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P Van Gelder

Publications and source records attributed to P Van Gelder.

At least 19 recordsLinked to original sources
PubMed1998

Coupling site-directed mutagenesis with high-level expression: large scale production of mutant porins from E. coli.

Combination of an origin repair mutagenesis system with a new mutS host strain increased the efficiency of mutagenesis from 46% to 75% mutant clones. Overexpression with the T7 expression system afforded large quantities of proteins from mutant strains. A series of E. coli BE host strains devoid of major outer membrane proteins was constructed, facilitating the purification of mutant porins to homogeneity. This allowed preparation of 149 porin mutants in E. coli used in detailed explorations of the structure and function of this membrane protein to high resolution.

Adenosine Triphosphatases↗
PubMed1997

Role of the constriction loop in the gating of outer membrane porin PhoE of Escherichia coli.

Porins form voltage-gated channels in the bacterial outer membrane. These proteins are composed of three identical subunits, each forming a 16-stranded beta-barrel. In this study, the role in voltage gating of a loop that forms a constriction within the pore was studied. The channel characteristics of mutant PhoE porins, in which the tip of the constriction loop was connected to the barrel wall, were determined. Whereas the properties of several mutant channels were changed, all of these channels could still be closed at high potential, showing that a gross movement of the constriction loop within the channel is not implicated in voltage gating.

Anti-Bacterial Agents↗
PubMed1997

Voltage sensing in the PhoE and OmpF outer membrane porins of Escherichia coli: role of charged residues.

The porins PhoE and OmpF form anion and cation-selective pores, respectively, in the outer membrane of Escherichia coli. Each monomer of these trimeric proteins consists of a 16-stranded beta-barrel, which contains a constriction at half the height of the channel. The functional significance of a transverse electrical field that is formed by charged amino acid residues within the constriction zone was investigated. For this purpose, the PhoE residues R37, R75, K18 and E110 were substituted by neutral amino acids. The mutant pores allowed an increased permeation of beta-lactam antibiotics across the outer membrane in vivo, although the single channel conductance, measured in planar lipid bilayers, was not increased or even slightly decreased. Replacement of the positively charged residues resulted in a decreased voltage sensitivity, whereas the substitution of a negatively charged residue resulted in an increased voltage sensitivity. Similar substitutions in OmpF caused the opposite effects, i.e. the substitution of positive and negative charges resulted in increased and decreased voltage sensitivity, respectively. Together, the results suggest that opposite charges, i.e. positive charges in anion-selective and negative charges in cation-selective porins, act as sensors for voltage gating.

Bacterial Outer Membrane Proteins↗
PubMed1997

Peak velocities of visually and nonvisually guided saccades in smooth-pursuit and saccadic tasks.

Smooth pursuit typically includes corrective catch-up saccades, but may also include such intrusive saccades away from the target as anticipatory or large overshooting saccades. We sought to differentiate catch-up from anticipatory and overshooting saccades by their peak velocities, to see whether the higher velocities of visually rather than nonvisually guided saccades in saccadic tasks may be found also in saccades in pursuit. In experiment 1, 12 subjects showed catch-up, anticipatory, and overshooting saccades to comprise 70.4% of all saccades in pursuit of periodic, 30 degrees/s constant-velocity targets. Catch-up saccades were faster than the others. Saccadic tasks were run as well, on 19 subjects, including the 12 whose pursuit data were analyzed, with target-onset, target-remaining (saccade to the remaining target when the other three extinguish), and antisaccade tasks. For 17 of the 19 subjects, antisaccade velocities were lower than for either target-onset or target-remaining tasks. Velocities for the target-remaining task were near those for target onset, indicating that target presence, not its onset, defines visually guided saccades. Error and reaction-time data suggest greater cognitive difficulty for target remaining than for target onset, so that the cognitive difficulty of typical nonvisually guided saccade tasks is not sufficient to produce their lowered velocity. To produce reliably, in each subject, catch-up and anticipatory saccades with comparable amplitude distributions, nine new subjects were asked in experiment 2 to make intentional catch-up and anticipatory saccades in pursuit, and were presented with embedded target jumps to elicit catch-up saccades, all with periodic target trajectories of 15 degrees/s and 30 degrees/s. Velocities of intentional anticipatory saccades were lower than velocities of intentional catch-up saccades, while velocities of intentional and embedded catch-up saccades were similar. Target-onset and remembered-target saccadic tasks were run, showing the expected higher velocity for the target-onset task in each subject. Both experiments demonstrate higher peak velocities for catch-up saccades than for anticipatory saccades, suggesting that cortical structures preferentially involved in nonvisually guided saccades may initiate the anticipatory and overshooting saccades in pursuit.

