Biomedical subjects

G C Roberts

Publications and source records attributed to G C Roberts.

At least 19 recordsLinked to original sources

PubMed2006

An examination of the factorial structure of the Norwegian version of the sport anxiety scale.

The purpose of the present research was to develop and validate a Norwegian version of the sport anxiety scale (SAS-N), a multidimensional sport performance trait anxiety inventory. The SAS consists of three unique dimensions measuring somatic anxiety, worry and concentration disruption, respectively. The translation-back translation method was used in development of the SAS-N, and athletes from different sports in Norway (N=282) participated in the study. The findings are similar to those of the English original, and lend support for the use of this instrument with Norwegian athlete subjects.

Adolescent↗

PubMed2005

Post-craniotomy analgesia: current practices in British neurosurgical centres--a survey of post-craniotomy analgesic practices.

BACKGROUND AND OBJECTIVE: An evaluation of post-craniotomy analgesia within the University Hospital of Wales Neurosurgical Unit, Cardiff, found that many patients were experiencing moderate to severe pain post-craniotomy. It was therefore decided to undertake a nationwide survey of analgesic practices in order to establish best practice guidelines and benchmark with other units. METHOD: A postal questionnaire was sent to the senior nurse of every Neurosurgical Directorate within the UK inquiring about the current, standard analgesic practices for post-craniotomy patients in their unit. RESULTS: Completed replies were received from 23 of the 33 centres (70%). Intramuscular (i.m.) codeine phosphate was found to be the principal first-line analgesic used post-craniotomy. Only three centres used morphine as the first-line analgesic and only one centre used patient controlled analgesia routinely. The majority of centres (82%) used balanced analgesia. Pain assessments were only carried out in 57% of centres and no centre used a validated pain assessment tool specifically for dysphasic patients. CONCLUSIONS: Codeine phosphate continues to be the mainstay of post-craniotomy analgesia, however, it is proposed that patient controlled analgesia with morphine is an efficacious and safe alternative.

Analgesia↗

PubMed2002

Elite athletes' experiences of the motivational climate: the coach matters.

Recent research investigating the effect of the motivational climate has found that even elite athletes benefit from a mastery climate. The purpose of the present study was to obtain a deeper understanding of the importance of the climate surrounding an athlete and the role of the coach for elite athletes by conducting in-depth interviews with a group of athletes (n=7). The athletes also completed the Perception of Success Questionnaire (POSQ) and Perception of Motivational Climate Questionnaire (PMCQ) to measure motivational indices. All the athletes were very high on task orientation and moderate to high on ego orientation. Most of the athletes perceived a high mastery climate and a low performance climate. The athletes emphasized the importance of the coach as the creator of the climate, as well as their preference for a supportive and caring climate. This speaks for an emphasis on a mastery climate for elite athletes.

Adult↗

PubMed2001

Role of the conserved phenylalanine 181 of NADPH-cytochrome P450 oxidoreductase in FMN binding and catalytic activity.

NADPH-cytochrome P450 oxidoreductase (P450 reductase, EC 1.6.2.4) is an essential component of the P450 monooxygenase complex and binds FMN, FAD, and NADPH cofactors. Residues Tyr140 and Tyr178 are known to be involved in FMN binding. A third aromatic side chain, Phe181, is also located in the proximity of the FMN ring and is highly conserved in FMN-binding proteins, suggesting an important functional role. This role has been investigated by site-directed mutagenesis. Substitution of Phe181 with leucine or glutamine decreased the cytochrome c reductase activity of the enzyme by approximately 50%. Ferricyanide reductase activity was unaffected, indicating that the FAD domain was unperturbed. The mutant FMN domains were expressed in Escherichia coli, and the redox potentials and binding energies of their complexes with FMN were determined. The affinity for FMN was decreased approximately 50-fold in the Leu181 and Gln181 mutants. Comparison of the binding energies of the wild-type and mutant enzymes in the three redox states of FMN suggests that Phe181 stabilizes the FMN-apoprotein complex. The amide 1H and 15N resonances of the Phe181Leu FMN domain were assigned; comparison of their chemical shifts with those of the wild-type domain indicated that the effect of the substitution on FMN affinity results from perturbation of two loops which form part of the FMN binding site. The results indicate that Phe181 cooperates with Tyr140 and Tyr178 to play a major role in the binding and stability of FMN.

