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R W Harrison

Publications and source records attributed to R W Harrison.

At least 55 records · Page 3Linked to original sources
PubMed1996

Functional identification of genes up- and down-regulated by glucocorticoids in AtT-20 pituitary cells using an enhancer trap.

The AtT-20/D1 mouse pituitary tumor cell line has been used to study glucocorticoid regulation of POMC. We have used an enhancer trap to determine whether other glucocorticoid-regulated genes exist in AtT-20 cells. An enhancer trap is a recombinant construction containing a selectable marker driven by a promoter that has been weakened by removal of its enhancers so that the transfected trap is only expressed if it comes under the influence of an endogenous enhancer. For a selectable marker, we used a fusion gene coding for hygromycin phosphotransferase (Hy) and herpes simplex thymidine kinase. Thus, expression of this gene conferred hygromycin resistance and ganciclovir sensitivity. Suppression resulted in ganciclovir resistance and hygromycin sensitivity. An enhancerless promoter was produced using a truncated, transcriptionally inactive, form of the POMC promoter. AtT-20/D1 cells were transfected with this construct and cultured in medium containing hygromycin to kill any cells not expressing the Hy gene. The survivors were cultured in medium containing ganciclovir and dexamethasone and cloned. Clones in which the transgene was down-regulated by dexamethasone survived and were designated AtT-20/NET (for negative enhancer trap). Northern blot analysis confirmed that the transgene was down-regulated by dexamethasone as expected and that in at least one instance, suppression of the transgene was more complete than suppression of the full-length POMC promoter. Southern blot analysis after restriction enzyme digestion showed that each cell clone contained a single copy of the transgene, and PCR analysis of the promoter region showed that insertion had occurred in two unique sites in at least two cell clones. Another plasmid construct was prepared that contained the selectable gene but lacked any promoter elements. After transfection of AtT-20 cells with this vector, up-regulated enhancers were trapped by selection in hygromycin and dexamethasone followed by ganciclovir alone and designated AtT-20/PET cells (for positive enhancer trap). Up-regulation of the selectable gene in AtT-20/PET cells was confirmed by Northern blot analysis of dexamethasone-treated cells. In summary, glucocorticoid-regulated enhancers have been identified in AtT-20/D1 cells by an enhancer trap strategy that uses sequential selection under conditions that test whether the transgene is active. These results indicate that in addition to the well characterized, down-regulated POMC gene, there are other glucocorticoid-regulated genes in AtT-20/D1 cells that are both up-regulated and down-regulated by glucocorticoids.

Animals↗
PubMed1995

Sugar specificity of human beta-cell glucokinase: correlation of molecular models with kinetic measurements.

Human beta-cell glucokinase recognition and phosphorylation of different sugars was investigated by steady-state kinetic analysis, measurements of substrate-induced intrinsic fluorescence changes, and molecular modeling and calculation of interaction energies. Measurements of kcat/Km showed that glucokinase phosphorylated the sugars in the order glucose = mannose > deoxyglucose > fructose = glucosamine. The mode of binding of these sugars to the open conformation of glucokinase was predicted from molecular modeling. Glucokinase is predicted to form similar interactions with the 6-OH, 4-OH, and 1-OH groups of all these sugars. The interactions of the 2-OH and 3-OH groups differ and depend on the type of sugar and reflect differences in cooperative behavior. For example, glucose and deoxyglucose exhibited cooperative behavior with Hill coefficients of 1.8 and 1.5, respectively, while mannose and fructose demonstrated Michaelis-Menten behavior. Galactose, allose, and 2,5-anhydroglucitol were not substrates under the assay conditions used, and the alpha- and beta-anomers of methylglucose were poor substrates with Km's greater than 1000 mM. Glucokinase exhibited an ATPase activity which was 1/2000th that of the rate of the kinase reaction, and unlike yeast hexokinase, it was not affected by the addition of lyxose. Glucosamine was a low affinity inhibitor as well as a substrate, while N-acetylglucosamine and mannoheptulose were high-affinity inhibitors. The change in intrinsic fluorescence that was induced by glucose, mannose, and mannoheptulose had the opposite sign for glucosamine, which implies a very different mode of binding from the other sugars. The calculated interaction energies of glucokinase with glucose, mannose, deoxyglucose, and fructose agree very well with the measured values of kcat/Km, which indicates that these sugars are recognized by binding to the open conformation of glucokinase.

