Search PubMed⌕ Search

Biomedical subjects

James L Thomas

Publications and source records attributed to James L Thomas.

At least 19 recordsLinked to original sources
PubMed2006

The microcontact imprinting of proteins: the effect of cross-linking monomers for lysozyme, ribonuclease A and myoglobin.

The performance of molecularly imprinted polymers (MIPs) is of interest to researchers in the field of analytical chemistry, and in the pharmaceutical and food industries. Because the choice of the functional monomer(s) plays a key role in the selectivity of a MIP, the synthesis of an effective, tight-binding MIP can be difficult and time-consuming, involving the evaluation of the binding performance of MIPs of many different compositions. In this study, we report an express method combining molecular imprinting and microcontact printing techniques to prepare a polymer thin film as an artificial antibody. In addition to the microcontact printing technique, isothermal titration of monomers to proteins stamps was investigated to screen the functional monomer for MIPs. Finally, the importance of the choice of cross-linking monomers in MIPs was studied, and these studies suggest that monomers containing an optimal length PEG spacer give higher imprinting effectiveness. Several model antigens (lysozyme, ribonuclease A and myoglobin) were adsorbed on a cover glasses that were pretreated with hexamethyldisilazane (HMDS). These protein stamps were then contacted with different monomer solutions (cross-linking monomers) on a glass slide substrate. Photopolymerization yielded the molecularly imprinted polymer. This technique, analogous to microcontact printing, allows for the rapid, parallel synthesis of MIPs of different compositions, and requires very small volumes of monomers (ca. 4 microL). The technique also avoids potential solubility problems with the molecular targets. Of several cross-linking monomers screened, tetraethyleneglycol dimethacrylate (TEGDMA) gave the most selective lysozyme binding, while polyethyleneglycol 400 dimethacrylate (PEG400DMA) were most selective for ribonuclease A and myoglobin.

Adsorption↗
PubMed2006

Rational proteomics V: structure-based mutagenesis has revealed key residues responsible for substrate recognition and catalysis by the dehydrogenase and isomerase activities in human 3beta-hydroxysteroid dehydrogenase/isomerase type 1.

Mammalian 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) is a member of the short chain dehydrogenase/reductase. It is a key steroidogenic enzyme that catalyzes the first step of the multienzyme pathway conversion of circulating dehydroepiandrosterone and pregnenolone to active steroid hormones. A three dimensional model of a ternary complex of human 3beta-HSD type 1 (3beta-HSD_1) with an NAD cofactor and androstenedione product has been developed based upon X-ray structures of the ternary complex of E. coli UDP-galactose 4-epimerase (UDPGE) with an NAD cofactor and substrate (PDB_AC: 1NAH) and the ternary complex of human type 1 17beta-hydroxysteroid dehydrogenase (17beta-HSD_1) with an NADP cofactor and androstenedione (PDB_AC: 1QYX). The dimeric structure of the enzyme was built from two monomer models of 3beta-HSD_1 by respective 3D superposition with A and B subunits of the dimeric structure of Streptococcus suis DTDP-D-glucose 4,6-dehydratase (PDB_AC: 1KEP). The 3D model structure of 3beta-HSD_1 has been successfully used for the rational design of mutagenic experiments to further elucidate the key substrate binding residues in the active site as well as the basis for dual function of the 3beta-HSD_1 enzyme. The structure based mutant enzymes, Asn100Ser, Asn100Ala, Glu126Leu, His232Ala, Ser322Ala and Asn323Leu, have been constructed and functionally characterized. The mutagenic experiments have confirmed the predicted roles of the His232 and Asn323 residues in recognition of the 17-keto group of the substrate and identified Asn100 and Glu126 residues as key residues that participate for the dehydrogenase and isomerization reactions, respectively.

Amino Acid Sequence↗
PubMed2006

Incorporation of styrene enhances recognition of ribonuclease A by molecularly imprinted polymers.