Amino Acid Substitution↗

PubMed2001

Dynamics of mononuclear cadmium beta-lactamase revealed by the combination of NMR and PAC spectroscopy.

The two metal sites in cadmium substituted beta-lactamase from Bacillus cereus 569/H/9 have been studied by NMR spectroscopy ((1)H, (15)N, and (113)Cd) and PAC spectroscopy ((111m)Cd). Distinct NMR signals from the backbone amides are identified for the apoenzyme and the mononuclear and binuclear cadmium enzymes. For the binuclear cadmium enzyme, two (113)Cd NMR signals (142 and 262 ppm) and two (111m)Cd PAC nuclear quadrupole interactions are observed. Two nuclear quadrupole interactions are also observed, with approximately equal occupancy, in the PAC spectra at cadmium/enzyme ratios < 1; these are different from those derived for the binuclear cadmium enzyme, demonstrating interaction between the two metal ion binding sites. In contrast to the observation from PAC spectroscopy, only one (113)Cd NMR signal (176 ppm) is observed at cadmium/enzyme ratios < 1. The titration of the metal site imidazole (N)H proton signals as a function of cadmium ion-to-enzyme ratio shows that signals characteristic for the binuclear cadmium enzyme appear when the cadmium ion-to-enzyme ratio is between 1 and 2, whereas no signals are observed at stoichiometries less than 1. The simplest explanation consistent with all data is that, at cadmium/enzyme ratios < 1, the single Cd(II) is undergoing exchange between the two metal sites on the enzyme. This exchange must be fast on the (113)Cd NMR time scale and slow on the (111m)Cd PAC time scale and must thus occur in a time regime between 0.1 and 10 micros.

Bacillus cereus↗

PubMed2001

Thiomandelic acid, a broad spectrum inhibitor of zinc beta-lactamases: kinetic and spectroscopic studies.

Resistance to beta-lactam antibiotics mediated by metallo-beta-lactamases is an increasingly worrying clinical problem. Candidate inhibitors include mercaptocarboxylic acids, and we report studies of a simple such compound, thiomandelic acid. A series of 35 analogues were synthesized and examined as metallo-beta-lactamase inhibitors. The K(i) values (Bacillus cereus enzyme) are 0.09 microm for R-thiomandelic acid and 1.28 microm for the S-isomer. Structure-activity relationships show that the thiol is essential for activity and the carboxylate increases potency; the affinity is greatest when these groups are close together. Thioesters of thiomandelic acid are substrates for the enzyme, liberating thiomandelic acid, suggesting a starting point for the design of "pro-drugs." Importantly, thiomandelic acid is a broad spectrum inhibitor of metallo-beta-lactamases, with a submicromolar K(i) value for all nine enzymes tested, except the Aeromonas hydrophila enzyme; such a wide spectrum of activity is unprecedented. The binding of thiomandelic acid to the B. cereus enzyme was studied by NMR; the results are consistent with the idea that the inhibitor thiol binds to both zinc ions, while its carboxylate binds to Arg(91). Amide chemical shift perturbations for residues 30-40 (the beta(3)-beta(4) loop) suggest that this small inhibitor induces a movement of this loop of the kind seen for other larger inhibitors.

Arginine↗

PubMed2001

Stopped-flow kinetic studies of flavin reduction in human cytochrome P450 reductase and its component domains.