Adenosine Triphosphate↗
PubMed1995

Human beta-cell glucokinase. Dual role of Ser-151 in catalysis and hexose affinity.

Glucokinase is distinguished from yeast hexokinase and low Km mammalian hexokinases by its low affinity for glucose and its cooperative behavior, even though glucose binding residues and catalytic residues are highly conserved in all of these forms of hexokinase. The roles of Ser-151 and Asn-166 as determinants of hexose affinity and cooperative behavior of human glucokinase have been evaluated by site-directed mutagenesis, expression and purification of the wild-type and mutant enzymes, and steady-state kinetic analysis. Mutation of Asn-166 to arginine increased apparent affinity for both glucose and ATP by a factor of 3. Mutation of Ser-151 to cysteine, alanine, or glycine lowered the Km for glucose by factors of 2-, 26-, and 40-fold, respectively, decreased Vmax, abolished cooperativity for glucose, and also decreased Km for mannose and fructose. The Ser-151 mutants had hexose Km values similar to those of yeast hexokinase, hexokinase I, and the recombinantly expressed COOH-terminal half of hexokinase I. However, the Ki values for the competitive inhibitors, N-acetylglucosamine and glucose-6-P, were unchanged, suggesting that Ser-151 is not important for inhibitor binding. Mutation of Ser-151 also increased the Km for ATP about 5-fold and abolished the enzyme's low ATPase activity, which indicates it is essential for ATP hydrolysis. The substrate-induced change in intrinsic fluorescence of S151A occurred at a much lower glucose concentration than that for wild-type enzyme. The results implicate a dual role for Ser-151 as a determinant of hexose affinity and catalysis, exclusive of the glucose-induced conformational change, and suggest that the low hexose affinity of glucokinase is dependent on interaction of Ser-151 with other regions of the protein.

Acetylglucosamine↗
PubMed1995

Selection of glucocorticoid-resistant mutations from an AtT-20 cell line containing a glucocorticoid-regulated selectable transgene.

AtT-20/IDG8 cells contain the stably transfected, selectable gene, neomycin phosphotransferase, under negative glucocorticoid regulation. Thus, when cultured in the simultaneous presence of the neomycin analogue, G418, and dexamethasone, AtT-20/IDG8 cells fail to grow. Our hypothesis was that mutated AtT-20/IDG8 cells capable of growth in such medium would have a defect in the glucocorticoid-mediated regulation of the neor gene. AtT-20/IDG8 cells were chemically mutagenized using ethyl-methane sulfonate and cloned in the presence of G418 and dexamethasone. Fourteen clones were obtained and loss of glucocorticoid control of neor expression was confirmed in them all. The naturally occurring gene, pro-opiomelanocortin, which is down-regulated by glucocorticoids in parent AtT-20/IDG8 cells, was down-regulated by dexamethasone in ten of the mutant lines, indicating that in those cells the receptor was functional in spite of aberrant regulation of neor. In the other four lines, pro-opiomelanocortin regulation was lost, also suggesting that a general transcription factor, such as the receptor, had been altered. These results indicate that multiple factors are involved in glucocorticoid-mediated gene regulation and that new, informative mutations can be produced after insertion of a regulated, selectable gene into a previously non-selectable cell line.

Animals↗
PubMed1995

The influence of temperature on lysozyme crystals. Structure and dynamics of protein and water.

