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Biomedical subjects

R J Clarke

Publications and source records attributed to R J Clarke.

At least 37 records · Page 2Linked to original sources
PubMed2001

Measurements of the inverse Faraday effect from relativistic laser interactions with an underdense plasma.

Magnetic fields in excess of 7 MG have been measured with high spatial and temporal precision during interactions of a circularly polarized laser pulse with an underdense helium plasma at intensities up to 1x10(19) W cm(-2). The fields, while of the form expected from the inverse Faraday effect for a cold plasma, are much larger than expected, and have a duration approaching that of the high intensity laser pulse ( <3 psec). These observations can be explained by particle-in-cell simulations in 3D. The simulations show that the magnetic field is generated by fast electrons which spiral around the axis of the channel created by the laser field.

Journal Article↗
PubMed2001

Influence of allosteric effectors on the kinetics and equilibrium binding of phosphoenolpyruvate (PEP) to phosphoenolpyruvate carboxylase (PEPC) from Zea mays.

Phosphoenolpyruvate carboxylase (PEPC) the carbon dioxide processing enzyme of C(4) plants, shows the features of an allosteric enzyme. Allosteric activators such as D-glucose-6-phosphate and glycine increase the affinity of PEPC for its substrate PEP at pH 8.0 and pH 7.0. Allosteric inhibitors like L-malate and L-aspartate predominantly decrease the affinity of the carboxylase for PEP at pH 7.0. This was demonstrated by determination of the enzymatic activity and stopped flow (SF) fluorimetry. The binding reaction of PEP to PEPC from Zea mays was measured using the fluorescence probe 2-p-toluidinonaphthalene-6-sulfonate (TNS). The kinetics are described by an allosteric mechanism with a fast reversible bimolecular binding step of PEP to a high affinity (tensed) form of PEPC, which is in equilibrium with its low affinity (relaxed) form. The influence of allosteric effectors on the conformational transition step is demonstrated in support of the description of the kinetics of PEPC by applying a concerted allosteric mechanism as introduced by Monod, Wyman and Changeux. In summary, we present data for the influence of allosteric activators on the kinetics of PEP binding to PEPC and on the concentration dependence of the isomerisation reaction between two allosteric forms of PEPC.

Algorithms↗
PubMed2001

Mg2+-induced tRNA folding.

Mg(2+)-induced folding of yeast tRNA(Phe) was examined at low ionic strength in steady-state and kinetic experiments. By using fluorescent labels attached to tRNA, four conformational transitions were revealed when the Mg(2+) concentration was gradually increased. The last two transitions were not accompanied by changes in the number of base pairs. The observed transitions were attributed to Mg(2+) binding to four distinct types of sites. The first two types are strong sites with K(diss) of 4 and 16 microM. The sites of the third and fourth types are weak with a K(diss) of 2 and 20 mM. Accordingly, the Mg(2+)-binding sites previously classified as "strong" and "weak" can be further subdivided into two subtypes each. Fluorescent transition I is likely to correspond to Mg(2+) binding to a unique strong site selective for Mg(2+); binding to this site causes only minor A(260) change. The transition at 2 mM Mg(2+) is accompanied by substantial conformational changes revealed by probing with ribonucleases T1 and V1 and likely enhances stacking of the tRNA bases. Fast and slow kinetic phases of tRNA refolding were observed. Time-resolved monitoring of Mg(2+) binding to tRNA suggested that the slow kinetic phase was caused by a misfolded tRNA structure formed in the absence of Mg(2+). Our results suggest that, similarly to large RNAs, Mg(2+)-induced tRNA folding exhibits parallel folding pathways and the existence of kinetically trapped intermediates stabilized by Mg(2+). A multistep scheme for Mg(2+)-induced tRNA folding is discussed.

Binding Sites↗
PubMed2001

Observation of a hot high-current electron beam from a self-modulated laser wakefield accelerator.

A highly relativistic electron beam produced by a 50 TW laser-plasma accelerator has been characterized by photonuclear techniques. The beam has large divergence that increases with plasma density. The electron yield also increases with plasma density and reaches up to 4x10(11) electrons ( >10 MeV), with beam current approaching the Alfvén limit. Effective electron temperatures exceeding 8 MeV are found, leading to an order of magnitude higher photonuclear activation yield than in solid target experiments with the same laser system.