The reduction by NADPH of the FAD and FMN redox centers in human cytochrome P450 reductase and its component domains has been studied by rapid-mixing, stopped-flow spectroscopy. Reduction of the isolated FAD-domain occurs in three kinetically resolvable steps. The first represents the rapid formation (>500 s(-)(1)) of a charge-transfer species between oxidized FAD and NADPH. This is followed by an isomerization ( approximately 200 s(-)(1)) to a second charge-transfer species, characterized by a more intense absorption in the long-wavelength region. The third step represents hydride transfer from NADPH to FAD and is accompanied by a change in the tryptophan fluorescence of the FAD-domain. Flavin reduction is reversible, and the observed rate of hydride transfer displays a complex dependence on NADPH concentration. Two-electron-reduced FAD-domain is active in electron transfer reactions with the isolated FMN domain through the formation of a weakly associating electron transfer complex. Reduction of the CPR by NADPH occurs without direct spectral evidence for the formation of charge-transfer species, although the presence of such species is inferred indirectly. Transfer of the first hydride ion leads to the accumulation of a blue di-semiquinoid species of the reductase, indicating rapid transfer of one electron to the FMN domain. The di-semiquinoid species decays on transfer of the second hydride ion. A third phase is seen following prolonged incubation with NADPH and is assigned to a series of equilibration reactions between different redox species of the enzyme as the system relaxes to its thermodynamically most stable state. As with the isolated FAD-domain, the first hydride transfer in the reductase shows a complex dependence on NADPH concentration. At high NADPH concentration, the observed rate of hydride transfer is slow (approximately 20 s(-1)), and this attenuated rate is attributed to the reversible formation of an less active complex resulting from the binding of a second molecule of NADPH. The kinetic data are discussed with reference to the potentiometric studies on the enzyme and its component domains presented in the preceding paper in this issue [Munro, A., Noble, M., Robledo, L., Daff, S., and Chapman, S. (2001) Biochemistry 40, 1956-1963].

Animals↗

PubMed2001

Structure-activity relationships in flexible protein domains: regulation of rho GTPases by RhoGDI and D4 GDI.

The guanine dissociation inhibitors RhoGDI and D4GDI inhibit guanosine 5'-diphosphate dissociation from Rho GTPases, keeping these small GTPases in an inactive state. The GDIs are made up of two domains: a flexible N-terminal domain of about 70 amino acid residues and a folded 134-residue C-terminal domain. Here, we characterize the conformation of the N-terminal regions of both RhoGDI and D4GDI using a series of NMR experiments which include (15)N relaxation and amide solvent accessibility measurements. In each protein, two regions with tendencies to form helices are identified: residues 36 to 58 and 9 to 20 in RhoGDI, and residues 36 to 57 and 20 to 25 in D4GDI. To examine the functional roles of the N-terminal domain of RhoGDI, in vitro and in vivo functional assays have been carried out with N-terminally truncated proteins. These studies show that the first 30 amino acid residues are not required for inhibition of GDP dissociation but appear to be important for GTP hydrolysis, whilst removal of the first 41 residues completely abolish the ability of RhoGDI to inhibit GDP dissociation. The combination of structural and functional studies allows us to explain why RhoGDI and D4GDI are able to interact in similar ways with the guanosine 5'-diphosphate-bound GTPase, but differ in their ability to regulate GTP-bound forms; these functional differences are attributed to the conformational differences of the N-terminal domains of the guanosine 5'-diphosphate dissociation inhibitors. Therefore, the two transient helices, appear to be associated with different biological effects of RhoGDI, providing a clear example of structure-activity relationships in a flexible protein domain.

Amides↗

PubMed2001

Engineering of a functional human NADH-dependent cytochrome P450 system.

