Memory for perceived and imagined pictures--an event-related potential study.

Event-related potentials (ERPs) and behavioural measures were used to investigate recognition memory and source-monitoring judgements about previously perceived and imagined pictures. At study, word labels of common objects were presented. Half of these were followed by a corresponding picture and the other half by an empty frame, signalling to the participants to mentally visualise an image. At test, participants in a source-monitoring task made a three-way discrimination between new words and words corresponding to previously perceived and imagined pictures. Participants in an old/new-recognition task indicated whether test words were previously presented or not. In both tasks, correctly identified old items elicited more positive-going ERPs than correctly judged new items. This widely distributed old/new effect was found to have an earlier onset and to be of a greater magnitude for imagined than for perceived items. Task (source versus item-memory) affected the old/new effects over prefrontal areas and the reaction times to remembered old items. The present findings are consistent with the view that a greater amount, or a different type, of information is necessary for accurate source-memory judgements than for correct recognition, and moreover, that different types of source-specifying information revive at different rates. In addition, the results add weight to the view that the late widespread ERP-old/new effect is sensitive to the quality or the amount of information retrieved from memory.

The folding and stability of human alpha class glutathione transferase A1-1 depend on distinct roles of a conserved N-capping box and hydrophobic staple motif.

An N-capping box and a hydrophobic staple motif are strictly conserved in the core of all known glutathione S-transferases (GST). In the present work, mutations of hGSTA1-1 enzyme residues forming these motifs have been generated. The analysis of S154A, D157A, and S154A/D157A capping mutants indicate that the removal of this local signal destabilizes the protein. The fact that the third helical residue D157A mutation (N-3) was much more destabilizing than the first helical residue S154A mutation (N-cap) suggests that the appropriate conformation of the conserved substructure formed by the alpha 6-helix and preceding loop (GST motif II) is crucial for the overall protein stability. The refolding study of GSTA1-1 variants supports the prediction that this subdomain could represent a nucleation site of refolding. The analysis of L153A, I158A, L153G, and L153A/I158A hydrophobic staple mutants indicate that the removal of this motif destabilizes the GSTA1-1 structure as well as its refolding transition state. The hydrophobic staple interaction favors essential inter-domain contacts and, thereby, in contrast to capping interactions, accelerates the enzyme reactivation. Its strict conservation in the GST system supports the suggestion that this local signal could represent an evolutionarily conserved determinant for rapid folding.

Structures of thermolabile mutants of human glutathione transferase P1-1.

An N-capping box motif (Ser/Thr-Xaa-Xaa-Asp) is strictly conserved at the beginning of helix alpha6 in the core of virtually all glutathione transferases (GST) and GST-related proteins. It has been demonstrated that this local motif is important in determining the alpha-helical propensity of the isolated alpha6-peptide and plays a crucial role in the folding and stability of GSTs. Its removal by site-directed mutagenesis generated temperature-sensitive folding mutants unable to refold at physiological temperature (37 degrees C). In the present work, variants of human GSTP1-1 (S150A and D153A), in which the capping residues have been substituted by alanine, have been generated and purified for structural analysis. Thus, for the first time, temperature-sensitive folding mutants of an enzyme, expressed at a permissive temperature, have been crystallized and their three-dimensional structures determined by X-ray crystallography. The crystal structures of human pi class GST temperature-sensitive mutants provide a basis for understanding the structural origin of the dramatic effects observed on the overall stability of the enzyme at higher temperatures upon single substitution of a capping residue.

Semantic processing without conscious identification: evidence from event-related potentials.

Three event-related potential (ERP) experiments examined whether semantic content can be accessed from visually presented words that cannot be consciously identified. Category labels were shown to participants, followed by masked, briefly exposed words that were either exemplars of the category or not exemplars. The task was to verify the category, by guessing if necessary, and to identify the word, naming it if possible. Exposure durations were selected to allow identification in approximately half the trials. For identified words, there was a marked difference in the ERP response between in-category and out-of-category words because of an N400 component. For unidentified words, there was a similar although smaller difference. Conscious identification was defined using a variety of approaches: verbal report, 6-alternative forced choice, and binary categorization (in the context of the regression method; A. G. Greenwald, M. R. Klinger, & E. S. Schuh, 1995). By any definition, ERPs for unidentified words showed evidence of semantic processing. In addition, there were differences in the neuronal populations recruited to process above-threshold versus below-threshold words, suggesting qualitative differences.