Lysozyme structures at six different temperatures in the range 95-295 K have been determined using X-ray crystallography at a resolution of 1.7 A. The crystals at lower temperatures had a 7.4% decrease in the unit-cell volume. The volume change was discontinuous with the volume being near 238 000 A(3) from 295 to 250 K and about 220 200 A(3) below 180 K. The thermal expansion of the protein has been analyzed and shows anisotropy, which is correlated with local atomic packing and secondary-structure elements. The lysozyme structure at low temperature is nearly the same as that at high temperature, with only small relative translations and rotations of structure elements including a hinge-bending rearrangement of two domains. Because of a considerable increase of lattice disorder at low temperature dynamical analysis of internal motion is difficult. The analysis of structural and dynamical properties of well ordered protein-bound water has been carried out.

Journal Article↗
PubMed1995

Analysis of six protein structures predicted by comparative modeling techniques.

The protein structures of six comparative modeling targets were predicted in a procedure that relied on improved energy minimization, without empirical rules, to position all new atoms. The structures of human nucleoside diphosphate kinase NM23-H2, HPr from Mycoplasma capricolum, 2Fe-2S ferredoxin from Haloarcula marismortui, eosinophil-derived neurotoxin (EDN), mouse cellular retinoic acid protein I (CRABP1), and P450eryf were predicted with root mean square deviations on C alpha atoms of 0.69, 0.73, 1.11, 1.48, 1.69, and 1.73 A, respectively, compared to the target crystal structures. These differences increased as the sequence similarity between the target and parent proteins decreased from about 60 to 20% identity. More residues were predicted than form the common region shared by the two crystal structures. In most cases insertions or deletions between the target and the related protein of known structure were not correctly positioned. One two residue insertion in CRABP1 was predicted in the correct conformation, while a nine residue insertion in EDN was predicted in the correct spatial region, although not in the correct conformation. The positions of common cofactors and their binding sites were predicted correctly, even when overall sequence similarity was low.

Amino Acid Sequence↗
PubMed1995

Model complexes of tumor necrosis factor-alpha with receptors R1 and R2.

The biological activities of tumor necrosis factor-alpha (TNF-alpha) are mediated by two different receptors, TNFR1 and TNFR2. To analyze the receptor binding site(s) of TNF-alpha, molecular models have been built of the complexes of TNF-alpha with the extracellular regions of receptors R1 and R2, based on the known crystal structures of TNF-alpha and lymphotoxin bound to R1. The model structure of R2 from residues 18-160 was built by analogy to the crystal structure of R1 in complex with lymphotoxin. The amino acid sequences of R1 and R2 show 27.5% identity over this region and were aligned with five insertions and three deletions. There are 18 conserved cysteines that form disulfides. R2 has lost one pair of cysteines compared with R1, but two new cysteines were modeled as forming a new disulfide bond. Both symmetric and asymmetric trimers of TNF-alpha were used to model the complexes with TNFR1 and R2. An analysis of differences in the model complexes showed good agreement with data on the differential binding of TNF mutants to its two receptors.

Amino Acid Sequence↗
PubMed1994

Site-directed mutagenesis studies on the determinants of sugar specificity and cooperative behavior of human beta-cell glucokinase.