A functional human NADH-dependent cytochrome P450 system has been developed by altering the cofactor preference of human NADPH cytochrome P450 reductase (CPR), the redox partner for P450s. This has been achieved by a single amino acid change of the conserved aromatic amino acid Trp-676, which covers the re-side of the FAD isoalloxazine ring in the nicotinamide-binding site. Of the mutations made, the substitution of Trp-676 with alanine (W676A) resulted in a functional NADH-dependent enzyme, which catalyzed the reduction of cytochrome c and ferricyanide as well as facilitated the metabolism of 7-ethoxyresorufin by CYP1A2. Kinetic analysis measuring cytochrome c activity revealed that the NADH-dependent k(cat) of W676A is equivalent (90%) to the NADPH-dependent k(cat) of the wild-type enzyme, with W676A having an approximately 1,000-fold higher specificity for NADH. The apparent K(M)(NADPH) and K(M)(NADH) values of W676A are 80- and 150-fold decreased, respectively. In accordance with structural data, which show a bipartite binding mode of NADPH, substitution of Trp-676 does not affect 2'-AMP binding as seen by the inhibition of both wild-type CPR and the W676A mutant. Furthermore, NADPH was a potent inhibitor of the W676A NADH-dependent cytochrome c reduction and CYP1A2 activity. Overall, the results show that Trp-676 of human CPR plays a major role in cofactor discrimination, and substitution of this conserved aromatic residue with alanine results in an efficient NADH-dependent cytochrome P450 system.

Amino Acid Substitution↗

PubMed2001

Influences on the ethical beliefs of graduate students concerning research.

Development of and influence on ethical beliefs were surveyed at a major research university campus. Courses were ranked by faculty and students as most important. Mentors were ranked eighth in a list of nine factors. Of the 1,152 returned student questionnaires, 97 (8.4%) made the effort to write comments, and of the 610 faculty questionnaires returned, 64 (10%) wrote comments. These comments were rich in detail and description.

Chi-Square Distribution↗

PubMed2001

Mentoring and the impact of the research climate.

In this article, we focus on the mentoring process, and we argue that the internal and external pressures extant at research universities may create a research culture that may be antithetical to appropriate mentoring. We developed a scale based on motivation theory to determine the perceived research culture in departments and research laboratories, and a mentoring scale to determine approaches to mentoring graduate students. Participants were 610 faculty members across 49 departments at a research oriented university. The findings were that a mastery-oriented research climate and an outcome-oriented research climate were manifested at the university. More importantly, each research climate had its own unique impact on how the faculty approached mentoring graduate students. A mastery research climate was related to a more supportive approach to mentoring than the outcome research climate. We concluded by suggesting that the outcome research climate may have an adverse effect on effective mentoring and on maintaining research ethics.

Analysis of Variance↗

PubMed2001

Structural consequences of site-directed mutagenesis in flexible protein domains: NMR characterization of the L(55,56)S mutant of RhoGDI.

The guanine dissociation inhibitor RhoGDI consists of a folded C-terminal domain and a highly flexible N-terminal region, both of which are essential for biological activity, that is, inhibition of GDP dissociation from Rho GTPases, and regulation of their partitioning between membrane and cytosol. It was shown previously that the double mutation L55S/L56S in the flexible region of RhoGDI drastically decreases its affinity for Rac1. In the present work we study the effect of this double mutation on the conformational and dynamic properties of RhoGDI, and describe the weak interaction of the mutant with Rac1 using chemical shift mapping. We show that the helical content of the region 45-56 of RhoGDI is greatly reduced upon mutation, thus increasing the entropic penalty for the immobilization of the helix, and contributing to the loss of binding. In contrast to wild-type RhoGDI, no interaction with Rac1 could be identified for amino-acid residues of the flexible domain of the mutant RhoGDI and only very weak binding was observed for the folded domain of the mutant. The origins of the effect of the L55S/L56S mutation on the binding constant (decreased by at least three orders of magnitude relative to wild-type) are discussed with particular reference to the flexibility of this part of the protein.

Cell Membrane↗

PubMed2001

Students' perceptions of the motivational climate, achievement beliefs, and satisfaction in physical education.

The present study examined the relationship between students' perceptions of the motivational climate and beliefs about the causes of success, preference for challenging tasks, and satisfaction in physical education. Responses of 50 female and 46 male students (M age = 12.08 years; SD = .72) showed that perceptions of a mastery-oriented motivational climate were related to the belief that motivation or effort caused success and satisfaction. In contrast, perceptions of a performance climate were related to the belief that deception caused success and related negatively to the students' preference for challenging tasks. Results of hierarchical regression analyses revealed that perceptions of the motivational climate explained a significant amount of unique variance in the students' responses after controlling for dispositional goal orientations. The results suggest that the teacher can influence the salience of a mastery-oriented climate and, in so doing, optimize a child's motivation in physical education.