Calibration↗
PubMed1997

Pore functioning of outer membrane protein PhoE of Escherichia coli: mutagenesis of the constriction loop L3.

Each monomer of the trimeric outer membrane porin PhoE of Escherichia coli consists of a 16-stranded beta-barrel with short turns at the periplasmic side and large loops at the cell surface. One of these loops, L3, is folded inside the beta-barrel and forms a constriction within the channel. Therefore, it is assumed to play an important role in the permeability properties of this general diffusion pore. Several site-directed mutations were introduced in loop L3 to investigate its function. The loop L3 contains a short alpha-helix and, at the tip of the loop, a highly conserved PEFGG sequence. The alpha-helix was deleted and the two glycines in the PEFGG sequence were either replaced by alanines or deleted. A serine residue, supposed to play an indirect role in the anion selectivity of the pore, was removed. The mutant porins were analysed both in vitro and in vivo. The results suggest that flexibility of the third loop is important for solute passage and that this flexibility is determined by the two glycine residues in the PEFGG sequence. Furthermore, the alpha-helix is probably important for the folding of the protein. The supposed involvement of Ser115 (Ser121A in OmpF nomenclature) in anion selectivity was confirmed.

Anti-Bacterial Agents↗
PubMed1996

Structural and functional characterization of a His-tagged PhoE pore protein of Escherichia coli.

The recent elucidation of the 3-D structure of the outer membrane protein PhoE of Escherichia coli provides an excellent tool for a detailed analysis of the structure-function relationship of this pore-forming protein. For this purpose, a fast and efficient method for the purification of mutant porins is needed. A histidine-tag was engineered between the signal sequence and the N terminus of mature PhoE. The recombinant PhoE protein was normally assembled into the outer membrane and could be purified by immobilized metal affinity chromatography. Part of the total amount of the trimers dissociated into folded monomers during purification. The histidine-tag did not change the electrophysical characteristics of the protein in lipid bilayers. Hence, the method is useful for the fast purification of mutant porins for functional and structural characterization.

Escherichia coli↗
PubMed1996

Demonstration of a folded monomeric form of porin PhoE of Escherichia coli in vivo.

The porins in the outer membranes of gram-negative bacteria are trimeric proteins. A folded monomeric form of the Escherichia coli porin PhoE, with a higher electrophoretic mobility than that of the denatured protein, has recently been detected in in vitro folding studies. To investigate the possible biological significance of the folded monomer, we attempted to detect this form in vivo. After pulse-labeling, folded monomers could be detected by immunoprecipitation. Furthermore, folded monomers were detected in a preparation of mutant PhoE porins, in which the subunit interactions were weakened by a E-66-->R substitution. Together, these results show that the folded monomer is not an in vitro folding artifact but an integral part of the native trimer.

Electrophoresis, Gel, Pulsed-Field↗
PubMed1996

Square-root relations between main saccadic parameters.

