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Biomedical subjects

T J Andrews

Publications and source records attributed to T J Andrews.

At least 19 recordsLinked to original sources
PubMed2001

Plastome-encoded bacterial ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) supports photosynthesis and growth in tobacco.

The efficiency with which crop plants use their resources of light, water, and fertilizer nitrogen could be enhanced by replacing their CO(2)-fixing enzyme, d-ribulose-1,5-bisphosphate carboxylase-oxygenase (RubisCO), with more efficient forms, such as those found in some algae, for example. This important challenge has been frustrated by failure of all previous attempts to substitute a fully functional, foreign RubisCO (efficient or inefficient) into higher plants. This failure could be caused by incompatibility between the plastid-encoded large subunits and the nucleus-encoded small subunits or by inability of the foreign RubisCO subunits to fold or assemble efficiently in the plastid. Mismatch between the regulatory requirements of the foreign RubisCO and conditions in the chloroplast also might render the substituted enzyme inactive but, previously, it has not been possible to test this. To answer the general question of whether a foreign RubisCO can support photosynthesis in a plant, we used plastid transformation to replace RubisCO in tobacco with the simple homodimeric form of the enzyme from the alpha-proteobacterium, Rhodospirillum rubrum, which has no small subunits and no special assembly requirements. The transplastomic plants so obtained are fully autotrophic and reproductive but require CO(2) supplementation, consistent with the kinetic properties of the bacterial RubisCO. This establishes that the activity of a RubisCO from a very different phylogeny can be integrated into chloroplast photosynthetic metabolism without prohibitive problems.

Carbon Dioxide↗
PubMed2001

CO(2) fixation by Rubisco: computational dissection of the key steps of carboxylation, hydration, and C-C bond cleavage.

Despite intensive experimental and computational studies, some important features of the mechanism of the photosynthetic CO(2)-fixing enzyme, Rubisco, are still not understood. To complement our previous investigation of the first catalytic step, the enolization of D-ribulose-1,5-bisphosphate (King et al., Biochemistry 1998, 44, 15414-15422), we present the first complete computational dissection of subsequent steps of the carboxylation reaction that includes the roles of the central magnesium ion and modeled residues of the active site. We investigated carboxylation, hydration, and C-C bond cleavage using the density functional method and the B3LYP/6-31G(d) level to perform geometry optimizations. The energies were determined by B3LYP/6-311+G(2d,p) single-point calculations. We modeled a fragment of the active site and substrate, taking into account experimental findings that the residues coordinated to the Mg ion, especially the carbamylated Lys-201, play critical roles in this reaction sequence. The carbamate appears to act as a general base, not only for enolization but also for hydration of the beta ketoacid formed by addition of CO(2) and, as well, cleavage of the C2-C3 bond of the hydrate. We show that CO(2) is added directly, without assistance of a Michaelis complex, and that hydration of the resultant beta ketoacid occurs in a separate subsequent step with a discrete transition state. We suggest that two conformations of the hydrate (gem-diol), with different metal coordination, are possible. The step with the highest activation energy during the carboxylation cycle is the C-C bond cleavage. Depending on the conformations of the gem-diol, different pathways are possible for this step. In either case, special arrangements of the metal coordination result in bond breaking occurring at remarkably low activation energies (between 28 and 37 kcal mol(-1)) which might be reduced further in the enzyme environment.

Binding Sites↗
PubMed2001

Stereoacuity thresholds in the presence of a reference surface.

With isolated binocular targets, the best depth discrimination is found in the fixation plane (Blakemore, C., Journal of Physiology 211 (1970) 599). More recent studies have suggested that stereoscopic thresholds are not always a simple function of absolute disparity, but depend on the relative disparities in the stimulus. Here, we explored the effects of relative disparity in more detail, taking particular care to control for the possibility that subjects might change their binocular eye position or exploit monocular information provided by additional reference cues. Subjects judged the depth of a vertical target line presented above a comparison line in a blank window within a fronto-parallel reference surface composed of randomly positioned dots. On individual trials, the reference surface was presented at one of three disparities (-10, 0 and +10 arc min). To control for changes in binocular eye position, exposure duration was 150 ms, and experimental conditions with different disparities of the reference surface and comparison line were randomly interleaved. To control for monocular cues, changes in threshold were determined with respect to a disparity noise condition that was in all ways identical to the reference plane condition, except that the disparities of the dots were randomly assigned between 10 and +10 arc min. Stereo-thresholds were lowered by a factor of about 2 when the surface was at the same depth as the comparison line. Thresholds were also lowered when the comparison disparity was close to the same depth as the reference surface, but were often raised when the comparison disparity had the opposite disparity sign. These results provide unequivocal evidence that the fundamental sensitivity of the disparity detecting system can be influenced by relative disparity cues that are not related to the task.

