Biomedical subjects

Edward Wright

Publications and source records attributed to Edward Wright.

9 recordsLinked to original sources

PubMed2007

Thermodynamic characterization of the interaction between CAR-RXR and SRC-1 peptide by isothermal titration calorimetry.

The constitutive androstane receptor (CAR) enhances transcription of specific target genes that regulate several metabolic pathways. CAR functions as an obligate heterodimer (CAR-RXR) with the retinoid X receptor (RXR). Also part of the active receptor complex is the steroid receptor coactivator-1 (SRC-1) which interacts with the receptor complex via specific receptor interaction domains (RIDs). A peptide derived from SRC-1 RID2 is used to study the thermodynamic properties of the interaction with the CAR-RXR ligand binding domain (LBD) complex. In the absence of ligands for both CAR and RXR, binding of coactivator peptide to the CAR-RXR heterodimer is characterized by a favorable enthalpy change and an unfavorable entropy change. The addition of the CAR agonist, TCPOBOP, increases the affinity for coactivator by decreasing the unfavorable entropy and increasing the favorable intrinsic enthalpy of the interaction. The RXR ligand, 9-cis-RA, generates a second SRC-1 site and increases the affinity by improving the entropic component of binding. There is an additional increase in affinity for one of the two sites in the presence of both ligands. The change in heat capacity (deltaCp) is also investigated. A 2-fold difference in deltaCp is observed between liganded and unliganded CAR-RXR. The observed thermodynamic parameters for binding of SRC-1 peptide to liganded and apo CAR-RXR as well as the difference in the deltaCp data provide evidence that the apo CAR-RXR heterodimer is conformationally mobile. The more favorable enthalpic contribution for TCPOBOP-bound CAR-RXR indicates that preformation of the binding site improves the complementarity of the coactivator-receptor interaction.

Alitretinoin↗

PubMed2006

Molecular determinants of affinity for aminoglycoside binding to the aminoglycoside nucleotidyltransferase(2'')-Ia.

One of the most commonly occurring aminoglycoside resistance enzymes is aminoglycoside 2''-O-nucleotidyltransferase [ANT(2'')]. In the present study molecular determinants of affinity and specificity for aminoglycoside binding to this enzyme are investigated using isothermal titration calorimetry (ITC). Binding of aminoglycosides is enthalpically driven accompanied by negative entropy changes. The presence of metal-nucleotide increases the affinity for all but one of the aminoglycosides studied but has no effect on specificity. The substituents at positions 1, 2', and 6' are important determinants of substrate specificity. An amino group at these positions leads to greater affinity. No correlation is observed between the change in affinity and enthalpy. At the 2' position greater affinity results from a more negative enthalpy for an aminoglycoside containing an amino rather than a hydroxyl at that position. At the 6' position the greater affinity for an aminoglycoside containing an amino substituent results from a less disfavorable entropic contribution. The thermodynamic basis for the change in affinity at position 1 could not be determined because of the weak binding of one of the aminoglycoside substrates, amikacin. The effect of increasing osmotic stress on affinity was used to determine that a net release of approximately four water molecules occurs when tobramycin binds to ANT(2''). No measurable net change in the number of bound water molecules is observed when neomycin binds the enzyme. Data acquired in this work provide the rationale for the ability of ANT(2'') to confer resistance against kanamycins but not neomycins.

Aminoglycosides↗

PubMed2005

Discovery of non-carbohydrate inhibitors of aminoglycoside-modifying enzymes.

Chemical modification and inactivation of aminoglycosides by many different enzymes expressed in pathogenic bacteria are the main mechanisms of bacterial resistance to these antibiotics. In this work, we designed inhibitors that contain the 1,3-diamine pharmacophore shared by all aminoglycoside antibiotics that contain the 2-deoxystreptamine ring. A discovery library of molecules was prepared by attaching different side chains to both sides of the 1,3-diamine motif. Several of these diamines showed inhibitory activity toward two or three different representative aminoglycoside-modifying enzymes (AGMEs). These studies yielded the first non-carbohydrate inhibitor N-cyclohexyl-N'-(3-dimethylamino-propyl)-propane-1,3-diamine (Compound G,H) that is competitive with respect to the aminoglycoside binding to the enzyme aminoglycoside-2''-nucleotidyltransferase-Ia (ANT2''). Another diamine molecule N-[2-(3,4-dimethoxyphenyl)-ethyl]-N'-(3-dimethylamino-propyl)-propane-1,3-diamine (Compound H,I) was shown to be a competitive inhibitor of two separate enzymes (aminoglycoside-3'-phosphotransferase-IIIa (APH3') and ANT2'') with respect to metal-ATP. Thermodynamic and structural-binding properties of the complexes of APH3' with substrates and inhibitor were shown to be similar to each other, as determined by isothermal titration calorimetry and NMR spectroscopy.

Aminoglycosides↗

PubMed2005

Enzyme-substrate interactions with an antibiotic resistance enzyme: aminoglycoside nucleotidyltransferase(2'')-Ia characterized by kinetic and thermodynamic methods.

