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Biomedical subjects

T R Lee

Publications and source records attributed to T R Lee.

At least 19 recordsLinked to original sources
PubMed2006

Slc11a1 (formerly Nramp1) polymorphisms and susceptibility to post-transplant lymphoproliferative disease following pediatric liver transplantation.

BACKGROUND: Polymorphisms of the solute carrier family 11 member 1 (Slc11a1) gene have previously been associated with susceptibility to infectious disease, anti-tumor defenses, and autoimmune diseases. We postulated that polymorphisms of the gene may also be associated with susceptibility to post-transplant lymphoproliferative disease (PTLD), a disease thought to be related to an impaired immune response to Epstein-Barr virus (EBV) in immunosuppressed patients. METHODS: Whole blood samples were obtained from 45 pediatric patients who underwent liver transplantation. Polymerase chain reaction (PCR) was used to amplify a 3' region of the gene that includes an exon 15 single-nucleotide substitution (referred to as D543N) and a 4-bp deletion polymorphism (referred to as 3'-UTR). PCR products were digested using AvaII and FokI restriction enzymes for the D543N and 3'-UTR polymorphisms, respectively. PTLD disease status and EBV virus serum titers of all patients were obtained from hospital records. RESULTS: Six of the 45 pediatric transplant recipients developed PTLD. An association was found between 3'-UTR polymorphisms of Slc11a1 and incidence of PTLD after liver transplantation (P = 0.005). In addition, post-transplant serum EBV titers were higher (P = 0.009) for recipients with certain Slc11a1 polymorphisms. No association was found between the D543N polymorphism and incidence of PTLD. CONCLUSION: 3'-UTR polymorphisms of the Slc11a1 gene appear to be associated with susceptibility to PTLD and the immune response to EBV in pediatric liver transplant recipients. Genotyping of pediatric patients undergoing liver transplantation may enable early identification of patients at high risk for developing high EBV titers and/or PTLD.

3' Untranslated Regions↗
PubMed2005

Positive regulation of hepatic carnitine palmitoyl transferase 1A (CPT1A) activities by soy isoflavones and L-carnitine.

BACKGROUND: Genistein increases CPT1A, a rate-limiting enzyme in the beta-oxidation pathway, enzyme activity by increasing CPT1A transcription in HepG2 cells and, consequently, suppresses high fat induced obesity in C57BL/6J mice. Genistein and daidzein are the most abundant isoflavones in soy. AIM OF STUDY: To investigate the effect of co-treatment of genistein and L-carnitine on CPT1A enzyme activity and to determine whether daidzein also increases CPT1A activity and to establish a cell line that can be used to screen chemicals to regulate CPT1A transcription. METHODS: The enzyme activities of CPT1A were determined after HepG2 cells were incubated with 10 microM genistein or 10 microM daidzein or 1 mM L-carnitine or in combination with 10 microM genistein and 1 mM L-carnitine or in combination with 10 microM daidzein and 1 mM L-carnitine. The mRNA expression levels of CPT1A were determined by real time PCR method after HepG2 cells were incubated with 10 microM genistein or 10 microM daidzein. A suggested CPT1A promoter region was cloned from human genomic DNA and the CPT1A promoter-luciferase reporter gene construct was made, and the promoter-reporter gene construct was transfected into human hepatoma cell line Huh7. RESULTS: The enzyme activity of CPT1A was at least 2.3- fold higher in L-carnitine and genistein co-treated HepG2 cells than either single-agent treated cells. Daidzein also significantly increased the mRNA expression of CPT1A as well as the enzyme activity of CPT1A. A stable Huh7 cell line, which was selected after Huh7 cells were transfected with CPT1A promoter luciferase reporter gene construct, was characterized by confirming that luciferase activity of the cell line can be regulated by genistein and daidzein as well as clofibrate, a well-known CPT1A mRNA up-regulating drug. CONCLUSIONS: Genistein and daidzein can up-regulate CPT1A enzyme activity through up-regulation of CPT1A transcription. Co-treatment of L-carnitine and genistein additively increases CPT1A enzyme activity in HepG2 cells. A stable Huh7 cell line transfected with the CPT1A promoter luciferase reporter gene was established and characterized.

Carcinoma, Hepatocellular↗
PubMed2005

Probing the local structure and mechanical response of nanostructures using force modulation and nanofabrication.