Tyrosine 50 at the subunit interface of dimeric human glutathione transferase P1-1 is a structural key residue for modulating protein stability and catalytic function.

The dimer interface in human GSTP1-1 has been altered by site-directed mutagenesis of Tyr50. It is shown that the effects of some mutations of this single amino acid residue are as detrimental for enzyme function as mutations of Tyr8 in the active site. The dimeric structure is a common feature of the soluble glutathione transferases and the structural lock-and-key motif contributing to the subunit-subunit interface is well conserved in classes alpha, mu, and pi. The key residue Tyr50 in GSTP1-1 was replaced with 5 different amino acids with divergent properties and the mutant proteins expressed and characterized. Mutant Y50F is an improved variant, with higher thermal stability and higher catalytic efficiency than the wild-type enzyme. The other mutants studied are also dimeric proteins, but have lower stabilities and catalytic activities that are reduced by a factor of 10(2)-10(4) from the wild-type value. Mutants Y50L and Y50T are characterized by a markedly increased K(M) value for GSH, while the effect is mainly due to decreased k(cat) values for mutants Y50A and Y50R. In conclusion, residue 50 in the interface governs both structural stability and catalytic function.

A conserved "hydrophobic staple motif" plays a crucial role in the refolding of human glutathione transferase P1-1.

The specific (i, i+5) hydrophobic staple interaction involving a helix residue and a second residue located in the turn preceding the helix is a recurrent motif at the N terminus of alpha-helices. This motif is strictly conserved in the core of all soluble glutathione transferases (GSTs) as well as in other protein structures. Human GSTP1-1 variants mutated in amino acid Ile(149) and Tyr(154) of the hydrophobic staple motif of the alpha6-helix were analyzed. In particular, a double mutant cycle analysis has been performed to evaluate the role of the hydrophobic staple motif in the refolding process. The results show that this local interaction, by restricting the number of conformations of the alpha6-helix relative to the alpha1-helix, favors the formation of essential interdomain interactions and thereby accelerates the folding process. Thus, for the first time it is shown that the hydrophobic staple interaction has a role in the folding process of an intact protein. In P(i) class GSTs, Tyr(154) appears to be of particular structural importance, since it interacts with conserved residues Leu(21), Asp(24), and Gln(25) of the adjacent alpha1-helix which contributes to the active site. Human GSTP1-1 variants L21A and Y154F have also been analyzed in order to distinguish the role of interdomain interactions from that of the hydrophobic staple. The experimental results reported here suggest that the strict conservation of the hydrophobic staple motif reflects an evolutionary pressure for proteins to fold rapidly.

Unfolding and refolding of human glyoxalase II and its single-tryptophan mutants.

Here the structure of human glyoxalase II has been investigated by studying unfolding at equilibrium and refolding. Human glyoxalase II contains two tryptophan residues situated at the N-terminal (Trp57) and C-terminal (Trp199) regions of the molecule. Trp57 is a non-conserved residue located within a "zinc binding motif" (T/SHXHX57DH) which is strictly conserved in all known glyoxalase II sequences as well as in metal-dependent beta-lactamase and arylsulfatase. Site-directed mutagenesis has been used to construct single-tryptophan mutants in order to characterize better the guanidine-induced unfolding intermediates. The denaturation at equilibrium of wild-type glyoxalase II, as followed by activity, intrinsic fluorescence and CD, is multiphasic, suggesting that different regions of varying structural stability characterize the native structure of glyoxalase II. At intermediate denaturant concentration (1.2 M guanidine) a molten globule state is attained. The reactivation of the denatured wild-type enzyme occurs only in the presence of Zn(II) ions. The results show that Zn(II) is essential for the maintenance of the native structure of glyoxalase II and that its binding to the apoenzyme occurs during an essential step of refolding. The comparison of unfolding fluorescence transitions of single-trypthophan mutants with that of wild-type enzyme indicates that the strictly conserved "zinc binding motif" is located in a flexible region of the active site in which Zn(II) participates in catalysis.

