Biomedical subjects

R J Clarke

Publications and source records attributed to R J Clarke.

At least 55 records · Page 3Linked to original sources

PubMed1997

Isoforms of 11beta-hydroxysteroid dehydrogenase in human granulosa-lutein cells.

To date, two isoforms of 11beta-hydroxysteroid dehydrogenase (11betaHSD) have been characterized: a low affinity, NADP+-dependent isoform (11betaHSD1) and a high affinity, NAD+-dependent isoform which metabolizes dexamethasone and is inhibited by cortisone (11betaHSD2). Having previously reported a relationship between ovarian 11betaHSD activities and conception in women undergoing in vitro fertilization (IVF-ET), the objective of the present study was to identify which isoforms of 11betaHSD metabolize glucocorticoids in cultures of human granulosa-lutein cells. In both intact cells and cell homogenates, two distinct 11betaHSD activities were identified with differing affinities for cortisol (Km = 490 nM and 2.6 microM). Even at low concentrations, cortisol oxidation was preferentially supported by NADP+ and was independent of NAD+. Although inhibited by the hemisuccinate ester of glycyrrhetinic acid, carbenoxolone, the predominant 11betaHSD activity in intact cells was resistant to end-product inhibition. Intact cells were also able to reduce [3H]cortisone (Km = 190 nM) but did not metabolize [3H]dexamethasone. 11BetaHSD1 mRNA was expressed in 23 of 28 cell cultures whereas 11betaHSD2 mRNA was not expressed in any of the 22 independent cultures studied by reverse transcriptase-polymerase chain reaction (RT-PCR). We conclude that human granulosa-lutein cells express both type 11betaHSD and a novel isoform of this enzyme. While the low affinity 11beta-dehydrogenase and 11-ketosteroid reductase activities exhibit properties consistent with 11betaHSD1, the high affinity 11beta-dehydrogenase differs from 11betaHSD2 in that it is NADP+-dependent, does not metabolize dexamethasone and is resistant to end-product inhibition.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1997

Effect of lipid structure on the dipole potential of phosphatidylcholine bilayers.

A fluorescent ratio method utilizing styrylpyridinium dyes has recently been suggested for the measurement of the membrane dipole potential. Up to now only qualititative measurements have been possible. Here the fluorescence excitation ratio of the dye di-8-ANEPPS has been measured in lipid vesicles composed of a range of saturated and unsaturated phosphatidylcholines. It has been found that the fluorescence ratio is inversely proportional to the surface area occupied by the lipid in its fully hydrated state. This finding allows, by extra- and interpolation, the packing density to be estimated of phosphatidylcholines for which X-ray crystallographic data are not yet available. Comparison of the fluorescence data with literature data of the dipole potential from electrical measurements on monolayers and bilayers allows a calibration curve to be constructed, so that a quantitative determination of the dipole potential using di-8-ANEPPS is possible. It has been found that the value of the dipole potential decreases with increasing unsaturation and, in the case of unsaturated lipids, with increasing length of the hydrocarbon chains. This effect can be explained by the effects of chain packing on the spacing between the headgroups. In addition to the effects of lipid structure on membrane fluidity, these measurements demonstrate the possibility of a direct electrical mechanism for lipid regulation of protein function, in particular of ion transport proteins.

Crystallography, X-Ray↗

PubMed1997

Optical detection of membrane dipole potential: avoidance of fluidity and dye-induced effects.

Fluorescent styrylpyridinium dyes have recently been suggested as probes of the membrane dipole potential and of the kinetics of electrogenic ion pumps. It is necessary, however, to be able to confidently attribute observed fluorescence changes to electrical effects alone and avoid interference from changes in membrane fluidity. Furthermore, the effect of the dyes themselves on the dipole potential must be investigated. The effect of membrane fluidity on the fluorescence excitation and emission spectra of the dyes RH421 and di-8-ANEPPS have been investigated in lipid vesicles by temperature scans between 15 and 60 degrees C. Both dyes show significant temperature-dependent shifts of their excitation spectra, the magnitude of which depend on the emission wavelength and on the lipid structure. In order to eliminate membrane fluidity effects, fluorescence must be detected at the red edge of the emission spectrum; in this case 670 nm. In order to avoid dye-induced shifts of the excitation spectra of membrane-bound dye, an excess molar ratio of lipid to dye of at least 200-fold is necessary. Fluorescence ratio measurements indicate qualitatively that dimyristoylphosphatidylcholine has a significantly higher dipole potential than that of dioleoylphosphatidylcholine.