Ribonuclease A (RNase A) is an RNA-cleaving enzyme characterized by its high conformational stability and strong catalytic activity. This enzyme is ubiquitous in living organisms and is difficult to inactivate. In polymerase chain reaction (PCR) RNase activity is removed by adding inhibitors. Molecularly imprinted polymers (MIPs) with high selectivity, high stability, low cost and facile synthesis could prove useful in extraction of target molecules, such as RNase A, from reaction mixtures. In this investigation, MIPs were synthesized from the monomers styrene and polyethyleneglycol 400 dimethacrylate (PEG400DMA) in several different ratios. Styrene as a functional monomer gave MIPs with a higher affinity for RNase A than other functional monomers tested, according to both enzyme-linked immnuosorbent assay (ELISA) and isothermal titration calorimetry (ITC). The optimum volume ratio of styrene/PEG400DMA was 20/100 at 25 degrees C, and this ratio maximized the rebinding efficiency of RNase A to MIPs. Isothermal titration calorimetry was also used, and could be useful to design the composition of molecularly imprinted polymers for various target molecules.

Adsorption↗
PubMed2005

Receptor aggregation by intermembrane interactions: a Monte Carlo study.

The lateral organization of receptors on cell surfaces is critically important to their function; many receptors transmit transmembrane signals when redistributed into clusters, while the response of others is potentiated by their aggregation. Cell-cell contact can play a crucial role in receptor aggregation, even when the bonds between receptors on one cell and ligands on the other are monovalent. Monte Carlo simulations on a two-membrane model were carried out to determine whether weak enthalpic interactions among receptors in one membrane, and among ligands in another, can work synergistically to give large-scale clustering when the two membranes are brought into contact. The simulations give support to such a clustering mechanism. In addition, because clustering is a cooperative process akin to a phase separation, individual receptors and ligands may undergo repeated binding and unbinding while in a clustered "phase," and a single ligand could interact with multiple different receptor partners. The results suggest a resolution of the dichotomy between serial triggering and aggregation models of T cell activation.

Cell Membrane↗
PubMed2005

Identification of key amino acids responsible for the substantially higher affinities of human type 1 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD1) for substrates, coenzymes, and inhibitors relative to human 3beta-HSD2.

The human type 1 (placenta, breast tumors, and prostate tumors) and type 2 (adrenals and gonads) isoforms of 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD1 and 3beta-HSD2) are encoded by two distinct genes that are expressed in a tissue-specific pattern. Our recent studies have shown that His156 contributes to the 14-fold higher affinity that 3beta-HSD1 exhibits for substrate and inhibitor steroids compared with human 3beta-HSD2 containing Tyr156 in the otherwise identical catalytic domain. Our structural model of human 3beta-HSD localizes His156 or Tyr156 in the subunit interface of the enzyme homodimer. The model predicts that Gln105 on one enzyme subunit has a higher probability of interacting with His156 on the other subunit in 3beta-HSD1 than with Tyr156 in 3beta-HSD2. The Q105M mutant of 3beta-HSD1 (Q105M1) shifts the Michaelis-Menten constant (Km) for 3beta-HSD substrate and inhibition constants (Ki) for epostane and trilostane to the much lower affinity profiles measured for wild-type 3beta-HSD2 and H156Y1. However, the Q105M2 mutant retains substrate and inhibitor kinetic profiles similar to those of 3beta-HSD2. Our model also predicts that Gln240 in 3beta-HSD1 and Arg240 in 3beta-HSD2 may be responsible for the 3-fold higher affinity of the type 1 isomerase activity for substrate steroid and cofactors. The Q240R1 mutation increases the isomerase substrate Km by 2.2-fold to a value similar to that of 3beta-HSD2 isomerase and abolishes the allosteric activation of isomerase by NADH. The R240Q2 mutation converts the isomerase substrate, cofactor, and inhibitor kinetic profiles to the 4-14-fold higher affinity profiles of 3beta-HSD1. Thus, key structural reasons for the substantially higher affinities of 3beta-HSD1 for substrates, coenzymes, and inhibitors have been identified. These structure and function relationships can be used in future docking studies to design better inhibitors of the 3beta-HSD1 that may be useful in the treatment of hormone-sensitive cancers and preterm labor.