The determinants of sugar specificity and cooperative behavior of human beta-cell glucokinase were studied by mutating several active site residues and performing a steady-state kinetic analysis of the purified mutant and wild-type enzymes after their expression in Escherichia coli. Asn-204, Glu-256, and Glu-290 were predicted from molecular modeling to interact with the 3-OH, 4-OH, 2-OH, and 1-OH groups of glucose. Mutation of these residues resulted in enzymes with decreased values of kcat and increased values of Km for glucose, mannose, and 2-deoxyglucose. Lys-56 is also predicted to make an interaction with the side chain of Glu-256 and its mutation increased the Km for glucose, deoxyglucose, mannose, and fructose by 4-, 4-, 3-, and 10-fold, respectively, and also increased the kcat for fructose by 5-fold. The Ki values for N-acetylglucosamine and mannoheptulose for the wild-type enzyme were 0.2 and 0.8 mM, respectively, and mutation of glucose binding residues to alanine resulted in an increase of about 3 orders of magnitude in these Ki values. Mutation of residues that directly hydrogen bond glucose hydroxyls (Asn-204, Glu-256, and Glu-290) to alanine resulted in enzymes that did not exhibit cooperative behavior, but mutation of Lys-56 or other residues that do not directly contact glucose had no effect on the Hill coefficient. Only glucose and deoxyglucose exhibited cooperative behavior. The results 1) confirm the predictions of the model that Asn-204, Glu-256, and Glu-290 are important residues involved in catalysis and hydrogen bonding glucose hydroxyl groups, 2) provide evidence for a role of Lys-56 in hexose binding, and 3) are consistent with the cooperative behavior of glucokinase being mediated by interactions of other regions of the protein with the highly conserved active site glucose binding residues.

Allosteric Regulation↗
PubMed1994

Kinetic and modeling studies of subsites S4-S3' of Moloney murine leukemia virus protease.

The substrate specificity of the Moloney murine leukemia virus protease (Mo-MuLV PR) was analyzed by using the oligopeptide substrate Val-Ser-Gln-Asn-Tyr decreases Pro-Ile-Val-Gln-NH2 and a series of analogs containing single amino acid substitutions in the P4-P3' positions. Mo-MuLV PR appears to act similarly to the human immunodeficiency virus (HIV) PRs, except for peptides having substitutions at P4 and P2 positions. Mo-MuLV PR shows a strong preference for the analogs having hydrophobic residues, such as Val or Ile at P4, and Ile and Leu at P2, in contrast to HIV-1 and HIV-2 PRs, which prefer smaller or more polar residues at both positions. We built a molecular model of Mo-MuLV PR on the basis of the crystal structure of the related HIV PR. Although the overall structure of Mo-MuLV PR is predicted to be close to that of HIV-1 PR, almost all of the residues forming the subsites are different. The increased hydrophobicity due to the Pro12 insertion and the presence of more aromatic residues in the S4 subsite of Mo-MuLV PR compared to HIV-1 and HIV-2 PRs can be correlated with the observed differences using P4-substituted analogs of VSQNYPIVQ. The preference of Mo-MuLV PR for larger hydrophobic residues at the P2 position can be correlated with the larger size of its S2 subsite, due in part to the presence of Val39, Ala57, and His84 in Mo-MuLV PR, instead of Ile32, Ile50, and Met76, respectively, as occurs in HIV-2 PR.

Amino Acid Sequence↗
PubMed1994

Prediction of new serine proteinase inhibitors.

We describe here the use of a rapid computational method to predict the relative binding strengths of a series of small-molecule ligands for the serine proteinase trypsin. Flexible molecular models of the ligands were docked to the proteinase using an all-atom potential set, without cutoff limits for the non-bonded and electrostatic energies. The binding-strength calculation is done directly in terms of a molecular mechanics potential. The binding of eighteen different compounds, including non-binding controls, has been successfully predicted. The measured Ki is correlated with the predicted energy. The correctness of the theoretical calculations is demonstrated with both kinetics measurements and X-ray structure determination of six enzyme-inhibitor complexes.

Computer Simulation↗
PubMed1994

Molecular dynamics simulations of HIV-1 protease with peptide substrate.