Cell Membrane↗

PubMed1997

Biological fate of sulphur mustard: identification of valine and histidine adducts in haemoglobin from casualties of sulphur mustard poisoning.

1. Analytical methods were developed for the detection of N-terminal valine and histidine adducts in haemoglobin alkylated with sulphur mustard. 2. N-(2-hydroxyethylthioethyl)-N-terminal valine was selectively cleaved from globin with the Edman reagent pentafluorophenyl isothiocyanate. The resulting thiohydantoin derivative was analysed by high resolution gc-ms using negative ion chemical ionization. An alternative procedure, involving acid hydrolysis of globin to its constituent amino acids and conversion of the adduct to its di-TBDMS derivative, was less sensitive. 3. N-(2-hydroxyethylthioethyl)histidine was analysed, after acid hydrolysis of globin, as its fluorenylmethyloxycarbonyl derivative by lc-ms-ms using electrospray ionisation and selected reaction monitoring. 4. N-(2-hydroxyethylthioethyl)valine and (2-hydroxyethylthioethyl)histidine were detected in globin isolated from a rat treated percutaneously with sulphur mustard, and in globin from five blood samples collected from human casualties of sulphur mustard poisoning. The adducts are proposed as biological markers of sulphur mustard poisoning. in addition to urinary metabolites and DNA adducts.

Alkylating Agents↗

PubMed1996

Interaction of the fluorescent probe RH421 with ribulose-1,5-bisphosphate carboxylase/oxygenase and with Na+,K(+)-ATPase membrane fragments.

Fluorescence titrations have shown that the voltage-sensitive probe RH421 interacts with the water-soluble protein ribulose-1,5-bisphosphate carboxylase/oxygenase and with Na+,K(+)-ATPase membrane fragments. The probe exhibits significantly different fluorescence excitation spectra in pure lipid and pure protein environments. Experiments with a range of polyamino acids showed interactions of the probe with tyrosine, lysine and arginine residues. At saturating RH421 concentrations (> or = microM) the probe quenches 60-75% of the total tryptophan fluorescence of the Na+,K(+)-ATPase preparation. Inhibition of the hydrolytic activity of the Na+,K(+)-ATPase occurs at RH421 concentrations in the micromolar range. This may be due to a probe-induced change in membrane fluidity. The sensitivity of the probe towards conformational changes of the Na+,K(+)-ATPase decreases hyperbolically as one increases the probe concentration. The decrease in sensitivity correlates well with association of the probe in the vicinity of membrane protein, as measured by tryptophan quenching. These results have important practical consequences for the application of RH421 as a voltage indicator in membrane preparations. Based on these and previously reported results, the fluorescent response of RH421 to the ATP-induced conformational change of the Na+,K+-ATPase is consistent with either a redistribution of dye from the liquid-crystalline lipid matrix into the vicinity of membrane protein or a reorganisation of the lipids surrounding the protein into a more rigid structure caused by the conformational change of the protein.

Cell Membrane↗

PubMed1996

Behavior of luminance neurons in the pretectal olivary nucleus during the pupillary near response.

In cats and monkeys, extrastriate visual areas that have been reported to be involved in the near triad of pupilloconstriction, convergence, and accommodation have well-defined projections to the pretectal olivary nucleus (PON), the retinorecipient pretectal nucleus mediating the pupillary light reflex in mammals. We have therefore used alert, behaving primates to investigate the possibility that PON neurons are involved in the pupillary near response in addition to the pupillary light reflex. Single-unit recording revealed that PON luminance neurons significantly increased their firing rate with increases in retinal illumination and the resultant pupilloconstriction. In contrast, their activity did not significantly increase during pupilloconstriction elicited by near viewing. Thus the behavior of PON luminance neurons is appropriate for their participation in the pupillary light reflex, but is inappropriate for any proposed role in the pupillary near response. This result strongly suggests that neurons in the primate PON are solely related to the pupillary light reflex and that the cortical projections to this pretectal nucleus are related to this reflex and do not play a role in the pupillary near response.

Accommodation, Ocular↗

PubMed1995

Time-resolved fluorescence investigations of the interaction of the voltage-sensitive probe RH421 with lipid membranes and proteins.