Aminoglycoside nucleotidyltransferase(2'')-Ia is one of the most often detected enzymes in aminoglycoside-resistant bacteria. Despite its prevalence, little biochemical and biophysical work has been reported for this enzyme. In the current study, substrate specificity and temperature dependence of k(cat) are determined by kinetic assays. Dissociation constants and thermodynamic properties of enzyme-substrate complexes are determined by isothermal titration calorimetry, electron paramagnetic resonance, and fluorescence spectroscopy. Kinetic studies show that aminoglycosides with 2'-NH(2) are better substrates (higher k(cat)/K(m)) than ones with 2'-OH when magnesium(II) is used as the catalytically required divalent cation. The activity is reduced 10-fold for substrates with 2'-NH(2) when manganese(II) replaces magnesium as the required metal. However, kanamycin A, which has a 2'-OH, shows a much smaller decrease in activity when manganese substitutes for magnesium as the divalent cation. Temperature dependence studies show the activation energy of catalysis to be 19.2 kcal/mol and the temperature optimum between 30 and 32 degrees C. The binding of the aminoglycoside substrate tobramycin to the enzyme occurs with a favorable enthalpy which compensates for a large entropic penalty to yield a negative DeltaG value for the complex formation. Enthalpy of binding is less exothermic in the presence of metal-nucleotide. However, due to the more favorable entropy, a more favorable DeltaG is observed for the formation of the enzyme-metal-nucleotide:aminoglycoside complex. Tobramycin binds to ANT(2' ') with a dissociation constant of 0.6 microM, which is further reduced by 3-fold when metal-nucleotide is present. Binding of ATP to the enzyme is determined to be very weak in the absence of a divalent cation, and becomes 2 orders of magnitude tighter when magnesium or manganese is present. Binding studies also show that, in addition to binding to the enzyme in the form of metal-nucleotide complex, a second catalytically required metal binds to an additional site on the enzyme.

Anti-Bacterial Agents↗

PubMed2005

Ets-2 repressor factor recruits histone deacetylase to silence human cytomegalovirus immediate-early gene expression in non-permissive cells.

Previous work from this laboratory has shown that expression of human cytomegalovirus (HCMV) immediate-early (IE) genes from the major immediate-early promoter (MIEP) is likely to be regulated by chromatin remodelling around the promoter affecting the acetylation state of core histone tails. The HCMV MIEP contains sequences that bind cellular transcription factors responsible for its negative regulation in undifferentiated, non-permissive cells. Ets-2 repressor factor (ERF) is one such factor that binds to such sequences and represses IE gene expression. Although it is not known how cellular transcription factors such as ERF mediate transcriptional repression of the MIEP, it is likely to involve differentiation-specific co-factors. In this study, the mechanism by which ERF represses HCMV IE gene expression was analysed. ERF physically interacts with the histone deacetylase, HDAC1, both in vitro and in vivo and this physical interaction between ERF and HDAC1 mediates repression of the MIEP. This suggests that silencing of viral IE gene expression, associated with histone deacetylation events around the MIEP, is mediated by differentiation-dependent cellular factors such as ERF, which specifically recruit chromatin remodellers to the MIEP in non-permissive cells.

Cell Culture Techniques↗

PubMed2004

Isolation of aminoglycoside nucleotidyltransferase (2'')-Ia from inclusion bodies as active, monomeric enzyme.

Aminoglycoside nucleotidyltransferase( 2'')-Ia (ANT( 2'') confers resistance to pathogenic bacteria against several aminoglycoside antibiotics including gentamicin, kanamycin, and tobramycin. The gene for this aminoglycoside-modifying enzyme has been cloned from a clinical isolate of Pseudomonas aeruginosa. This gene was inserted into an overexpression vector, the vector was then transformed into Escherichia coli BL21(DE3), and the protein has been isolated in the form of inclusion bodies. Optimal refolding conditions have been determined to be direct dilution of solubilized inclusion bodies into 0.1M Tris-HCl, pH 8.5, 0.2M KCl, 0.4M l-arginine, and 5mM reduced glutathione at 4 degrees C. The refolded enzyme is monomeric in solution and has similar kinetic properties and substrate selectivity to the enzyme isolated in soluble form.

Cloning, Molecular↗

PubMed2003

In vitro wear of various orthotic device materials.

STATEMENT OF PROBLEM: Orthotic devices are advocated to decrease occlusal attrition caused by bruxism but tend to wear with time. PURPOSE: This study investigated the wear rate of various materials used to fabricate orthotic devices. MATERIAL AND METHODS: Five experimental groups (n=8) were studied: Splint Biocryl autopolymerized (SBA), Splint Biocryl autopolymerized plus additional heat and pressure (SBHP), Forestacryl autopolymerized (FA), Forestacryl autopolymerized plus additional heat and pressure (FHP), and Quick Splint 15-minute (QS), light-polymerized composite. Specimens were mounted to the base of a universal testing machine. A wear device using steatite balls and a load of 9.1 kg was positioned against the specimens, submerged in a 37 degrees C water bath and subjected to 2500 reciprocal cycles. Wear, in micrometers, was calculated as the maximum peak to valley measurement (Ry) using profilometry. Data were subjected to analysis of variance (ANOVA) and Tukey's HSD (alpha=.05). RESULTS: Mean acrylic wear in micrometers was as follows: FA 6.8 +/-3.0; FHP 7.1 +/- 1.8; SBA 20.4 +/- 5.6; SBHP 23.7 +/- 7.8; and QS 23.8 +/- 6.9. One-way ANOVA detected significant differences between groups (P<.001); the Tukey honestly significant difference test determined that FA and FHP specimens were significantly more resistant to wear than all other specimens (P=.007). CONCLUSION: Differences in in vitro wear resistance among various orthotic device materials exist. The in vitro wear resistance among other autopolymerizing materials appears to be related to proprietary differences.

Acrylic Resins↗

PubMedDate not supplied

Here comes the future.

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Diffusion of Innovation↗

PubMedDate not supplied

A manufacturer's view.

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Blood Pressure Determination↗