Nanostructures of self-assembled monolayers (SAMs) are designed and produced using coadsorption and nanografting techniques. Because the structures of these artificially engineered domains are predesigned and well-characterized, a systematic investigation is possible to study the mechanical responses to force modulation under atomic force microscope tips. Force modulation imaging reveals characteristic contrast sensitivity to changes in molecular-level packing, molecule chain lengths, domain boundaries, and surface chemical functionalities in SAMs. By means of actively tuning the driving frequency, the resonances at the tip-surface contact are selectively activated. Therefore, specific surface features, such as the edges of the domains and nanostructures or desired chemical functionalities, can be selectively enhanced in the amplitude images. These observations provide a new and active approach in materials characterization and the study of nanotribology using atomic force microscopy.

Journal Article↗
PubMed2001

The cis-trans isomerization of 1,2,5,6-tetrasilacycloocta-3,7-dienes: analysis by mechanistic probes and density functional theory.

A series of alkyl- and aryl-substituted derivatives of cis,cis-1,2,5,6-tetrasilacycloocta-3,7-diene were prepared. Isomerization of these compounds to the corresponding trans,trans-1,2,5,6-tetrasilacycloocta-3,7-dienes by exposure to Ru and Zr hydride complexes was explored. Experimental probes of the isomerization were consistent with a stepwise mechanism involving metal hydride addition/elimination rather than one involving radical intermediates. Analysis of the low energy conformers of the various cis and trans isomers of 1,1,2,2,5,5,6,6-octamethyl-1,2,5,6-tetrasilacycloocta-3,7-diene using density functional theory suggested the following trend in stability: trans,trans > cis,trans > cis,cis. The calculated trend in stability was consistent with the experimentally observed unidirectional isomerization of the carbon-carbon double bonds from all cis to all trans and supports a cis,trans isomer as a tenable intermediate.

Journal Article↗
PubMed2001

Kinetics of the cis,cis to trans,trans isomerization of 1,1,2,2,5,5,6,6-octamethyl-1,2,5,6-tetrasilacycloocta-3,7-diene.

The kinetics of the ruthenium-promoted cis,cis to trans,trans isomerization of 1,1,2,2,5,5,6,6-octamethyl-1,2,5,6-tetrasilacycloocta-3,7-diene were investigated. Incubation of a ruthenium alkylidene complex, (Cy(3)P)RuCl(2)(==CHPh)Ru(p-cymene)Cl(2), in CD(2)Cl(2) for 5 days at 40 degrees C afforded a catalytically active ruthenium species that was shown to be responsible for promoting the isomerization. The isomerization was observed to proceed in two steps: (1) conversion of the starting cis,cis isomer to a proposed cis,trans intermediate and (2) subsequent conversion of the intermediate to the product trans,trans isomer. Kinetic studies demonstrated that the two steps are first-order with respect to the concentrations of the cis,cis isomer, the intermediate, and the ruthenium alkylidene complex. The data were further consistent with a mechanism involving bimolecular hydride addition-elimination during the two isomerization steps.

Journal Article↗
PubMed2001

From consensus sequence peptide to high affinity ligand, a "library scan" strategy.

A wide variety of proteins have been shown to recognize and bind to specific amino acid sequences on other proteins. These sequences can be readily identified using combinatorial peptide libraries. However, peptides containing these preferred sequences ("consensus sequence peptides") typically display only modest affinities for the consensus sequence-binding site on the intact protein. In this report, we describe a parallel synthesis strategy that transforms consensus sequence peptides into high affinity ligands. The work described herein has focused on the Lck SH2 domain, which binds the consensus peptide acetyl-Tyr(P)-Glu-Glu-Ile-amide with a K(D) of 1.3 micrometer. We employed a strategy that creates a series of spatially focused libraries that challenge specific subsites on the target protein with a diverse array of functionality. The final lead compound identified in this study displayed a 3300-fold higher affinity for the Lck SH2 domain than the starting consensus sequence peptide.

Amino Acid Sequence↗
PubMed2001

Nonhypoglycemic effects of thiazolidinediones.