Fluorescence lifetimes and fluorescence anisotropy decays of the voltage-sensitive styryl-pyridinium dye RH421 have been measured in the presence of dimyristoylphosphatidylcholine vesicles, the water soluble enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco), and Na+,K(+)-ATPase-containing membrane fragments. The effect of an intramembrane electric field on the photophysical properties of the dye was investigated by the binding of the hydrophobic ion tetraphenylborate (TPB) to the membrane. TPB was found to significantly increase the average fluorescence lifetime and the order of membrane-bound dye. The increase in fluorescence lifetime is consistent with reorientation of the dye further into the membrane interior. The increase in order may be attributed to an electric field-induced alignment of the dye molecules. From the values of the rotational diffusion constant experimentally determined, the expected response time of the dye to an applied electric field can be calculated for a reorientational mechanism to be on the order of tens of nanoseconds. Experiments with rubisco showed that the dye interacts strongly with the protein. In this case, the dye is so tightly bound that it has almost no independent motion and rotates virtually solely with the protein. The rate of rotational motion of the dye in the presence of Na+,K(+)-ATPase-containing membrane fragments is similar to that in pure lipid membranes. The order parameter of the dye in the Na+,K(+)-ATPase membrane fragments is close to the maximum value. This is most probably due to the high density of protein molecules, which restricts the range of motion of the dye.

Binding Sites↗

PubMed1995

Sterkfontein member 2 foot bones of the oldest South African hominid.

Four articulating hominid foot bones have been recovered from Sterkfontein Member 2, near Johannesburg, South Africa. They have human features in the hindfoot and strikingly apelike traits in the forefoot. While the foot is manifestly adapted for bipedalism, its most remarkable characteristic is that the great toe (hallux) is appreciably medially diverged (varus) and strongly mobile, as in apes. Possibly as old as 3.5 million years, the foot provides the first evidence that bipedal hominids were in southern Africa more than 3.0 million years ago. The bones probably belonged to an early member of Australopithecus africanus or another early hominid species.

Animals↗

PubMed1995

Projections of the lateral terminal accessory optic nucleus of the common marmoset (Callithrix jacchus).

The connections of the lateral terminal nucleus (LTN) of the accessory optic system (AOS) of the marmoset monkey were studied with anterograde 3H-amino acid light autoradiography and horseradish peroxidase retrograde labeling techniques. Results show a first and largest LTN projection to the pretectal and AOS nuclei including the ipsilateral nucleus of the optic tract, dorsal terminal nucleus, and interstitial nucleus of the superior fasciculus (posterior fibers); smaller contralateral projections are to the olivary pretectal nucleus, dorsal terminal nucleus, and LTN. A second, major bundle produces moderate-to-heavy labeling in all ipsilateral, accessory oculomotor nuclei (nucleus of posterior commissure, interstitial nucleus of Cajal, nucleus of Darkschewitsch) and nucleus of Bechterew; some of the fibers are distributed above the caudal oculomotor complex within the supraoculomotor periaqueductal gray. A third projection is ipsilateral to the pontine and mesencephalic reticular formations, nucleus reticularis tegmenti pontis and basilar pontine complex (dorsolateral nucleus only), dorsal parts of the medial terminal accessory optic nucleus, ventral tegmental area of Tsai, and rostral interstitial nucleus of the medial longitudinal fasciculus. Lastly, there are two long descending bundles: (1) one travels within the medial longitudinal fasciculus to terminate in the dorsal cap (ipsilateral >> contralateral) and medial accessory olive (ipsilateral only) of the inferior olivary complex. (2) The second soon splits, sending axons within the ipsilateral and contralateral brachium conjunctivum and is distributed to the superior and medial vestibular nuclei. The present findings are in general agreement with the documented connections of LTN with brainstem oculomotor centers in other species. In addition, there are unique connections in marmoset monkey that may have developed to serve the more complex oculomotor behavior of nonhuman primates.

Animals↗

PubMed1995

Luminance neurons in the pretectal olivary nucleus mediate the pupillary light reflex in the rhesus monkey.