Effects of nicotine in a bimodal attention task.

Fifteen male users of oral snuff participated in an experiment where we used an auditory-visual vigilance task to study nicotine effects on P300 and response parameters. Quantitative EEG was also studied. Fifteen male nonusers served as controls. We found some decrease of response times and a tendency towards improved signal detection. P300 parameters were not affected in this study. Quantitative EEG analysis indicated an expected increase of arousal, as the activity within the alpha band shifted towards higher frequencies.

An evolutionary approach to the design of glutathione-linked enzymes.

Studies of protein structure provide information about principles of protein design that have come into play in natural evolution. This information can be exploited in the redesign of enzymes for novel functions. The glutathione-binding domain of glutathione transferases has similarities with structures in other glutathione-linked proteins, such as glutathione peroxidases and thioredoxin (glutaredoxin), suggesting divergent evolution from a common ancestral protein fold. In contrast, the binding site for glutathione in human glyoxalase I is located at the interface between the two identical subunits of the protein. Comparison with the homologous, but monomeric, yeast glyoxalase I suggests that new domains have originated through gene duplications, and that the oligomeric structure of the mammalian glyoxalase I has arisen by 'domain swapping'. Recombinant DNA techniques are being used for the redesign of glutathione-linked proteins in attempts to create binding proteins with novel functions and catalysts with tailored specificities. Enzymes with desired properties are selected from libraries of variant structures by use of phage display and functional assays.

Structure-activity relationships and thermal stability of human glutathione transferase P1-1 governed by the H-site residue 105.

Human glutathione transferase P1-1 (GSTP1-1) is polymorphic in amino acid residue 105, positioned in the substrate binding H-site. To elucidate the role of this residue an extensive characterization of GSTP1-1/Ile105 and GSTP1-1/Val105 was performed. Mutant enzymes with altered volume and hydrophobicity of residue 105, GSTP1-1/Ala105 and GSTP1-1/Trp105, were constructed and included in the study. Steady-state kinetic parameters and specific activities were determined using a panel of electrophilic substrates, with the aim of covering different types of reaction mechanisms. Analysis of the steady-state kinetic parameters indicates that the effect of the substitution of the amino acid in position 105 is highly dependent on substrate used. When 1-chloro-2,4-dinitrobenzene was used as substrate a change in the side-chain of residue 105 seemed primarily to cause changes in the KM value, while the kcat value was not distinctively affected. With other substrates, such as 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and ethacrynic acid both kcat and KM values were altered by the substitution of amino acid 105. The constant for formation of the sigma-complex between 1,3, 5-trinitrobenzene and glutathione was shown to be dependent upon the volume of the amino acid in position 105. The nature of the amino acid in position 105 was also shown to affect the thermal stability of the enzyme at 50 degrees C, indicating an important role for this residue in the stabilization of the enzyme. The GSTP1-1/Ile105 variant was approximately two to three times more stable than the Val105 variant as judged by their half-lives. The presence of glutathione in the incubation buffer afforded a threefold increase in the half-lives of the enzymes. Thus, the thermal stability of the enzyme and depending on substrate, both KM values and turnover numbers are influenced by substitutions in position 105 of GSTP1-1.

Differences in the catalytic efficiencies of allelic variants of glutathione transferase P1-1 towards carcinogenic diol epoxides of polycyclic aromatic hydrocarbons.

