of dehydroepiandrosterone is 13-fold greater, and the maximal turnover rate (K(cat)) is 2-fold greater for wild-type 2 3beta-HSD compared with the wild-type 1 3beta-HSD activity. The H156Y mutant of the type 1 enzyme has substrate kinetic constants for 3beta-HSD activity that are very similar to those of the wild-type 2 enzyme. Dixon analysis shows that epostane inhibits the 3beta-HSD activity of the wild-type 1 enzyme with 14-17-fold greater affinity compared with the wild-type 2 and H156Y enzymes. The Y154F and K158Q mutants exhibit no 3beta-HSD activity, have substantial isomerase activity, and utilize substrate with K(m) values similar to those of wild-type 1 isomerase. The Y269S and K273Q mutants have low, pH-dependent 3beta-HSD activity, exhibit only 5% of the maximal isomerase activity, and utilize the isomerase substrate very poorly. From these studies, a structural basis for the profound differences in the substrate and inhibition kinetics of the wild-type 1 and 2 3beta-HSD, plus a catalytic role for the Tyr(154) and Lys(158) residues in the 3beta-HSD reaction have been identified. These advances in our understanding of the structure/function of human type 1 and 2 3beta-HSD/isomerase may lead to the design of selective inhibitors of the type 1 enzyme not only in placenta to control the onset of labor but also in hormone-sensitive breast, prostate, and choriocarcinoma tumors to slow their growth.

3-Hydroxysteroid Dehydrogenases↗
PubMed2002

Differences in substrate and inhibitor kinetics of human type 1 and type 2 3beta-hydroxysteroid dehydrogenase are explained by the type 1 mutant, H156Y.

Two distinct genes encode the human type 1 (placenta, mammary gland) and type 2 (adrenal, gonad) isoforms of 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD). We have produced the Y154F, H156Y, and K158Q mutant enzymes in the Y154-P-H156-S-K158 motif of the human type 1 3beta-HSD/isomerase. The H156Y mutant was created to produce a chimera of the type 2 enzyme motif (Y154-P-Y156-S-K158) in the type 1 enzyme. The wild-type (WT) 1 and 2 plus the mutant enzymes were expressed and purified. The Km for dehydroepiandrosterone and Ki for epostane measured with both the H156Y mutant and WT 2 are 13-fold to 17-fold greater than those values obtained with the WT 1 3beta-HSD. The Y154F and K158Q mutants exhibit no 3beta-HSD but have significant isomerase activity. Thus, H156 in WT 1 vs. Y156 in WT 2 accounts for the substantially higher affinity of WT 1 3beta-HSD activity for these substrate and inhibitor steroids relative to the WT 2 enzyme.

Androstenols↗
PubMed2002

Single-cell measurements of polyamidoamine dendrimer binding.

The binding of fluorescein-labeled polyamidoamine dendritic polymers (dendrimers) to the surface of individual fibroblast cells has been quantitatively measured using confocal fluorescence microscopy. Because the binding of these polymers is not strong, significant fluorescence from polymers in solution complicates the measurement, even using confocal optics. To overcome this difficulty, we have developed a new method employing a cell-entrapped marker dye to determine the extracellular volume in each voxel. This allows for a correct subtraction of fluorescence due to polymers in solution. We have found that, as expected, the larger dendrimers bound more tightly than the smaller generations, which may partially account for their increased efficacy in DNA transfection applications. The binding varied significantly from cell to cell, and an analysis of the cell surface area showed that this variability was not caused simply by variations in cell size.

Animals↗
PubMedDate not supplied

ACFAS Universal Foot and Ankle Scoring System: First Metatarsophalangeal Joint and First Ray (module 1).

The American College of Foot and Ankle Surgeons presents the Universal Evaluation Scoring System to evaluate parameters related to foot and ankle surgery. This instrument was developed primarily to allow investigators a means of consistency in clinical assessments. The project was developed in four sections or modules. The first of these modules, First Metatarsophalangeal Joint and First Ray, is presented. This project is unique in that it is the first clinical scoring system of the foot and ankle to become validated by statistical analysis.

Biomechanical Phenomena↗