Molecular dynamics simulations of human immunodeficiency virus (HIV)-1 protease with a model substrate were used to test if there is a stable energy minimum for a proton that is equidistant from the four delta oxygen atoms of the two catalytic aspartic acids. The crystal structure of HIV-1 protease with a peptidic inhibitor was modified to model the peptide substrate Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln for the starting geometry. A proton was positioned between the two closet oxygen atoms of the two catalytic aspartic acids, and close to the carbonyl oxygen of the scissile bond in the substrate. All crystallographic water molecules were included. Two molecular dynamics simulations were run: 30 ps with united-atom potentials and 40 ps using the more accurate all-atom potentials. The molecular dynamics used a new algorithm that increased the speed and allowed the elimination of a cut-off for non-bonded interactions and the inclusion of an 8 A shell of water molecules in the calculations. The overall structure of the protease dimer, including the catalytic aspartic acids, was stable during the course of the molecular dynamics simulations. The substrate and a water molecule, that is an important component of the binding site, were stable during the simulation using all-atom potentials, but more mobile when united-atom potentials were used. A Poincare map representation showed that the positions of the proton and its coordinating oxygen atoms were stable for 93% of both simulations, although many of the buried and poorly accessible water molecules exchanged with solvent. The proton has a stable minimum energy position and maintains coordination with all four delta oxygen atoms of the two catalytic aspartic acids and the carbonyl oxygen of the scissile bond of the substrate. Therefore, a loosely bound hydrogen ion at this position will not be rapidly exchanged with solvent, and will rebond to either a catalytic aspartic acid or possibly the substrate. The implications for the reaction mechanism are discussed.

Amino Acid Sequence↗
PubMed1994

The Fourier-Green's function and the rapid evaluation of molecular potentials.

Two tasks must be accomplished when calculating the binding modalities and binding energies of two molecules in solution: the calculation of the interaction energy and the calculation of the effects of solvation. It is the competition between the energy of binding and the energy of remaining solvation which determines the binding properties. It is necessary to calculate (or at least approximate in some manner) the partition function in order to make a theoretical estimate of these effects. An efficient algorithm for performing the energy evaluations necessary for this calculation is presented in this paper. The fast Fourier transform (FFT) is used in combination with a polar factorization of the potentials to calculate the interaction energy at all relative translations between two molecules of fixed orientation. Thermodynamic quantities, including the partition function, internal and free energies can then be estimated from a set of these calculations covering the orientation space.

Algorithms↗
PubMed1994

Do cholesterol-lowering agents affect brain activity? A comparison of simvastatin, pravastatin, and placebo in healthy volunteers.

1. The effects of simvastatin and pravastatin on measures of central nervous system activity were investigated in a double-blind, placebo-controlled, randomised crossover study. 2. Twenty-five healthy volunteers sequentially took 40 mg day-1 simvastatin, 40 mg day-1 pravastatin or placebo for 4 weeks, separated by a 4-6 week washout phase. 3. CNS measures included EEG evoked potentials, power spectral analysis, Leeds Sleep Questionnaire, Hospital Anxiety Depression (HAD) Scale, and Digit Symbol Substitution Test (DSST); biochemical measures included plasma cholesterol, liver enzymes (gamma-GT, AST, ALT) and creatine kinase. 4. Mean cholesterol concentrations with both drugs were significantly lower than with placebo, and the cholesterol-lowering effect was greater with simvastatin. There were no significant differences between treatment in EEG evoked potentials, HAD Scale, or DSST scores. On the sleep measure, subjects reported significantly greater difficulty in getting to sleep while on simvastatin than on pravastatin, but neither score differed from placebo. No significant correlations were observed between sleep ratings and either plasma cholesterol concentrations or EEG evoked potentials. 5. The study showed that, while both drugs reduced plasma cholesterol concentrations, neither exerted significant effects, compared with placebo, on EEG evoked potentials, mood, sleep, or cognitive performance after 4 weeks of chronic administration in healthy volunteers.

Adult↗
PubMed1994

Dosimetry for asymmetric x-ray fields.