Previous studies have identified allelic variants of the human glutathione transferase (GST) Pi gene and showed that the two different encoded proteins with isoleucine (GSTP1-1/I-105) or valine (GSTP1-1/V-105) at position 105, respectively, differ significantly in their catalytic activities with model substrates. Moreover, recent epidemiological studies have demonstrated that individuals differing in the expression of these allelic variants also differ in susceptibility to tumour formation in certain organs, including such in which polycyclic aromatic hydrocarbons (PAH) may be etiological factors. In the present study the catalytic efficiencies (kcat/Km) of these GSTP1-1 variants were determined with a number of stereoisomeric bay-region diol epoxides, known as the ultimate mutagenic and carcinogenic metabolites of PAH, including those from chrysene, benzo[a]pyrene and dibenz[a,h]anthracene. In addition, GSTP1-1 mutants in which amino residue 105 is alanine (GSTP1-1/A-105) or tryptophan (GSTP1-1/W-105) have been constructed and characterized. GSTP1-1/V-105 was found to be more active than GSTP1-1/I-105 in conjugation reactions with the bulky diol epoxides of PAH, being up to 3-fold as active towards the anti- and syn-diol epoxide enantiomers with R-absolute configuration at the benzylic oxiranyl carbon. Comparing the four enzyme variants, GSTP1-1/A-105 generally demonstrated the highest kcat/Km value and GSTP1-1/W-105 the lowest with the anti-diol epoxides. A close correlation was observed between the volume occupied by the amino acid residue at position 105 and the value of kcat/Km. With the syn-diol epoxides, such a correlation was observed with alanine, valine and isoleucine, whereas tryptophan was associated with increased kcat/Km values. The mutational replacement of isoleucine with alanine or tryptophan at position 105 did not alter the enantio selectivity of the GSTP1-1 variants compared with the naturally occurring allelic variants GSTP1-1/I-105 and GSTP1-1/V-105. Since the amino acid at position 105 forms part of the substrate binding site (H-site) the effect of increasing bulkiness is expected to cause restricted access of the diol epoxide and proper alignment of the two reactants for efficient glutathionylation. In conclusion, the present study indicates that individuals who are homozygous for the allele GSTP1* B (coding for GSTP1-1/V-105) display a higher susceptibility to malignancy because of other factors than a decreased catalytic efficiency of GSTP1-1/V-105 in the detoxication of carcinogenic diol epoxides of benzo[a]pyrene or structurally related PAH.

The conserved N-capping box in the hydrophobic core of glutathione S-transferase P1-1 is essential for refolding. Identification of a buried and conserved hydrogen bond important for protein stability.

The second domain of cytosolic glutathione S-transferases (GSTs) contains a strictly conserved N-capping box motif (Ser/Thr-Xaa-Xaa-Asp) at the beginning of alpha6-helix in the hydrophobic core of the molecule. Considering the specific function attributed to capping box residues in the helix nucleation, we decided to investigate, by site-directed mutagenesis, the role that this motif could have in the folding and stability of human GSTP1-1. Both capping box mutants, S150A and D153A, were significantly more thermolabile than wild-type GSTP1-1, indicating that the local destabilization of the alpha6-helix determined by a single capping residue mutation affects the overall stability of the protein. The results also show that, in addition to capping interactions, an important role in the stability of the final structure of the protein is played by a buried and conserved hydrogen bond formed between the side chain of Asp-153 and the amide NH of Ile-144 located in the long loop preceding alpha6-helix. Reactivation experiments in vitro indicate that the N-capping box is essential for refolding of the denatured protein at a physiological temperature. The results suggest that during folding this buried and conserved motif, making a definite set of native-like contacts, determines the formation of a specific folding nucleus that probably represents a transition state of the folding process.

Importance of electrostatic interactions in the rapid binding of polypeptides to GroEL.

The question of how chaperones rapidly bind non-native proteins of very different sequence and function has been examined by determining the effect of ionic strength on the refolding of barnase on GroEL, and on the thermal denaturation of barnase in the presence of GroEL and SecB. Both chaperones bind the denatured state of barnase, so lowering the T(m) value. The refolding of barnase in the presence of GroEL is multiphasic, the slowest phase corresponding to the refolding of a singly bound molecule of barnase in the complex with GroEL. The fastest phase is related to the association of barnase and GroEL. At high ratios of GroEL to barnase and low ionic strength (less than 200 mM) this fast phase corresponds to the observed rate of binding. The rate of association of barnase and GroEL was found to be highly dependent on ionic strength, and at high ionic strength (greater than 600 mM) the majority of barnase molecules escaped binding and refolded free in solution. The data are consistent with an initial, transient, ionic interaction between barnase and GroEL, before hydrophobic binding occurs, allowing diffusion-controlled association and slow dissociation of unfolded polypeptide.

Folding of barnase in the presence of the molecular chaperone SecB.