Journal Article↗
PubMed2001

The dipole potential of phospholipid membranes and methods for its detection.

The dipole potential is an electrical potential within phospholipid membranes, which arises because of the alignment of dipolar residues of the lipids and/or water dipoles in the region between the aqueous phases and the hydrocarbon-like interior of the membrane. For a fully saturated phosphatidylcholine membrane, its value is believed to be in the range 220-280 mV, positive in the membrane interior. This results in an enormous electric field strength within the membrane of 10(8)-10(9) Vm(-1). The dipole potential is thus likely to have great significance in controlling the conformation of ion-translocating membrane proteins and so in regulating enzyme function. Because of its location within the membrane, quantification of the dipole potential is extremely difficult and presents a great challenge to the experimentalist and theoretician alike. Both electrical and spectroscopic methods developed for the determination of the dipole potential on lipid bilayers and monolayers are presented and possible causes for differences in the values derived are discussed.

Cell Membrane↗
PubMed2001

Rate limitation of the Na(+),K(+)-ATPase pump cycle.

The kinetics of Na(+)-dependent phosphorylation of the Na(+),K(+)-ATPase by ATP were investigated via the stopped-flow technique using the fluorescent label RH421 (saturating [ATP], [Na(+)], and [Mg(2+)], pH 7.4, and 24 degrees C). The well-established effect of buffer composition on the E(2)-E(1) equilibrium was used as a tool to investigate the effect of the initial enzyme conformation on the rate of phosphorylation of the enzyme. Preincubation of pig kidney enzyme in 25 mM histidine and 0.1 mM EDTA solution (conditions favoring E(2)) yielded a 1/tau value of 59 s(-1). Addition of MgCl(2) (5 mM), NaCl (2 mM), or ATP (2 mM) to the preincubation solution resulted in increases in 1/tau to values of 129, 167, and 143 s(-1), respectively. The increases can be attributed to a shift in the enzyme conformational equilibrium before phosphorylation from the E(2) state to an E(1) or E(1)-like state. The results thus demonstrate conclusively that the E(2) --> E(1) transition does in fact limit the rate of subsequent reactions of the pump cycle. Based on the experimental results, the rate constant of the E(2) --> E(1) transition under physiological conditions could be estimated to be approximately 65 s(-1) for pig kidney enzyme and 90 s(-1) for enzyme from rabbit kidney. Taking into account the rates of other partial reactions, computer simulations show these values to be consistent with the turnover number of the enzyme cycle (approximately 48 s(-1) and approximately 43 s(-1) for pig and rabbit, respectively) calculated from steady-state measurements. For enzyme of the alpha(1) isoform the E(2) --> E(1) conformational change is thus shown to be the major rate-determining step of the entire enzyme cycle.

Adenosine Triphosphate↗
PubMed2001

Electrogenic properties of the Na+,K+-ATPase probed by presteady state and relaxation studies.

Electrical measurements on planar lipid bilayers, patch/voltage clamp experiments, and spectroscopic investigations involving a potential sensitive dye are reviewed. These experiments were performed to analyze the kinetics of charge translocation of the Na+,K+-ATPase. High time resolution was achieved by applying caged ATP, voltage-jump, and stopped-flow techniques, respectively. Kinetic parameters and the electrogenicity of the relevant transitions in the Na+,K+-ATPase reaction cycle are discussed.

Electric Conductivity↗
PubMed2001

Do socioeconomic disadvantages persist into old age? Self-reported morbidity in a 29-year follow-up of the Whitehall Study.

OBJECTIVES: This study examined (1) the relation of employment grade in middle age to self-reported poor health and functional limitations in old age and (2) whether socioeconomic status at approximately the time of retirement modifies health differentials in old age. METHODS: Survivors of the Whitehall Study cohort of men were resurveyed. Respondents were aged 40 to 69 years when they were originally screened in 1967 to 1970. RESULTS: Compared with senior administrators, men in clerical or manual (low-grade) jobs in middle age had quadruple the odds of poor physical performance in old age, triple the odds of poor general health, and double the odds of poor mental health and disability. At most, 20% of these differences were explained by baseline health or risk factors. Men who moved from low to middle grades before retirement were less likely than those who remained in low grades to have poor mental health. CONCLUSIONS: Socioeconomic status in middle age and at approximately retirement age is associated with morbidity in old age.