Confidence Intervals↗
PubMed2001

Form I Rubiscos from non-green algae are expressed abundantly but not assembled in tobacco chloroplasts.

Non-green algae have Rubiscos that are phylogenetically distinct from their counterparts in green algae and higher plants. Some non-green-algal Rubiscos are more specific for CO2, relative to O2, than higher-plant Rubiscos, sometimes coupled with lower Michaelis constants for CO2. If these Rubiscos could be substituted for the higher-plant enzyme, and if they functioned successfully in the higher-plant chloroplast and were regulated appropriately, they would improve the CO2 use and quantum efficiency of higher-plant photosynthesis. To assess the feasibility of expressing non-green algal Rubiscos in higher-plant chloroplasts, we inserted the rbcLS operons from the rhodophyte Galdieria sulphuraria and the diatom Phaeodactylum tricornutum into the inverted repeats of the plastid genome of tobacco, leaving the tobacco rbcL gene unaltered. Homoplasmic transformants were selected. The transgenes directed the synthesis of abundant amounts of transcripts and both subunits of the foreign Rubiscos. In some circumstances, leaves of the transformants with the P. tricornutum Rubisco contained as much foreign Rubisco protein as endogenous tobacco Rubisco (>30% of the soluble leaf protein). However, the subunits of the foreign Rubiscos were not properly folded and/or assembled. All the foreign large subunits and most of the foreign small subunits were recovered in the insoluble fractions of leaf extracts. Edman sequencing yielded the expected N-terminal sequences for the foreign small subunits but the N-termini of the foreign large subunits were blocked. Accumulation of large amounts of denatured foreign Rubisco in the leaves, particularly of the P. tricornutum transformants, caused a reduction in the amount of tobacco Rubisco present, with concomitant reductions in leaf CO2 assimilation and plant growth.

Amino Acid Sequence↗
PubMed2001

The gene for the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit relocated to the plastid genome of tobacco directs the synthesis of small subunits that assemble into Rubisco.

To assess the extent to which a nuclear gene for a chloroplast protein retained the ability to be expressed in its presumed preendosymbiotic location, we relocated the RbcS gene for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to the tobacco plastid genome. Plastid RbcS transgenes, both with and without the transit presequence, were equipped with 3' hepta-histidine-encoding sequences and psbA promoter and terminator elements. Both transgenes were transcribed abundantly, and their products were translated into small subunit polypeptides that folded correctly and assembled into the Rubisco hexadecamer. When present, either the transit presequence was not translated or the transit peptide was cleaved completely. After assembly into Rubisco, transplastomic small subunits were relatively stable. The hepta-histidine sequence fused to the C terminus of a single small subunit was sufficient for isolation of the whole Rubisco hexadecamer by Ni(2)+ chelation. Small subunits produced by the plastid transgenes were not abundant, never exceeding approximately 1% of the total small subunits, and they differed from cytoplasmically synthesized small subunits in their N-terminal modifications. The scarcity of transplastomic small subunits might be caused by inefficient translation or assembly.

Amino Acid Sequence↗
PubMed2000

The transition between the open and closed states of rubisco is triggered by the inter-phosphate distance of the bound bisphosphate.

d-Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) catalyses the central CO(2)-fixing reaction of photosynthesis in a complex, multiple-step process. Several structures of rubisco complexed with substrate analogues, inhibitors and products have been determined by X-ray crystallography. The structures fall into two well-defined and distinct states. The active site is either "open" or "closed". The timing and mechanism of the transition between these two states have been uncertain. We solved the crystal structure of unactivated (metal-free) rubisco from tobacco with only inorganic phosphate bound and conclude that phosphate binding per se does not trigger closure, as it does in the similarly structured enzyme, triosephosphate isomerase. Comparison of all available rubisco structures suggests that, instead, the distance between the terminal phosphates (P1 and P2) of the bisphosphate ligand is the trigger: if that distance is less than 9.1 A, then the active site closes; if it is greater than 9.4 A then the enzyme remains open. Shortening of the inter-phosphate distance results from the ligand binding in a more curved conformation when O atoms of the ligand's sugar backbone interact either with the metal, if it is present, or with charged groups in the metal-binding site, if the metal is absent. This shortening brings the P1 phosphate into hydrogen bonding contact with Thr65. Thr65 exists in two discrete states related by a rotation of the backbone psi torsion angle. This rotation is coupled to domain rotation and hence to active site closure. Rotation of the side-chain of Thr65 also affects the C-terminal strand of large subunit which packs against Loop 6 after closure. The position of the C-terminal strand in the closed state is stabilised by multiple polar interactions with a distinctive highly-charged latch site involving the side-chain of Asp473. In the open state, this latch site may be occupied instead by phosphorylated anions.