In humans and other primates, an increase in luminance in either eye elicits bilateral pupilloconstriction that is essentially equal in both eyes. Current models of the neural substrate for this clinically important light reflex propose that a retinorecipient pretectal nucleus projects bilaterally to the Edinger-Westphal nucleus (EW), which contains the parasympathetic, preganglionic neurons controlling pupilloconstriction. Based on single-unit recording studies in anesthetized cats and rats, it has been further suggested that luminance neurons in only one pretectal nucleus, the pretectal olivary nucleus, mediate this reflex. However, to our knowledge, there have been no comparable electrophysiological studies in primates of the pupillary light reflex or the pretectal luminance neurons that mediate this reflex. To address this issue, single-unit recording and electrical microstimulation studies were carried out in the pretectum of alert, trained, rhesus monkeys. These studies demonstrated that the primate pretectum contains luminance neurons with the characteristics appropriate for mediating the pupillary light reflex and that these neurons are located in one retinorecipient pretectal nucleus, the pretectal olivary nucleus. Electrical microstimulation at the site of these neurons often elicited pupilloconstriction. Our results provide clear evidence for the involvement of the pretectum, and more specifically the pretectal olivary nucleus, in mediating the pupillary light reflex in primates.

Adult↗

PubMed1995

Voltage sensitivity of the fluorescent probe RH421 in a model membrane system.

The voltage sensitivity of the fluorescent styrylpyridinium dye RH421 has been investigated in dimyristoylphosphatidylcholine vesicles by inducing an intramembrane electric field through the binding of the hydrophobic ion tetraphenylborate (TPB). To assess the probability of electrochromic and solvatochromic mechanisms for the dye response, the ground-state dipole moment of the dye in chloroform solution was determined from dielectric constant measurements to be 12 (+/- 1) Debye, and the change in dipole moment upon excitation was calculated from measurements of the Stokes shift in solvents of varying polarity to be 25 (+/- 11) Debye. As well as causing absorbance and fluorescence changes of membrane-bound dye, the TPB-induced electrical field was found to reduce significantly the pKa of the dye. The pH at which experiments are carried out is, thus, an important factor in determining the amplitude of the voltage-induced absorbance and fluorescence changes. The observed absorbance changes induced by the field are inconsistent with a pure electrochromic mechanism. A reorientation/solvatochromic mechanism, whereby the electrical field reorients the dye molecules so that they experience a change in polarity of their lipid environment is likely to make a significant contribution to both the spectral changes and to the field effect on the acid-base properties of the dye.

Biophysical Phenomena↗

PubMed1995

Single-unit activity in the primate nucleus reticularis tegmenti pontis related to vergence and ocular accommodation.

1. In the present study we used single-unit recording techniques in alert rhesus monkeys to investigate a precerebellar nucleus, the nucleus reticularis tegmenti pontis (NRTP), for neurons related to vergence and ocular accommodation. 2. In the medial NRTP, we identified 32 cells with activity that linearly increased with increases in the amplitude of the near response and 33 cells with activity that linearly increased with increases in the amplitude of the far response. These near and far response neurons were often encountered close to neurons displaying saccade-related activity, but their activity was related neither to saccadic nor to smooth pursuit eye movements. Micro-stimulation at the site of near or far response neurons often produced changes in vergence angle and accommodation. 3. The NRTP is known to receive cortical afferents and to have reciprocal connections with the cerebellum; therefore it is likely that the near and far response neurons in the medial NRTP form part of a cerebropontocerebellar pathway modulating or controlling vergence and ocular accommodation.

Accommodation, Ocular↗

PubMed1995

The pupillary light reflex pathway of the primate.

BACKGROUND: Many studies of the pupillary light reflex pathway in mammals have indicated that the pretectum is important for this reflex. However, no single retinorecipient pretectal nucleus has been unequivocally identified as being involved in the light reflex pathway. In this study, anatomical studies in the rhesus monkey were carried out to identify the relevant retinorecipient pretectal nucleus and to better define the central pathway of this reflex. METHODS: An injection of Wheatgerm Agglutinin/Horseradish peroxidase, a neuroanatomical tracer, was placed under physiological guidance into the Edinger-Westphal nucleus. Intravitreal injection of the same tracer in another animal was used to define the pretectal retinal terminal fields. RESULTS: Following injection of tracer in the Edinger-Westphal nucleus, retrogradely labeled cells were found in only one retinorecipient nucleus, the pretectal olivary nucleus. Most labeled cells were located contralateral to the injection site. A few labeled cells were located ipsilaterally. Intravitreal injection of tracer resulted in anterograde labeling of all the retinorecipient pretectal nuclei, including the pretectal olivary nucleus. The retinal terminal field in the pretectal olivary nucleus coincided with the location of the cells that were retrogradely labeled by the injection of tracer into the Edinger-Westphal nucleus. CONCLUSIONS: These results demonstrate that there is a direct projection from the pretectum to the Edinger-Westphal nucleus, that it arises from only one retinorecipient pretectal nucleus, the pretectal olivary nucleus, and that cells in the pretectal olivary nucleus almost all appear to project to the contralateral Edinger-Westphal nucleus.