Activities of Daily Living↗
PubMed2000

Stratigraphy, artefact industries and hominid associations for Sterkfontein, member 5.

A revised stratigraphy for the early hominid site of Sterkfontein (Gauteng Province, South Africa) reveals a complex distribution of infills in the main excavation area between 2.8 and 1.4 m.y.a, as well as deposits dating to the mid to late Pleistocene. New research now shows that the Member 4 australopithecine breccia (2.8-2.6 Ma) extends further west than was previously thought, while a late phase of Member 4 is recognized in a southern area. The artefact-bearing breccias were defined sedimentologically as Member 5, but one supposed part of these younger breccias, the StW 53 infill, lacks in situ stone tools, although it does appear to post-date 2.6 Ma when artefacts first appear in the archaeological record. The StW 53 hominid, previously referred to Homo habilis, is here argued to be Australopithecus. The first artefact-bearing breccia of Member 5 is the Oldowan Infill, estimated at 2-1.7 Ma. It occupies a restricted distribution in Member 5 east and contains an expedient, flake-based tool industry associated with a few fossils of Paranthropos robustus. An enlarged cave opening subsequently admitted one or more Early Acheulean infills associated in Member 5 west with Homo ergaster. The artefacts attest to a larger site accumulation between ca. 1.7 and 1.4 Ma, with more intensive use of quartzite over quartz and a subtle but important shift to large flakes and heavier-duty tools. The available information on palaeoenvironments is summarized, showing an overall change from tropical to sub-tropical gallery forest, forest fringe and woodland conditions in Member 4 to more open woodland and grassland habitats in the later units, but with suggestions of a wet localized topography in the Paranthropus -bearing Oldowan Infill.

Animals↗
PubMed2000

P(3)-[2-(4-hydroxyphenyl)-2-oxo]ethyl ATP for the rapid activation of the Na(+),K(+)-ATPase.

P(3)-[2-(4-hydroxyphenyl)-2-oxo]ethyl ATP (pHP-caged ATP) has been investigated for its application as a phototrigger for the rapid activation of electrogenic ion pumps. The yield of ATP after irradiation with a XeCl excimer laser (lambda = 308 nm) was determined at pH 6.0-7.5. For comparison, the photolytic yields of P(3)-[1-(2-nitrophenyl)]ethyl ATP (NPE-caged ATP) and P(3)-[1, 2-diphenyl-2-oxo]ethyl ATP (desyl-caged ATP) were also measured. It was shown that at lambda = 308 nm pHP-caged ATP is superior to the other caged ATP derivatives investigated in terms of yield of ATP after irradiation. Using time-resolved single-wavelength IR spectroscopy, we determined a lower limit of 10(6) s(-1) for the rate constant of release of ATP from pHP-caged ATP at pH 7.0. Like NPE-caged ATP, pHP-caged ATP and desyl-caged ATP bind to the Na(+), K(+)-ATPase and act as competitive inhibitors of ATPase function. Using pHP-caged ATP, we investigated the charge translocation kinetics of the Na(+),K(+)-ATPase at pH 6.2-7.4. The kinetic parameters obtained from the electrical measurements are compared to those obtained with a technique that does not require caged ATP, namely parallel stopped-flow experiments using the voltage-sensitive dye RH421. It is shown that the two techniques yield identical results, provided the inhibitory properties of the caged compound are taken into account. Our results demonstrate that under physiological (pH 7.0) and slightly basic (pH 7.5) or acidic (pH 6. 0) conditions, pHP-caged ATP is a rapid, effective, and biocompatible phototrigger for ATP-driven biological systems.

Adenosine Triphosphate↗
PubMed1999

Hofmeister effects of anions on the kinetics of partial reactions of the Na+,K+-ATPase.