Anions↗
PubMed2000

Ambiguity in the perception of moving stimuli is resolved in favour of the cardinal axes.

The aim of this study was to determine whether there is a link between the statistical properties of natural scenes and our perception of moving surfaces. Accordingly, we devised an ambiguous moving stimulus that could be perceived as moving in one of three directions of motion. The stimulus was a circular patch containing three square-wave drifting gratings. One grating was always either horizontal or vertical; the other two had component directions of drift at 120 degrees to the first (and to each other), producing four possible stimulus geometries. These were presented in a pseudorandom sequence. In brief presentations, subjects always perceived two of the gratings to cohere and move as a pattern in one direction, and the third grating to move independently in the opposite direction (its component direction). Although there were three equally plausible axes (one cardinal and two oblique) along which the coherent and independent motions could occur, subjects routinely saw motion along one of the cardinal axes. Thus, the visual system preferentially combines the two oblique gratings to form a pattern that drifts in the opposite direction to the cardinal grating. It was only when the contrast of one of the oblique gratings was changed that an oblique axis of motion was perceived. This perceptual anisotropy can be related to naturally occurring bias in the visual environment, notably the predominance of horizontal and vertical contours in our visual world.

Anisotropy↗
PubMed2000

Photosynthetic electron sinks in transgenic tobacco with reduced amounts of Rubisco: little evidence for significant Mehler reaction.

Transgenic tobacco (Nicotiana tabacum L. cv. W38) plants with an antisense gene directed against the mRNA of the small subunit of Rubisco were used to investigate the role of O2 as an electron acceptor during photosynthesis. The reduction in Rubisco has reduced the capacity for CO2-fixation in these plants without a similar reduction in electron transport capacity. Concurrent measurements of chlorophyll fluorescence and CO2 assimilation at different CO2 and O2 partial pressures showed close linear relationships between chloroplast electron transport rates calculated from chlorophyll fluorescence and those calculated from CO2-fixation. These relationships were similar for wild-type and transgenic plants, indicating that the reduced capacity for CO2 fixation in the transgenic plants did not result in extra electron transport not associated with the photosynthetic carbon reduction (PCR) or photorespiratory carbon oxidation (PCO) cycle. This was further investigated with mass spectrometric measurements of 16O2 and 18O2 exchange made concurrently with measurements of chlorophyll fluorescence. In all tobacco lines the rates of 18O2 uptake in the dark were similar to the 18O2 uptake rates at very high CO2 partial pressures in the light. Rates of oxygenase activity calculated from 18O2 uptake at the compensation point were linearly related to the Rubisco content of leaves. The ratios of oxygenase to carboxylase rates were calculated from measurements of 16O2 evolution and 18O2 uptake at the compensation point. These ratios were lower in the transgenic plants, consistent with their higher CO2 compensation points. It is concluded that although there may be some electron transport to O2 to balance conflicting demands of NADPH to ATP requirements, this flux must decrease in proportion with the reduced demand for ATP and NADPH consumption in the transgenic lines. The altered balance between electron transport and Rubisco capacity, however, does not result in rampant electron transport to O2 or other electron transport acceptors in the absence of PCR and PCO cycle activity.

Adenosine Triphosphate↗
PubMed2000

The role of chloroplast electron transport and metabolites in modulating Rubisco activity in tobacco. Insights from transgenic plants with reduced amounts of cytochrome b/f complex or glyceraldehyde 3-phosphate dehydrogenase.