SecB is a molecular chaperone dedicated to interact exclusively with proteins destined for translocation across membranes. We find that SecB interacts with barnase during its folding in a similar manner to its interaction with GroEL. On mixing acid-denatured barnase with SecB in a stopped-flow spectrofluorimeter under conditions that favour refolding, we observe a series of fluorescence changes, corresponding to the binding of the denatured protein and the subsequent refolding of multiply and singly bound forms. The different phases were assigned using a combination of kinetics and mutant proteins. The refolding of barnase when bound to SecB is strongly retarded but never blocked. Multiply bound barnase is less tightly bound and refolds with a higher rate constant than singly bound barnase. Up to 4 mol of denatured barnase bind to 1 mol of tetrameric SecB.

Heterologous expression, purification and characterization of rat class theta glutathione transferase T2-2.

Rat glutathione transferase (GST) T2-2 of class Theta (rGST T2-2), previously known as GST 12-12 and GST Yrs-Yrs, has been heterologously expressed in Escherichia coli XLI-Blue. The corresponding cDNA was isolated from a rat hepatoma cDNA library, ligated into and expressed from the plasmid pKK-D. The sequence is the same as that of the previously reported cDNA of GST Yrs-Yrs. The enzyme was purified using ion-exchange chromatography followed by affinity chromatography with immobilized ferric ions, and the yield was approx. 200 mg from a 1 litre bacterial culture. The availability of a stable recombinant rGST T2-2 has paved the way for a more accurate characterization of the enzyme. The functional properties of the recombinant rGST T2-2 differ significantly from those reported earlier for the enzyme isolated from rat tissues. These differences probably reflect the difficulties in obtaining fully active enzyme from sources where it occurs in relatively low concentrations, which has been the case in previous studies. 1-Chloro-2,4-dinitrobenzene, a substrate often used with GSTs of classes Alpha, Mu and Pi, is a substrate also for rGST T2-2, but the specific activity is relatively low. The Km value for glutathione was determined with four different electrophiles and was found to be in the range 0.3 mM-0.8 mM. The Km values for some electrophilic substrates were found to be in the micromolar range, which is low compared with those determined for GSTs of other classes. The highest catalytic efficiency was obtained with menaphthyl sulphate, which gave a Kcat/Km value of 2.3 x 10(6) s-1.M-1 and a rate enhancement over the uncatalysed reaction of 3 x 10(10).

Restitution of neurophysiological functions, performance, and subjective symptoms after moderate insulin-induced hypoglycaemia in non-diabetic men.

The restoration of cognitive function was studied in 10 healthy men aged 26 years (25.5 +/- 1.2 years; mean +/- SD) after insulin-induced hypoglycaemia (arterialized blood glucose 2.5 +/- 0.4 mmol l-1) for 62 +/- 8 min. Another group of six men participated in a single blind sham study for comparison. The hypoglycaemic event caused a significant increase (p = 0.006) in serum adrenaline levels. Ratings of adrenergically mediated symptoms increased during hypoglycaemia (p = 0.006), as did neuroglycopenic symptoms (p = 0.002), although neuroglycopenia ratings increased in both studies. During hypoglycaemia, P300 amplitudes in a relatively demanding visual search task decreased (p = 0.02), whereas easier tasks were unaffected. The amplitudes were restored after 40 min of normoglycaemia. Reaction time deteriorated after restoration of normoglycaemia, suggesting an effect of hypoglycaemia on learning. Thus, hypoglycaemia at a blood glucose level that is common among patients treated with insulin causes clear cognitive dysfunction, although restoration of the cognitive dysfunction to normal was fast.

Catalysis of amide proton exchange by the molecular chaperones GroEL and SecB.

Hydrogen-deuterium exchange of 39 amide protons of Bacillus amyloliquefaciens ribonuclease (barnase) was analyzed by two-dimensional nuclear magnetic resonance in the presence of micromolar concentrations of the molecular chaperones GroEL and SecB. Both chaperones bound to native barnase under physiological conditions and catalyzed exchange of deeply buried amide protons with solvent. Such exchange required complete unfolding of barnase, which occurred in the complex with the chaperones. Subsequent collapse of unfolded barnase to the exchange-protected folding intermediate was markedly slowed in the presence of GroEL or SecB. Thus, both chaperones have the potential to correct misfolding in proteins by annealing.