PURPOSE: To derive and evaluate two equations in which saccade duration and peak velocity are proportional to the square root of saccade amplitude. METHODS: A population of horizontal visually guided saccades in a range of amplitudes from 1.5 degrees to 30 degrees was recorded by means of electro-oculography in eight normal adult subjects. The peak velocity-amplitude data of this population were fitted to four models: inverse linear, exponential, power law, and square root. To approximate the duration-amplitude relation, the square root was tested against the linear and power law models. For each model, the best-fit values of its parameters were estimated by the method of least squares. RESULTS: When the entire population was used, all tested models displayed comparable goodness of fit, but when different subranges of this population were used, only the square root equations appeared to be robust and acceptably accurate. CONCLUSIONS: In a restricted range of saccade amplitudes from 1.5 degrees to 30 degrees, the square root model has some advantages over the others commonly used: to express peak velocity and duration as functions of amplitude, it requires the estimation of only two parameters, whereas the others require four. Because of its robustness, this model can be used to evaluate populations of saccadic eye movements with different ranges of amplitudes. The two parameters of the model equations allow a simple and clear physical interpretation.

Adult↗
PubMed1995

Anticipatory saccades in smooth pursuit: task effects and pursuit vector after saccades.

The dramatic improvement in smooth pursuit performance seen while analyzing the pursuit target has been ascribed to attention enhancement. With a periodic constant velocity target trajectory we ran a concurrent listening condition instead, to see if this mild distraction would degrade performance. Performance improved somewhat with the listening task, suggesting that displacing attentional effort from pursuit accuracy, rather than increasing it, brings better pursuit performance. Catch-up saccades were evenly distributed across tracking, listening, and target analysis conditions, but anticipatory and overshooting saccades were almost eliminated with target analysis. Thus the poor pursuit seems to have been caused by anticipatory and overshooting saccades, produced erroneously in the attempt to perform purposive smooth pursuit. Pursuit velocity immediately following anticipatory saccades was reduced such that the target would catch up with the point of gaze when it reached the endpoint of its trajectory, indicating a predictive goal other than instantaneous target foveation and velocity match.

Adult↗
PubMed1994

Detergent-induced folding of the outer-membrane protein PhoE, a pore protein induced by phosphate limitation.

The folding of the in vitro synthesized outer-membrane protein PhoE, a protein induced by phosphate limitation, has previously been studied using immunoprecipitation experiments with monoclonal antibodies that recognize conformational epitopes [De Cock, H., Hendricks, R., de Vrije, T. & Tommassen, J. (1990) J. Biol. Chem. 265, 4646-4651]. A folded monomer of the protein was detected in this way, while the addition of outer membranes was required to induce trimerization. In this study, we demonstrate that the folding of the in vitro synthesized PhoE protein did not occur spontaneously, but was dependent on the detergent that was present in the immunoprecipitation buffer. A remarkable specificity of phenyl-containing detergents on the efficient in vitro folding of PhoE molecules was observed. Furthermore, trimerization was detected in the absence of outer membranes when such detergents were present. However, the rate of trimerization was increased by the addition of crude cell envelopes containing outer membranes. The outer membranes probably enhanced trimerization by concentrating the folded PhoE molecules.

Cell Membrane↗
PubMed1992

Low cerebellar metabolism in medicated patients with chronic schizophrenia.

Because of the frequent association of cerebellar structural defects with schizophrenia, the authors reanalyzed the metabolic brain images of patients with chronic schizophrenia to assess if they had abnormalities in cerebellar metabolism. They used carbon-11-2-deoxyglucose and positron emission tomography to study 18 medicated patients with chronic schizophrenia and 12 normal comparison subjects. Patients with schizophrenia showed significantly lower absolute and relative metabolism in the cerebellum than normal subjects.

Adult↗
PubMed1990

An acceleration illusion caused by underestimation of stimulus velocity during pursuit eye movements: Aubert-Fleischl revisited.