Leaf metabolites, adenylates, and Rubisco activation were studied in two transgenic tobacco (Nicotiana tabacum L. cv W38) types. Plants with reduced amounts of cytochrome b/f complex (anti-b/f) have impaired electron transport and a low transthylakoid pH gradient that restrict ATP and NADPH synthesis. Plants with reduced glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) have a decreased capacity to use ATP and NADPH in carbon assimilation. The activation of the chloroplast NADP-malate dehydrogenase decreased in anti-b/f plants, indicating a low NADPH/NADP(+) ratio. The whole-leaf ATP/ADP in anti-b/f plants was similar to wild type, while it increased in anti-GAPDH plants. In both plant types, the CO(2) assimilation rates decreased with decreasing ribulose 1, 5-bisphosphate concentrations. In anti-b/f plants, CO(2) assimilation was further compromised by reduced carbamylation of Rubisco, whereas in anti-GAPDH plants the carbamylation remained high even at subsaturating ribulose 1,5-bisphosphate concentrations. We propose that the low carbamylation in anti-b/f plants is due to reduced activity of Rubisco activase. The results suggest that light modulation of activase is not directly mediated via the electron transport rate or stromal ATP/ADP, but some other manifestation of the balance between electron transport and the consumption of its products. Possibilities include the transthylakoid pH gradient and the reduction state of the acceptor side of photosystem I and/or the degree of reduction of the thioredoxin pathway.

Adenosine Monophosphate↗
PubMed2000

Investigation of endothelial hyperreactivity in the obese Zucker rat in-situ: reversal by vitamin E.

The obese Zucker rat, a popular model of insulin resistance allied with oxidant stress, is associated with either normal or paradoxically enhanced endothelial vasodilator function compared with its lean litter mate. We have investigated hindquarter endothelium-dependent vasodilation in the obese Zucker rat in-situ and have examined its relationship with oxidant stress. In perfused hindquarter preparations equivalently preconstricted with phenylephrine, vasodilator responses to the endothelium-dependent agent acetylcholine (0.03-1000 pmol) were greater in obese (pD2 = 11.03+/-0.19) compared with lean (pD2 = 10.53+/-0.13) animals (P < 0.01, two-way analysis of variance). In contrast, maximal vasodilation to the nitric oxide (NO) donor sodium nitroprusside (100 nmol) was similar in obese (59.6+/-19.8%) and lean (51.9+/-2.6%) preparations (P > 0.05). However, this exaggerated vasodilator reactivity to acetylcholine in obese animals was abolished following four-week dietary supplementation with the lipophilic antioxidant vitamin E (obese pD2 = 10.74+/-0.18; lean pD2 = 10.74+/-0.08). This antioxidant-mediated effect was associated with a reduction (P < 0.02, two-way analysis of variance) and an enhancement (P < 0.01, two-way analysis of variance) in endothelium-dependent vasodilator responses in obese and lean hindlimb preparations, respectively. Our data therefore now point to a differential modulation of hindquarter endothelium-dependent vasodilation in the obese and lean Zucker rat by the prevailing oxidant tone, resulting in an agonist-stimulated endothelial vasodilator hyperreactivity in obese animals.

Acetylcholine↗
PubMed1999

Idiosyncratic characteristics of saccadic eye movements when viewing different visual environments.

Eye position was recorded in different viewing conditions to assess whether the temporal and spatial characteristics of saccadic eye movements in different individuals are idiosyncratic. Our aim was to determine the degree to which oculomotor control is based on endogenous factors. A total of 15 naive subjects viewed five visual environments: (1) The absence of visual stimulation (i.e. a dark room); (2) a repetitive visual environment (i.e. simple textured patterns); (3) a complex natural scene; (4) a visual search task; and (5) reading text. Although differences in visual environment had significant effects on eye movements, idiosyncrasies were also apparent. For example, the mean fixation duration and size of an individual's saccadic eye movements when passively viewing a complex natural scene covaried significantly with those same parameters in the absence of visual stimulation and in a repetitive visual environment. In contrast, an individual's spatio-temporal characteristics of eye movements during active tasks such as reading text or visual search covaried together, but did not correlate with the pattern of eye movements detected when viewing a natural scene, simple patterns or in the dark. These idiosyncratic patterns of eye movements in normal viewing reveal an endogenous influence on oculomotor control. The independent covariance of eye movements during different visual tasks shows that saccadic eye movements during active tasks like reading or visual search differ from those engaged during the passive inspection of visual scenes.

Adult↗
PubMed1999

Directed mutation of the Rubisco large subunit of tobacco influences photorespiration and growth.