The thiazolidinediones are a new class of compounds for treatment of type 2 diabetes. Troglitazone became available in the United States in 1997 but was withdrawn from the market in March 2000 because it caused severe idiosyncratic liver injury. Rosiglitazone and pioglitazone have been available since 1999. Because these drugs directly improve insulin resistance and decrease plasma insulin levels (a risk factor for coronary artery disease), they may decrease risk for cardiovascular disease in patients with type 2 diabetes. Research on the non-glucose lowering effects of troglitazone and, to a lesser extent, of rosiglitazone and pioglitazone have demonstrated changes in several cardiovascular risk factors associated with the insulin resistance syndrome. These beneficial effects include a decrease in blood pressure, correction of diabetic dyslipidemia, improvement of fibrinolysis, and decrease in carotid artery intima-media thickness. Other in vitro effects related to the ability of these agents to bind a newly described class of receptors (peroxisome proliferator-activated receptors) may also have implications for atherosclerosis. However, these drugs increase low-density lipoprotein (LDL) cholesterol levels and may favorably change LDL particle size and susceptibility to oxidation (although the implications of the latter changes are not dear). Furthermore, these drugs tend to cause weight gain. The authors' enthusiasm for these drugs has diminished somewhat because of reported adverse events, including rare liver failure. Nevertheless, because of the mechanism of action of the thiazolidinediones, clinical trials designed to determine whether they (or similar "insulin sensitizers") decrease cardiovascular events in people with type 2 diabetes will be of interest.

Body Weight↗
PubMed2001

Transcriptional regulation of the Bacillus subtilis bscR-CYP102A3 operon by the BscR repressor and differential induction of cytochrome CYP102A3 expression by oleic acid and palmitate.

The adjacent yrhI and yrhJ genes were identified by the Bacillus subtilis genome sequencing project. We now report that yrhJ (renamed CYP102A3) encodes a cytochrome P450 and that yrhI (renamed bscR) encodes a repressor that negatively regulates the transcription of the bscR-CYP102A3 operon. The transcriptional initiation site of bscR has been mapped by primer extension analysis. An 18-bp perfect palindromic sequence centered 65.5 bp downstream from the transcriptional initiation site of bscR has been identified as the binding site for BscR by gel mobility shift assays. Base substitutions in the 18-bp inverted repeat resulted in derepression of the bscR-xylE transcriptional fusion in vivo. bscR-xylE fusion studies and Northern blot analysis revealed that oleic acid and palmitate could induce the expression of the bscR-CYP102A3 operon to a considerable extent. However, only oleic acid was capable of preventing the binding of BscR to its operator DNA in vitro, suggesting that the induction of CYP102A3 expression by oleic acid and palmitate in B. subtilis might be mediated through different mechanisms.

Amino Acid Sequence↗
PubMed2001

Academic goals, achievement, and age at first sexual intercourse: longitudinal, bidirectional influences.

This study examined bidirectional relationships between age at first sexual intercourse and academic goals and achievement. It was hypothesized that lower educational goals and achievement would be associated with initiating sexual intercourse at a younger age, and that initiating sexual activity early would be associated with a decrease in subsequent academic achievement and goals. In longitudinal data spanning 11 years, evidence was found for bidirectional effects. One interpretation of these results is that adolescents with high educational goals and achievement delay having intercourse because of the perceived risks (e.g., pregnancy and sexually transmitted diseases may jeopardize their plans for the future). Conversely, adolescents who engage in sexual intercourse at young ages might undergo a change in attitudes, including reduced interest in academic achievement and goals. The specific educational variables most strongly related to adolescent sexual intercourse in this study differed substantially by race and gender.

Achievement↗
PubMed2000

Molecular rulers: an assessment of distance and spatial relationships of Src tyrosine kinase Sh2 and active site regions.

The three-dimensional structures of the inactive conformations of Hck and Src, members of the Src protein-tyrosine kinase family, have recently been described. In both cases, the catalytic domain lies on the opposite face of the enzyme from the SH2 and SH3 domains. The active conformation of these enzymes has not yet been described. Given the known role of the SH2 and SH3 domains in promoting substrate binding, enzyme activation likely reorients the relative spatial arrangement between the SH2/SH3 domains and the active site region. We describe herein a series of "molecular rulers" and their use in assessing the topological and spatial relationships of the SH2 and active site regions of the Src protein-tyrosine kinase. These synthetic compounds contain sequences that are active site-directed (-Glu-Glu-Ile-Ile-(F(5))Phe-, where (F(5))Phe is pentafluorophenylalanine) and SH2-directed (-Tyr(P)-Glu-Glu-Ile-Glu-), separated by a sequence of variable length. The most potent bivalent compound, acetyl-Glu-Glu-Leu-Leu-(F(5))Phe-(GABA)(3)-Tyr(P)-Glu-Glu-Ile-Glu-amide (where GABA is gamma-aminobutyric acid), displays a >120-fold enhancement in inhibitory potency relative to the simple monovalent active site-directed species, acetyl-Glu-Glu-Leu-Leu-(F(5))Phe-amide. The short linker length (3 GABA residues) between the active site- and SH2-directed peptide fragments suggests that the corresponding domains on the Src kinase can assume a nearly contiguous spatial arrangement in the active form of the enzyme.