The effects of lyotropic anions, particularly perchlorate, on the kinetics of partial reactions of the Na+,K+-ATPase from pig kidney were investigated by two different kinetic techniques: stopped flow in combination with the fluorescent label RH421 and a stationary electrical relaxation technique. It was found that 130 mM NaClO4 caused an increase in the Kd values of both the high- and low-affinity ATP-binding sites, from values of 7.0 (+/- 0.6) microM and 143 (+/- 17) microM in 130 mM NaCl solution to values of 42 (+/- 3) microM and 660 (+/- 100) microM in 130 mM NaClO4 (pH 7.4, 24 degrees C). The half-saturating concentration of the Na+-binding sites on the E1 conformation was found to decrease from 8-10 mM in NaCl to 2.5-3.5 mM in NaClO4 solution. The rate of equilibration of the reaction, E1P(Na+)3 left arrow over right arrow E2P + 3Na+, decreased from 393 (+/- 51) s-1 in NaCl solution to 114 (+/- 15) s-1 in NaClO4. This decrease is attributed predominantly to an inhibition of the E1P(Na+)3 --> E2P(Na+)3 transition. The effects can be explained in terms of electrostatic interactions due to perchlorate binding within the membrane and/or protein matrix of the Na+,K+-ATPase membrane fragments and alteration of the local electric field strength experienced by the protein. The kinetic results obtained support the conclusion that the conformational transition E1P(Na+)3 --> E2P(Na+)3 is a major charge translocating step of the pump cycle.

Adenosine Triphosphate↗
PubMed1999

Influence of anions and cations on the dipole potential of phosphatidylcholine vesicles: a basis for the Hofmeister effect.

Anions and cations have long been recognized to be capable of modifying the functioning of various membrane-related physiological processes. Here, a fluorescent ratio method using the styrylpyridinium dyes, RH421 and di-8-ANEPPS, was applied to determine the effect of a range of anions and cations on the intramembrane dipole potential of dimyristoylphosphatidylcholine vesicles. It was found that certain anions cause a decrease in the dipole potential. This could be explained by binding within the membrane, in support of a hypothesis originally put forward by A. L. Hodgkin and P. Horowicz [1960, J. Physiol. (Lond.) 153:404-412.] The effectiveness of the anions in reducing the dipole potential was found to be ClO4- > SCN- > I- > NO3- > Br- > Cl- > F- > SO42-. This order could be modeled by a partitioning of ions between the membrane and the aqueous phase, which is controlled predominantly by the Gibbs free energy of hydration. Cations were also found to be capable of reducing the dipole potential, although much less efficiently than can anions. The effects of the cations was found to be trivalent > divalent > monovalent. The cation effects were attributed to binding to a specific polar site on the surface of the membrane. The results presented provide a molecular basis for the interpretation of the Hofmeister effect of lyotropic anions on ion transport proteins.

Anions↗
PubMed1998

A one-million-year-old Homo cranium from the Danakil (Afar) Depression of Eritrea.

One of the most contentious topics in the study of human evolution is that of the time, place and mode of origin of Homo sapiens. The discovery in the Northern Danakil (Afar) Depression, Eritrea, of a well-preserved Homo cranium with a mixture of characters typical of H. erectus and H. sapiens contributes significantly to this debate. The cranium was found in a succession of fluvio-deltaic and lacustrine deposits and is associated with a rich mammalian fauna of early to early-middle Pleistocene age. A magnetostratigraphic survey indicates two reversed and two normal magnetozones. The layer in which the cranium was found is near the top of the lower normal magnetozone, which is identified as the Jaramillo subchron. Consequently, the human remains can be dated at approximately 1 million years before present.

Animals↗
PubMed1998

Dephosphorylation kinetics of pig kidney Na+,K+-ATPase.

The kinetics of K+-stimulated dephosphorylation of the Na+,K+-ATPase were investigated at pH 7.4, 24 degrees C, and an ATP concentration of 1.0 mM via the stopped-flow technique using the fluorescent label RH421. Two different mixing procedures were used: (a) premixing with ATP to allow phosphorylation to go to completion, followed by mixing with KCl; and (b) simultaneous mixing with ATP and KCl. Using mixing procedure (a), the dephosphorylation rate constant of enzyme complexed with K+ ions could be determined directly to be </=366 s-1 and the rate constant for spontaneous dephosphorylation (without K+) </=60 s-1. The K+ concentration dependence of the observed reciprocal time constant showed half-saturation at a K+ concentration of 2.4-2.6 mM with positive cooperativity involved in the occupation of the K+ binding sites on the E2P conformation of the enzyme. Using mixing procedure (b), it was found that at saturating K+ concentrations the dephosphorylation of the enzyme is rate-limited by its phosphorylation, which occurs with a rate constant of approximately 190 s-1 (1). These results show that all reactions occurring after phosphorylation and prior to dephosphorylation, i.e., the E1P to E2P conformational transition as well as Na+ release and K+ binding steps, must be fast (>190 s-1).