3-Hydroxysteroid Dehydrogenases↗
PubMed2004

Factors affecting responsivity of unilamellar liposomes to 20 kHz ultrasound.

Ultrasound is commonly used in the preparation of unilamellar liposome dispersions and is often considered for cell membrane disruption for drug delivery or DNA transfection applications. To better understand the physical and chemical properties of lipid membranes that render them susceptible to ultrasonic permeabilization, the roles of temperature, lipid composition (cholesterol and PEG-lipid content), and liposome size have been studied. The results of these studies suggest that lipid packing is very important to ultrasound responsiveness; surprisingly, cohesive energy and tensile strength are not. Taken together, the experimental results implicate a defect-mediated permeabilization mechanism, rather than pore formation or membrane tearing. The implications of this work for drug release from liposomes and ultrasound-mediated DNA transfection are discussed.

Lipids↗
PubMed2004

Structure/function aspects of human 3beta-hydroxysteroid dehydrogenase.

Separate genes encode the human type 1 (placenta, breast tumors, other peripheral tissues) and type 2 (gonad, adrenal) isoforms of 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD1, 3 beta-HSD2). Mutagenesis of 3 beta-HSD1 produced the Y154F, H156Y and K158Q mutant enzymes in the probable Y(154)-P-H(156)-S-K(158) catalytic motif. The H(156)Y mutant of the 3 beta-HSD1 created a chimera of the 3 beta-HSD2 motif (Y(154)-P-Y(156)-S-K(158)) in 3 beta-HSD1. The D241N, D257L, D258L and D265N mutants are in the potential isomerase site of the 3 beta-HSD1 enzyme. Homology modeling with UDP-galactose-4-epimerase predicted that Asp(36) in the Rossmann-fold domain is responsible for the NAD(H) specificity of human 3 beta-HSD1, and our D36A/K37R mutant tested that assignment. The H(156)Y mutant of the 3 beta-HSD1 enzyme shifted the substrate (DHEA) kinetics to the 14-fold higher K(m) value measured for the 3 beta-HSD2 activity. From Dixon analysis, epostane inhibited the 3 beta-HSD1 activity with 17-fold greater affinity compared to 3 beta-HSD2 and H(156)Y. The mutants of Tyr(154) and Lys(158) exhibited no dehydrogenase activity and appear to be catalytic 3 beta-HSD residues. The D257L and D258L mutations eliminated isomerase activity, suggesting that Asp(257) or Asp(258) may be catalytic residues for isomerase activity. The D36A/K37R mutant shifted the cofactor preference of both 3 beta-HSD and isomerase from NAD(H) to NADP(H). In addition to characterizing catalytic residues, these studies have identified the structural basis (His(156)) for an exploitable difference in the substrate and inhibition kinetics of 3 beta-HSD1 and 3 beta-HSD2. Hence, it may be possible to selectively inhibit human 3 beta-HSD1 to slow the growth of hormone-sensitive breast tumor cells and control placental steroidogenesis near term to prevent premature labor.

3-Hydroxysteroid Dehydrogenases↗
PubMed2004

The higher affinity of human type 1 3beta-hydroxysteroid dehydrogenase (3beta-HSD1) for substrate and inhibitor steroids relative to human 3beta-HSD2 is validated in MCF-7 tumor cells and related to subunit interactions.