When the eyes pursue a fixation point that sweeps across a moving background pattern, and the fixation point is suddenly made to stop, the ongoing motion of the background pattern seems to accelerate to a higher velocity. Experiment I showed that this acceleration illusion is not caused by the sudden change in (i) the relative velocity between background and fixation point, (ii) the velocity of the retinal image of the background pattern, or (iii) the motion of the retinal image of the rims of the CRT screen on which the experiment was carried out. In experiment II the magnitude of the illusion was quantified. It is strongest when background and eyes move in the same direction. When they move in opposite directions it becomes less pronounced (and may disappear) with higher background velocities. The findings are explained in terms of a model proposed by the first author, in which the perception of object motion and velocity derives from the interaction between retinal slip velocity information and the brain's 'estimate' of eye velocity in space. They illustrate that the classic Aubert-Fleischl phenomenon (a stimulus seems to be moving slower when pursued with the eyes than when moving in front of stationary eyes) is a special case of a more general phenomenon: whenever we make a pursuit eye movement we underestimate the velocity of all stimuli in our visual field which happen to move in the same direction as our eyes, or which move slowly in the direction opposite to our eyes.

Acceleration↗
PubMed1988

The infection-stage-related IgG response to Toxoplasma gondii studied by immunoblotting.

The technique of SDS-polyacrylamide gel electrophoresis and immunoblotting was used to study the evolution of the IgG antibody response to Toxoplasma gondii antigens in sequential sera of acutely infected patients. The results show that the IgG immunoblot pattern can be a useful marker for the stage of T. gondii infection. A 35-kD antigen elicited the first IgG response soon after exposure. During the course of infection additional bands appeared consecutively, following a constant sequence, to evolve to a late-stage-specific pattern with about 13-15 major bands. This pattern, which was reached at least 4 months after infection, was also found in the immunoblots of 28 patients with a chronic (latent) T. gondii infection.

Acute Disease↗
PubMed1987

Phenomenological correlates of metabolic activity in 18 patients with chronic schizophrenia.

Using [11C]-deoxy-D-glucose and positron emission tomography (PET), the authors measured brain metabolism in 18 patients with chronic schizophrenia to assess which of the metabolic measures from two test conditions was more closely related to the patients' differing clinical characteristics. The two conditions were resting and activation, and an eye tracking task was used. Patients with more negative symptoms showed lower global metabolic rates and more severe hypofrontality than did the patients with fewer negative symptoms. Differences among the patients were distinguished by the task: sicker patients failed to show a metabolic activation response. These findings suggest that cerebral metabolic patterns reflect clinical characteristics of schizophrenic patients.

Adult↗
PubMed1986

Brain organization in schizophrenia.

Brain metabolism was measured with positron emission tomography and [11C]deoxyglucose during baseline and during a visual task in 12 normal subjects and 18 schizophrenic patients. Global measures of metabolism for 11 brain regions were transformed into relative values by dividing them by the metabolic value for whole brain. Factor analysis was accomplished on the matrix of intercorrelations among the relative regional values for the normal and for the schizophrenic patients under baseline and under the task. Four factors that revealed independently varying metabolism in frontal, occipital, left-versus-right hemisphere, and subcortical structures were obtained. The frontal and subcortical factors discriminated between normal subjects and schizophrenic patients, whereas the occipital factor discriminated between baseline and task. Although activity in these individual regions varied significantly, it was the pattern of differences in regional metabolic activity that best discriminated between diagnostic groups and testing conditions.

Adult↗
PubMed1985

A comparison of the effects of atenolol and metoprolol on attention.

Additional side-effects of two beta-blockers, atenolol (hydrophilic) and metoprolol (lipophilic), were measured in 18 normal male volunteers in a randomized double-blind placebo-controlled crossover study. A saccadic eye movement reaction time (RT) task, a manual RT version of the Stroop Colour-Word Task, the Span of Apprehension Task, and the Continuous Performance Task were presented on a graphics display screen. Standard cognitive effects were replicated on all measures. Atenolol was noticeably, but not significantly, superior to metoprolol in visual alerting and in angular breadth of visual information processing, both with saccadic RT.

Adult↗