Achievement↗

PubMed2000

Trp-676 facilitates nicotinamide coenzyme exchange in the reductive half-reaction of human cytochrome P450 reductase: properties of the soluble W676H and W676A mutant reductases.

The kinetics of flavin reduction in two mutant forms of human cytochrome P450 reductase have been studied by stopped-flow spectroscopy with absorption and fluorescence detection. The mutant enzymes were altered at the position of Trp-676, which, by analogy with the structure of rat CPR, is close to the isoalloxazine ring of the enzyme-bound FAD. We show that mutant CPRs in which Trp-676 has been changed to histidine (W676H) and alanine (W676A) can be reduced by NADPH only to the two-electron level in single mixing stopped-flow experiments. The concentration dependence of the rate of hydride transfer indicates that the second, noncatalytic NADPH-binding site present in wild-type CPR is retained in the mutant enzymes. Detailed studies of W676H CPR indicate that further reduction of the enzyme beyond the two electron level is prevented due to the slow release of NADP(+) from the active site following the first hydride transfer from NADPH, owing to the stability of a reduced enzyme-NADP(+) charge-transfer complex. Reduction to the four-electron level is achieved in a sequential mixing stopped-flow experiment. In this procedure, W676H CPR is reacted first with a stoichiometric amount of NADPH, and then, following a delay of 100 ms, with excess NADPH. The data indicate that occupancy of the noncatalytic coenzyme site also hinders NADP(+) release from reduced enzyme. Fluorescence stopped-flow studies of the W676H and wild-type CPR enzymes reveal that the complex signals associated with reduction of wild-type CPR by NADPH are attributable to changes in the environment of residue W676. From these studies, a model is proposed for nicotinamide binding in wild-type CPR. In this model W676 serves as a trigger to release NADP(+) from the active site following hydride transfer. In the W676H enzyme, the slow release of NADP(+) is a consequence of the combined effects of (i) removing W676 by mutagenesis (thus removing the trigger for displacement) and (ii) the binding of NADPH in the noncatalytic site, thus trapping NADP(+) in the catalytic site.

Alanine↗

PubMed2000

Interaction of the lantibiotic nisin with mixed lipid bilayers: a 31P and 2H NMR study.

Nisin is a positively charged antibacterial peptide which binds to the negatively charged membranes of Gram-positive bacteria. The initial interaction of the peptide with model membranes of neutral (phosphatidylcholine) and negatively charged (phosphatidylcholine/phosphatidylglycerol) model lipid membranes was studied using nonperturbing solid state magic angle spinning (MAS) (31)P NMR and (2)H wide-line NMR. In the presence of nisin, the coexistence of two bilayer lipid environments was observed both in charged and in neutral membranes. One lipid environment was found to be associated with lipid directly interacting with nisin and one with noninteracting lipid. Solid state (31)P MAS NMR results show that the acidic membrane lipid component partitions preferentially into the nisin-associated environment. Deuterium NMR ((2)H NMR) of the selectively headgroup-labeled acidic lipid provides further evidence of a strong interaction between the charged lipid component and the peptide. The segregation of acidic lipid into the nisin-bound environment was quantified from (2)H NMR measurements of selectively headgroup-deuterated neutral lipid. It is suggested that the observed lipid partitioning in the presence of nisin is driven, at least initially, by electrostatic interactions. (2)H NMR measurements from chain-perdeuterated neutral lipids indicate that nisin perturbs the hydrophobic region of both charged and neutral bilayers.

Amino Acid Sequence↗

PubMed2000

Mapping the binding site for the GTP-binding protein Rac-1 on its inhibitor RhoGDI-1.