The gene for the large subunit of Rubisco was specifically mutated by transforming the chloroplast genome of tobacco (Nicotiana tabacum). Codon 335 was altered to encode valine instead of leucine. The resulting mutant plants could not grow without atmospheric CO2 enrichment. In 0.3% (v/v) CO2, the mutant and wild-type plants produced similar amounts of Rubisco but the extent of carbamylation was nearly twice as great in the mutants. The mutant enzyme's substrate-saturated CO2-fixing rate and its ability to distinguish between CO2 and O2 as substrates were both reduced to 25% of the wild type's values. Estimates of these parameters obtained from kinetic assays with the purified mutant enzyme were the same as those inferred from measurements of photosynthetic gas exchange with leaves of mutant plants. The Michaelis constants for CO2, O2, and ribulose-1,5-bisphosphate were reduced and the mutation enhanced oxygenase activity at limiting O2 concentrations. Consistent with the reduced CO2 fixation rate at saturating CO2, the mutant plants grew slower than the wild type but they eventually flowered and reproduced apparently normally. The mutation and its associated phenotype were inherited maternally. The chloroplast-transformation strategy surmounts previous obstacles to mutagenesis of higher-plant Rubisco and allows the consequences for leaf photosynthesis to be assessed.

Amino Acid Substitution↗
PubMed1999

Interindividual variation in human visual performance.

The responses of 20 young adult emmetropes with normal color vision were measured on a battery of visual performance tasks. Using previously documented tests of known reliability, we evaluated orientation discrimination, contrast sensitivity, wavelength sensitivity, vernier acuity, direction-of-motion detection, velocity discrimination, and complex form identification. Performance varied markedly between individuals, both on a given test and when the scores from all tests were combined to give an overall indication of visual performance. Moreover, individual performances on tests of contrast sensitivity, orientation discrimination, wavelength discrimination, and vernier acuity covaried, such that proficiency on one test predicted proficiency on the others. These results indicate a wide range of visual abilities among normal subjects and provide the basis for an overall index of visual proficiency that can be used to determine whether the surprisingly large and coordinated size differences of the components of the human visual system (Andrews, Halpern, & Purves, 1997) are reflected in corresponding variations in visual performance.

Adult↗
PubMed1998

Quantum chemical analysis of the enolization of ribulose bisphosphate: the first hurdle in the fixation of CO2 by Rubisco.

A study, using ab initio quantum chemical methods, of the first step in the reaction mechanism of Rubisco, the enolization of the substrate, ribulose bisphosphate, is reported. This is the first such study that takes into account the likely roles of critical features within the active site. On the basis of molecular dynamics relaxation of the complex between activated enzyme and substrate using X-ray crystallographic structures as starting coordinates, a 29-atom fragment that mimicked the active site was constructed. States along a proposed reaction pathway were calculated using density functional theory and Moller-Plesset second-order perturbation theory. The results are consistent with the postulate that the base that abstracts the C3 proton of ribulose bisphosphate is the metal-stabilized carbamate of Lys-201 formed during the activation process. The calculations suggest that the active-site residue, Lys-175, is charged before enolization commences and they indicate a possible means by which the enzyme directs the incoming CO2 to attack the C2 carbon atom of the enediol, rather than the chemically very similar C3 atom.

Binding Sites↗
PubMed1997

Similarities in normal and binocularly rivalrous viewing.

We report here a series of observations-most of which the reader can experience directly-showing that distinct components of patterned visual stimuli (orthogonal lines of a different hue) vary in perception as sets. Although less frequent and often less complete, these perceptual fluctuations in normal viewing are otherwise similar to the binocular rivalry experienced when incompatible scenes are presented dichoptically.

Humans↗
PubMed1997

The perception of transparent three-dimensional objects.

When the proximal and distal elements of wire-frame cubes are conflated, observers perceive illusory structures that no longer behave veridically. These phenomena suggest that what we normally see depends on visual associations generated by experience. The necessity of such learning may explain why the mammalian visual system is subject to a prolonged period of plasticity in early life, when novel circuits are made in enormous numbers.

Aging↗
PubMed1997

Correlated size variations in human visual cortex, lateral geniculate nucleus, and optic tract.

We have examined several components of the human visual system to determine how the dimensions of the optic tract, lateral geniculate nucleus (LGN), and primary visual cortex (V1) vary within the same brain. Measurements were made of the cross-sectional area of the optic tract, the volumes of the magnocellular and parvocellular layers of the LGN, and the surface area and volume of V1 in one or both cerebral hemispheres of 15 neurologically normal human brains obtained at autopsy. Consistent with previous observations, there was a two- to threefold variation in the size of each of these visual components among the individuals studied. Importantly, this variation was coordinated within the visual system of any one individual. That is, a relatively large V1 was associated with a commensurately large LGN and optic tract, whereas a relatively small V1 was associated with a commensurately smaller LGN and optic tract. This relationship among the components of the human visual system indicates that the development of its different parts is interdependent. Such coordinated variation should generate substantial differences in visual ability among humans.

Adult↗