High-level bacterial expression of human glutathione transferase P1-1 encoded by semisynthetic DNA.

A cDNA clone, lambda GTHP1del, encoding glutathione transferase (GST) P1-1, was isolated from a human K562 erythroleukemia cell line cDNA library. The coding sequence was lacking the codons for the N-terminal 34 amino acids. A DNA segment was designed in order to obtain the missing portion and a structure representing the entire protein. The synthetic DNA sequence was constructed to achieve efficient base pairing with Escherichia coli 16S ribosomal RNA, avoidance of internal secondary structure, and optimal codon usage for high-level protein expression in accord with the known preferences in E. coli. The truncated GST P1-1 cDNA sequence and the synthetic segment were ligated into a plasmid to give an inducible expression system. Among the resulting clones a limited number was selected by immunodetection for highest yield of GST P1-1. Maximal expression was obtained from a spontaneously mutated sequence with altered as well as deleted bases as compared to the original construct. This clone, pKXHP1, allowed heterologous expression in E. coli in yields of > 200 mg enzyme per liter culture medium. The physicochemical and catalytic properties of the recombinant protein were indistinguishable from those of the enzyme purified from human placenta.

Functional significance of arginine 15 in the active site of human class alpha glutathione transferase A1-1.

Arg15 is a conserved active-site residue in class Alpha glutathione transferases. X-ray diffraction studies of human glutathione transferase A1-1 have shown that N epsilon of this amino acid residue is adjacent to the sulfur atom of a glutathione derivative bound to the active site, suggesting the presence of a hydrogen bond. The phenolic hydroxyl group of Tyr9 also forms a hydrogen bond to the sulfur atom of glutathione, and removal of this hydroxyl group causes partial inactivation of the enzyme. The present study demonstrates by use of site-directed mutagenesis the functional significance of Arg15 for catalysis. Mutation of Arg15 into Leu reduced the catalytic activity by 25-fold, whereas substitution by Lys caused only a threefold decrease, indicating the significance of a positively charged residue at position 15. Mutation of Arg15 into Ala or His caused a substantial reduction of the specific activity (200 or 400-fold, respectively), one order of magnitude more pronounced than the effect of the Tyr9-->Phe mutation. Double mutations involving residues 9 and 15 demonstrated that the effects of mutations at the two positions were additive except for the substitution of His for Arg15, which appeared to cause secondary structural effects. The pKa value of the phenolic hydroxyl of Tyr9 was determined by UV absorption difference spectroscopy and was found to be 8.1 in the wild-type enzyme. The corresponding pKa values of mutants R15K, R15H and R15L were 8.5, 8.7 and 8.8, respectively, demonstrating the contribution of the guanidinium group of Arg15 to the electrostatic field in the active site. Addition of glutathione caused an increased pKa value of Tyr9; this effect was not obtained with S-methylglutathione. These results show that Tyr9 is protonated when glutathione is bound to the enzyme at physiological pH values. The involvement of an Arg residue in the binding and activation of glutathione is a feature that distinguishes class Alpha glutathione transferases from members in other glutathione transferase classes.

EEG topography of acute ethanol effects in resting and activated normals.

Acute effects of ethanol on spectral characteristics of the EEG were studied using 18 recording sites and topographic mapping. The EEG was recorded both at rest and during a mental arithmetic task. Healthy young male volunteers were randomly assigned to an ethanol (n = 22) or a placebo (n = 15) group. The ethanol group received a total dose of 1.0 g/kg, divided into two equal doses given 75 minutes apart. and measurement sessions took place at baseline and after each dose. The placebo group underwent a similar schedule. Power in the theta, alpha and beta bands all increased in the ethanol group, but only the theta and beta bands clearly separated ethanol from placebo. Alpha increases were seen in the placebo group as well. The ethanol-induced changes were greater in the left hemisphere than in the right, having the effect of attenuating the right-over-left asymmetry seen at baseline. Differences between ethanol and placebo were more marked in the mentally activated condition, since the changes seen at rest were inhibited by the activation in the placebo group, but not in the ethanol group. The results indicate (1) that ethanol induces a less differentiated pattern of activity within the brain at rest, and (2) that it impairs the capacity to activate the brain under the challenge of a mental task.