Adenosine Triphosphate↗
PubMed1998

Kinetics of Na(+)-dependent conformational changes of rabbit kidney Na+,K(+)-ATPase.

The kinetics of Na(+)-dependent partial reactions of the Na+,K(+)-ATPase from rabbit kidney were investigated via the stopped-flow technique, using the fluorescent labels N-(4-sulfobutyl)-4-(4-(p-(dipentylamino)phenyl)butadienyl)py ridinium inner salt (RH421) and 5-iodoacetamidofluorescein (5-IAF). When covalently labeled 5-IAF enzyme is mixed with ATP, the two labels give almost identical kinetic responses. Under the chosen experimental conditions two exponential time functions are necessary to fit the data. The dominant fast phase, 1/tau 1 approximately 155 s-1 for 5-IAF-labeled enzyme and 1/tau 1 approximately 200 s-1 for native enzyme (saturating [ATP] and [Na+], pH 7.4 and 24 degrees C), is attributed to phosphorylation of the enzyme and a subsequent conformational change (E1ATP(Na+)3-->E2P(Na+)3 + ADP). The smaller amplitude slow phase, 1/tau 2 = 30-45 s-1, is attributed to the relaxation of the dephosphorylation/rephosphorylation equilibrium in the absence of K+ ions (E2P<==>E2). The Na+ concentration dependence of 1/tau 1 showed half-saturation at a Na+ concentration of 6-8 mM, with positive cooperatively involved in the occupation of the Na+ binding sites. The apparent dissociation constant of the high-affinity ATP-binding site determined from the ATP concentration dependence of 1/tau 1 was 8.0 (+/- 0.7) microM. It was found that P3-1-(2-nitrophenyl)ethyl ATP, tripropylammonium salt (NPE-caged ATP), at concentrations in the hundreds of micromolar range, significantly decreases the value of 1/tau 1, observed. This, as well as the biexponential nature of the kinetic traces, can account for previously reported discrepancies in the rates of the reactions investigated.

Adenosine Triphosphate↗
PubMed1997

Stopped-flow kinetic investigations of conformational changes of pig kidney Na+,K+-ATPase.

The kinetics of Na+-dependent partial reactions of the Na+,K+-ATPase were investigated via the stopped-flow technique using the fluorescent labels RH421 and BIPM. After the enzyme is mixed with MgATP, both labels give almost identical kinetic responses. Under the chosen experimental conditions two exponential time functions are necessary to fit the data. The dominant fast phase, 1/tau1 approximately 180 s-1 (saturating [ATP] and [Na+], pH 7.4 and 24 degrees C), is attributed to phosphorylation of the enzyme and a subsequent conformational change (E1ATP(Na+)3 --> E2P(Na+)3 + ADP). The rate of the phosphorylation reaction measured by the acid quenched-flow technique was 190 s-1 at 100 microM ATP, suggesting that phosphorylation controls the kinetics of the RH421 signal and that the conformational change is very fast (>/=600 s-1). The rate of the RH421 signal was optimal at pH 7.5. The Na+ concentration dependence of 1/tau1 showed half-saturation at a Na+ concentration of 8-10 mM with positive cooperativity involved in the occupation of the Na+ binding sites. The apparent dissociation constant of the high affinity ATP binding site determined from the ATP concentration dependence of 1/tau1 was 7.0 (+/-0.6) microM, while the apparent Kd for the low affinity site and the rate constant for the E2 to E1 conformational change evaluated in the absence of Mg2+ were 143 (+/-17) microM and </= 28 s-1. At RH421 concentrations in the micromolar range, a decrease in the value of 1/tau1 is observed. On the basis of rapid quenched-flow measurements, this inhibition can be attributed to a reaction step subsequent to phosphorylation. This accounts for previously observed kinetic discrepancies between RH421 and BIPM.

Adenosine Triphosphate↗