Amino Acid Sequence↗
PubMed2000

SH2-directed ligands of the Lck tyrosine kinase.

Two separate libraries, prepared via parallel synthesis, were employed to identify low-molecular-weight SH2-targeted ligands for the Lck tyrosine protein kinase. These libraries were constructed to furnish non-amino acid analogues of the (1) Glu-Glu and (2) Ile residues of the Lck SH2 domain peptide ligand Ac-pTyr-Glu-Glu-Ile-amide. The lead compound acquired in this study exhibits a dissociation constant for the Lck SH2 domain that is comparable to that displayed by Ac-pTyr-Glu-Glu-Ile-amide. These results demonstrate that the standard amino acid residues Glu-Glu-Ile can be completely replaced with non-amino acid moieties without loss of SH2 affinity.

Amides↗
PubMed1996

Rate-determining steps for tyrosine phosphorylation by the kinase domain of v-fps.

The rate-determining steps in the phosphorylation of four tyrosine-containing peptides by the kinase domain of the nonreceptor tyrosine protein kinase v-fps were measured using viscosometric methods. The peptides were phosphorylated by a fusion protein of glutathione-S-transferase and the kinase domain of v-fps (GST-kin) and the initial velocities were determined by a coupled enzyme assay. Peptides I (EEEIYEEIE), II (EAEIYEAIE), and III (DADIYDAID) were phosphorylated by GST-kin with similar kinetic constants. The viscosogens, glycerol and sucrose, were found to have intermediate effects on kcat and no effect on kcat/Kpeptide for the phosphorylation of these three peptides. The data are interpreted according to the Stokes-Einstein equation and a simple three-step mechanism involving substrate binding, phosphoryl group transfer, and net product release. Two competitive inhibitors (EAEIFEAIE and DADIFDAID) exhibited K1 values that are 6-10-fold higher than the Kpeptide values for their analogous peptide substrates. The data imply that peptides I-III are in rapid equilibrium with the enzyme and that kcat is partially limited by both phosphoryl group transfer (40-100 s-1) and product release (17-22 s-1). GST-kin phosphorylates peptide IV (R5AENLEYamide) with a low Km (100 microM) and a kcat that is 40-fold lower than that for peptide I. No effect of solvent viscosity was observed for the phosphorylation of this peptide on either kcat or kcat/Kpeptide. This suggests that highly viscous solutions do not perturb structure and that the rate-determining step for this poor substrate is phosphoryl group transfer. The data indicate that the kinase domain of v-fps phosphorylates its best substrate with a chemical rate constant that is at least 5-fold lower than that for the serine-specific cAMP-dependent protein kinase and its best substrate LRRASLG (Adams & Taylor, 1992). Interestingly, both enzymes exhibit a similar affinity for their substrates and both enzymes release their products at a similar rate. This implies that the differences in catalytic efficiency between serine- and tyrosine-specific protein kinases lie exclusively in the rate constants for phosphoryl group transfer and not in substrate absorption or product desorption.

Amino Acid Sequence↗
PubMed1996

Is protein kinase substrate efficacy a reliable barometer for successful inhibitor design?

We have addressed the question of whether protein kinase substrate efficacy is a reliable barometer for successful inhibitor design by assessing the dependence of kcat and kcat/Km for eight separate alcohol-bearing residues on solvent viscosity. We have found that the Km for three structurally distinct primary alcohol-containing peptides overestimates the affinity that these species exhibit for the cAMP-dependent protein kinase. In all three cases, the rate-determining step is product release, and substrate binding is best described as rapid equilibrium. In contrast, peptides containing the following phosphorylatable residues all provide Km values that are accurate assessments of substrate affinity for the protein kinase: a secondary alcohol, a simple phenol, and a primary alcohol with a relatively long side chain. In the latter three instances, the rate-determining step is phosphoryl transfer. Finally, two aromatic alcohol-containing residues that possess lipophilic side chains exhibit Michaelis constants that underestimate enzyme affinity. These results demonstrate that while it may be tempting to employ structural elements from the most efficient substrates (e.g. primary alcohols) for inhibitor design, less effective substrates may serve as a more accurate assessment of inhibitory success.

Alcohols↗
PubMed1995

Precision substrate targeting of protein kinases v-Abl and c-Src.