Conventional linear accelerators have four field-defining jaws or collimators. Usually, one set of the two opposing jaws moves concurrently to define the field width and the other set defines the field length. The resultant square or rectangular field will have the field centerline coincide with the collimator axis. However, some modern linacs have independent collimators or jaws that can be set asymmetrically. In this case, one of the two opposing jaws can be closed down independently of the other one to define an asymmetric field of smaller dimension. The field center now does not coincide with the collimator axis. Asymmetric collimators have found many clinical applications, but have complicated the dosimetry for physicists. Data acquisition and treatment planning implementations are tedious and complicated. An algorithm has been developed to correct for the reduced dose in the smaller asymmetric field. The approach used is similar in principle to the Day's equivalent field calculation. The difference in dose between an asymmetric and a symmetric radiation field is accounted for by a correction factor that is a function of the asymmetric and symmetric field sizes, off axis distance, and depth of measurement. The correction method presented here applies only to the closing down of one independent jaw. Beam profiles for asymmetric fields are measured for both the 6 and 10 MV photon beams.(ABSTRACT TRUNCATED AT 250 WORDS)

Humans↗
PubMed1994

Molecular model of human beta-cell glucokinase built by analogy to the crystal structure of yeast hexokinase B.

Recent studies have shown that mutations in human beta-cell glucokinase that impair the activity of this key regulatory enzyme of glycolysis can cause early-onset non-insulin-dependent diabetes mellitus (NIDDM). The amino acid sequence of human glucokinase has 31% identity with yeast hexokinase, a related enzyme for which the crystal structure has been determined. This homology has allowed us to model the three-dimensional structure of human glucokinase by analogy to the crystal structure of yeast hexokinase B. This model of human glucokinase provides a basis for understanding the effects of mutations on its enzymatic activity. Residues in the active site and on the surface of the binding cleft for glucose are highly conserved in both enzymes. Regions far from the active site are predicted to differ in conformation, and 10 insertions or deletions that range in size from 1 to 7 residues are located on the protein surface between elements of secondary structure. The model structure suggests that human glucokinase binds glucose in a similar manner to yeast hexokinase. The glucose-binding site contains a conserved aspartic acid, two conserved glutamic acids, and two conserved asparagines that form hydrogen bond interactions with the hydroxyls of the glucose similar to those observed in other sugar-binding proteins. Mutation of residues in the predicted glucose-binding site has been found to greatly reduce enzymatic activity. This model will be useful for future structure/function studies of glucokinase.

Amino Acid Sequence↗
PubMed1993

An analysis of autologous glucocorticoid receptor protein regulation in AtT-20 cells also reveals differential specificity of the BuGR2 monoclonal antibody.

When the anti-glucocorticoid receptor monoclonal antibody (BuGR2) was initially incorporated either into a new immunoassay strategy or into a traditional sedimentation analysis technique, both methods failed to reveal any change in the cellular content or distribution of BuGR2-reactive antigen following glucocorticoid treatment of AtT-20 cells. Furthermore, the immunoassay also generated strong positive signals with cytosol and nuclear extracts from a receptor-negative cell line (E8.2) derived from L929 cells. However, when the BuGR2 antibody was incorporated into a combined immunoprecipitation/Western blot analysis of AtT-20 cell extracts, only the glucocorticoid receptor protein produced a signal on the Western blot, even though other proteins had been specifically immunoprecipitated by BuGR2 antibody and were clearly present on the Western blot membrane. Applying the latter approach to AtT-20 cells chronically treated with glucocorticoid, we observed not only that the receptor protein rapidly and persistently (1-96 h) accumulated in the nucleus, but also that its total cellular content was first depleted (24 h) and then was progressively replenished (48-96 h). From these studies in AtT-20 cells we conclude: (i), the BuGR2 antibody can exhibit differential immunospecificity dependent upon whether antigen mixtures are denatured or not; (ii), glucocorticoid receptor protein resided almost exclusively in the nucleus during four days of glucocorticoid treatment and (iii), the same treatment regimen resulted in total receptor protein levels being regulated in a biphasic pattern. Together, these results suggest that receptor regulation in AtT-20 cells is a complex event, and that, since steroid was constantly present during our experiments, other factors are involved in regulation of receptor levels.

Animals↗