BACKGROUND: Members of the Rho family of small GTP-binding proteins, such as Rho, Rac and Cdc42, have a role in a wide range of cell responses. These proteins function as molecular switches by virtue of a conformational change between the GTP-bound (active) and GDP-bound (inactive) forms. In addition, most members of the Rho and Rac subfamilies cycle between the cytosol and membrane. The cytosolic guanine nucleotide dissociation inhibitors, RhoGDIs, regulate both the GDP/GTP exchange cycle and the membrane association/dissociation cycle. RESULTS: We have used NMR spectroscopy and site-directed mutagenesis to identify the regions of human RhoGDI-1 that are involved in binding Rac-1. The results emphasise the importance of the flexible regions of both proteins in the interaction. At least one specific region (residues 46-57) of the flexible N-terminal domain of RhoGDI, which has a tendency to form an amphipathic helix in the free protein, makes a major contribution to the binding energy of the complex. In addition, the primary site of Rac-1 binding on the folded domain of RhoGDI involves the beta4-beta5 and beta6-beta7 loops, with a slight movement of the 3(10) helix accompanying the interaction. This binding site is on the same face of the protein as the binding site for the isoprenyl group of post-translationally modified Rac-1, but is distinct from this site. CONCLUSIONS: Isoprenylated Rac-1 appears to interact with three distinct sites on RhoGDI. The isoprenyl group attached to the C terminus of Rac-1 binds in a pocket in the folded domain of RhoGDI. This is distinct from the major site on this domain occupied by Rac-1 itself, which involves two loops at the opposite end to the isoprenyl-binding site. It is probable that the flexible C-terminal region of Rac-1 extends from the site at which Rac-1 contacts the folded domain of RhoGDI to allow the isoprenyl group to bind in the pocket at the other end of the RhoGDI molecule. Finally, the flexible N terminus of RhoGDI-1, and particularly residues 48-58, makes a specific interaction with Rac-1 which contributes substantially to the binding affinity.

Amino Acid Sequence↗

PubMed2000

The relationship between motivational climate, perceived ability and sources of distress among elite athletes.

Situational factors as well as individual differences are assumed to play an important role in perceptions of stress. One factor that may affect an elite athlete's perception of stress may be his or her perceived motivational climate. To examine the relative importance of dispositional and situational factors on the perceptions of distress (i.e. negative stress), we assessed goal orientations, perception of motivational climate and sources of distress among Norwegian participants at the 1994 Winter Olympic Games in Lillehammer. Perception of a performance climate was associated with cognitive sources of distress, the coach, and team aspects as a source of distress. Furthermore, a performance climate was a significant predictor of high total distress. The athletes with lower perceptions of ability perceived the coach to be more a source of distress than athletes with a high perception of ability. A perception of a mastery climate was negatively associated with the coach as a source of distress. These findings indicate that, to reduce the perception of distress, the coach should focus on creating a mastery climate for elite athletes.

Age Factors↗

PubMed1999

The role of tyrosine-9 and the C-terminal helix in the catalytic mechanism of Alpha-class glutathione S-transferases.

Glutathione S-transferases (GSTs) play a key role in the metabolism of drugs and xenobiotics. To investigate the catalytic mechanism, substrate binding and catalysis by the wild-type and two mutants of GST A1-1 have been studied. Substitution of the 'essential' Tyr(9) by phenylalanine leads to a marked decrease in the k(cat) for 1-chloro-2,4-dinitrobenzene (CDNB), but has no affect on k(cat) for ethacrynic acid. Similarly, removal of the C-terminal helix by truncation of the enzyme at residue 209 leads to a decrease in k(cat) for CDNB, but an increase in k(cat) for ethacrynic acid. The binding of a GSH analogue increases the affinity of the wild-type enzyme for CDNB, and increases the rate of the enzyme-catalysed conjugation of this substrate with the small thiols 2-mercaptoethanol and dithiothreitol. This suggests that GSH binding produces a conformational change which is transmitted to the binding site for the hydrophobic substrate, where it alters both the affinity for the substrate and the catalytic-centre activity ('turnover number') for conjugation, perhaps by increasing the proportion of the substrate bound productively. Neither of these two effects of GSH analogues are seen in the C-terminally truncated enzyme, indicating a role for the C-terminal helix in the GSH-induced conformational change.

Amino Acid Substitution↗