The active site substrate specificities of v-Abl and c-Src are compared and contrasted. Both enzymes catalyze the phosphorylation of a broad assortment of peptide-bound aliphatic and aromatic alcohols, such as achiral and simple straight chain residues. In addition, both protein kinases exhibit a "dual specificity" with respect to the ability to utilize D- and L-configurational isomers as substrates. However, c-Src and v-Abl are extremely inefficient as catalysts for certain structural arrangements, including secondary alcohols and primary alcohols containing large substituents in close proximity to the hydroxyl moiety. In addition to these similarities, these enzymes also display noteworthy differences in catalytic behavior. Whereas c-Src exhibits a modest preference for aromatic versus aliphatic alcohols, v-Abl does not. Most dramatic is the ability of c-Src to utilize short chain alcohols as substrates, an activity virtually absent from the catalytic repertoire of v-Abl. The implications of these observations are 2-fold. First, because both enzymes are able to accommodate a wide variety of structural variants within their respective active site regions, there exists a substantial degree of flexibility with respect to inhibitor design. Second, because these enzymes exhibit disparate active site specificities, it is possible that other tyrosine-specific protein kinases will display unique substrate specificities as well. Consequently, it may ultimately be possible to exploit these differences to generate inhibitors that precisely target specific protein kinases.

Amino Acid Sequence↗
PubMed1995

The extraordinary active site substrate specificity of pp60c-src. A multiple specificity protein kinase.

We report the first active site substrate specificity analysis of a tyrosine-specific protein kinase, namely pp60c-src. Like the cAMP-dependent protein kinase and protein kinase C, pp60c-src will phosphorylate an assortment of achiral residues attached to active site-directed peptides. Furthermore, pp60c-src phosphorylates both aromatic and aliphatic alcohols. However, the substrate specificity of pp60c-src is much broader than that of the two previously examined serine/threonine-specific protein kinases. We have previously shown that both the cAMP-dependent protein kinase and protein kinase C will utilize a wide array of non-amino acid residues as substrates, as long as the distance between the hydroxyl moiety and the adjacent peptide backbone is comparable with that present in serine and threonine (Kwon, Y.-G., Mendelow, M., and Lawrence, D. S. (1994) J. Biol. Chem. 269, 4839-4844). In marked contrast, pp60c-src does not discriminate against substrates on the basis of chain length, catalyzing the phosphorylation of residues that contain anywhere from 2-12 carbons between the alcohol functional group and the adjacent peptide bond. In addition, pp60c-src phosphorylates L-serine in an active site-directed peptide. The possible structural basis for the multiple specificity of pp60c-src is discussed. Finally, the active site specificity of pp60c-src is not just limited to L-amino acid residues, but also extends into the realm of D-amino acids as well.

Alcohols↗
PubMed1994

Phenol kinase activity of the serine/threonine-specific cAMP-dependent protein kinase: steric and electronic effects.

We have found that the cAMP-dependent protein kinase catalyzes the phosphorylation of a wide variety of peptide-based aromatic alcohols, thereby greatly amplifying the range of compounds recognized as substrates by this enzyme. This newly discovered enzyme-catalyzed reaction is sensitive to both steric and electronic effects. Substituents on the aromatic ring that are positioned para to the hydroxyl moiety lower the observed Km, presumably via a favorable interaction with an adjacent hydrophobic pocket. In contrast, electron-withdrawing substituents have a slight adverse effect on the kinetics of phosphoryl transfer, an observation which is consistent with the notion that the rate of substrate turnover is dependent upon the nucleophilicity of the phosphorylatable hydroxyl moiety. As a corollary, electron-donating groups on the aromatic nucleus promote the rate of phosphoryl transfer to such an extent that the observed Vmax values approach those exhibited by aliphatic alcohols. This suggests that analogously appended electron-donating groups on tyrosine moieties could dramatically improve the modest Vmax values that are typical for tyrosine kinase-catalyzed reactions.

Amino Acid Sequence↗
PubMed1993

The active site substrate specificity of the cAMP-dependent protein kinase.

cAMP-dependent protein kinase substrates have been synthesized employing an unusually efficient method that allows the alcohol-bearing residue to be incorporated into the peptide after solid phase peptide synthesis. These peptide substrates have been utilized to map the active site substrate specificity of the protein kinase. Only alpha- or beta-substituted alcohol-bearing residues containing the proper absolute configuration are phosphorylated by the enzyme. However, the cAMP-dependent protein kinase will phosphorylate achiral residues. The implications of the observed protein kinase substrate specificity with respect to inhibitor design are discussed.

Alcohols↗