Two distinct genes encode the tissue-specific expression of the two isoforms of human 3beta-hydroxysteroid dehydrogenase: 3beta-HSD1 (placenta, mammary gland, breast tumors) and 3beta-HSD2 (gonads, adrenals). Purified 3beta-HSD1 utilizes DHEA as a substrate with 13-fold lower Km than 3beta-HSD2. Using homogenates of human MCF-7 Tet-off breast tumor cells stably transfected with human 3beta-HSD1 or 3beta-HSD2, DHEA is utilized as substrate by 3beta-HSD1 (Km = 4.8 microM) much more avidly than by 3beta-HSD2 (Km = 43 microM). In addition, the 3beta-HSD inhibitor, epostane, binds to purified 3beta-HSD1 with a 17-fold higher affinity compared to 3beta-HSD2. In the MCF-7 cells, two 3beta-HSD inhibitors block 3beta-HSD1 activity (Ki = 0.06 microM, epostane; 0.08 microM, trilostane) with 12- to 14-fold higher affinities compared to the inhibition of 3beta-HSD2 (Ki = 0.85 microM, epostane; 1.01 microM, trilostane). Thus, the substantially higher affinities of human 3beta-HSD1 for substrate and inhibitor steroids measured using the pure isoenzymes have been validated using microsome-bound 3beta-HSD1 and 3beta-HSD2 in the MCF-7 cells. Similar to our previously reported H156Y mutant of 3beta-HSD1, the Q105M mutant of 3beta-HSD1 shifts the substrate and inhibitor kinetic profiles to those of wild-type 3beta-HSD2. However, the Q105M mutant of 3beta-HSD2 retains the substrate and inhibitor kinetic profiles of wild-type 3beta-HSD2. Our structural homology model of human 3beta-HSD predicts that Gln105 on one enzyme subunit hydrogen-binds to His156 on the other subunit of the enzyme homodimer. The higher affinity of 3beta-HSD1 for the steroids may be related to different subunit interactions in the quaternary structures of the two isoenzymes. It may be possible to exploit these kinetic differences to selectively inhibit the conversion of DHEA ultimately to estradiol by 3beta-HSD1 and slow the growth of breast tumor cells.

3-Hydroxysteroid Dehydrogenases↗
PubMed2004

Engineered liposomes for potential alpha-particle therapy of metastatic cancer.

UNLABELLED: Disseminated, metastatic cancer is frequently incurable. Targeted alpha-particle emitters hold great promise as therapeutic agents for disseminated disease. (225)Ac is a radionuclide generator that has a 10-d half-life and results in alpha-emitting daughter elements ((221)Fr, (217)At, (213)Bi) that lead to the emission of a total of 4 alpha-particles. The aim of this study was to develop approaches for stable and controlled targeting of (225)Ac to sites of disseminated tumor metastases. Liposomes with encapsulated (225)Ac were developed to retain the potentially toxic daughters at the tumor site. METHODS: (225)Ac was passively entrapped in liposomes. To experimentally test the retention of actinium and its daughters by the liposomes, the gamma-emissions of (213)Bi were measured in liposome fractions, which were separated from the parent liposome population and the free radionuclides, at different times. Under equilibrium conditions the decay rate of (213)Bi was used to determine the concentration of (225)Ac. Measurements of the kinetics of (213)Bi activity were performed to estimate the entrapment of (213)Bi, the last alpha-emitting daughter in the decay chain. RESULTS: Stable pegylated phosphatidylcholine-cholesterol liposomes of different sizes and charge were prepared. Multiple (more than 2) (225)Ac atoms were successfully entrapped per liposome. (225)Ac retention by zwitterionic liposomes was more than 88% over 30 d. Retention by cationic liposomes was lower. A theoretical calculation showed that for satisfactory (213)Bi retention (>50%), liposomes of relatively large sizes (>650 nm in diameter) are required. (213)Bi retention was experimentally verified to be liposome-size dependent. For large liposomes, the measured (213)Bi retention was lower than theoretically predicted (less than 10%). CONCLUSION: This work supports the hypothesis that it may be possible to develop (225)Ac-based therapies by delivering multiple (225)Ac atoms in liposomes. Improvements in the retention of (225)Ac daughters will likely be necessary to fulfill this potential. Because of the size of the liposomal structures required to contain the daughters, the approach is ideally suited for locoregional therapy (e.g., intraperitoneal, intrahepatic artery, or intrathecal).

Actinium↗
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↗