Animals↗

PubMed1994

Application of gas chromatography-mass spectrometry and gas chromatography-tandem mass spectrometry to the analysis of chemical warfare samples, found to contain residues of the nerve agent sarin, sulphur mustard and their degradation products.

Samples of clothing, grave debris, soil and munition fragments, collected from the Kurdish village of Birjinni, were analysed by GC-MS with selected ion monitoring (SIM) for traces of chemical warfare agents and their degradation products. Positive analyses were confirmed, where possible, by full scan mass spectra, or at low concentrations by additional GC-MS-SIM analysis using chemical ionisation, by higher resolution GC-MS-SIM, and by GC-tandem mass spectrometry using multiple reaction monitoring. Sulphur mustard and/or thiodiglycol were detected in six soil samples; isopropyl methylphosphonic acid and methylphosphonic acid, the hydrolysis products of the nerve agent sarin, were detected in six different soil samples. Trace amounts of intact sarin were detected on a painted metal fragment associated with one of these soil samples. The results demonstrate the application of different GC-MS and GC-MS-MS techniques to the unequivocal identification of chemical warfare agent residues in the environment at concentrations ranging from low ppb to ppm (w/w). They also provide the first documented unequivocal identification of nerve agent residues in environmental samples collected after a chemical attack.

Chemical Warfare Agents↗

PubMed1993

Static and dynamic studies of the potential-sensitive membrane probe RH421 in dimyristoylphosphatidylcholine vesicles.

The dynamics of the potential-sensitive styryl dye RH421 in dimyristoylphosphatidylcholine vesicles have been investigated above and below the main phase transition temperature using iodine-laser temperature-jump relaxation spectrophotometry and time-resolved fluorescence lifetime measurements. Equilibrium fluorescence titrations have shown that the affinity of the dye for the membrane is much higher in the liquid-crystalline state than in the gel state. The interaction can be described by either a partition or a binding model and a theory is presented providing a relation between these two approaches. In the liquid-crystalline state bound dye exhibits steady-state fluorescence relaxation processes in the submicrosecond and millisecond time range following a temperature jump. Time-resolved fluorescence measurements show a variation in the fluorescence lifetime across the emission spectrum, suggesting an excited-state process occurring on the subnanosecond time scale. These processes are most likely related to dye and/or lipid reorientation following the temperature jump or excitation pulse. Temperature-dependent changes in the fluorescence excitation spectrum of bound dye suggest that the dye exists in at least two different sites within the membrane.

Calorimetry, Differential Scanning↗

PubMed1993

A theoretical description of non-steady-state diffusion of hydrophobic ions across lipid vesicle membranes including effects of ion-ion interactions in the aqueous phase.

A theoretical model of hydrophobic ion diffusion across vesicular membranes is presented, which is based upon activated rate theory. The model is applicable to the sudden addition of hydrophobic ions to a vesicle suspension, for example in a stopped-flow experiment. The time course of diffusion is calculated by numerical integration of differential rate equations for the ion concentrations and electrical potential differences across the membrane. The model utilizes the three-capacitor model of the membrane and an extended Debye-Hückel theory, taking into account non-neutrality on each side of the membrane. At low ionic strengths good agreement is found between the infinite time diffusion potential and the equilibrium Nernst potential. At large excess of inert electrolyte discrepancies are found, but under such conditions the membrane potential is negligible due to screening.

Cell Membrane↗

PubMed1993

ACE inhibition versus calcium antagonism in the treatment of mild to moderate hypertension: a multicentre study. Ireland-Netherlands Lisinopril-Nifedipine Study Group.

The efficacy of lisinopril 10-40 mg once daily was compared with that of nifedipine tablets 20-40 mg twice daily in a multicentre double-blind randomized parallel group study of 16 weeks duration involving 127 patients with mild to moderate hypertension. The groups randomized to lisinopril or to nifedipine were not significantly different with respect to any demographic variable. An analysis of the pooled data from all centres demonstrated a significantly greater fall in both lying and standing systolic blood pressure (SBP) on lisinopril than on nifedipine treatment (difference between treatments 7.70 +/- 3.34 mmHg; P = 0.02 and 10.2 +/- 3.30 mmHg; P = 0.003 for lying and standing SBP, respectively). However, this difference may be accounted for by the slightly higher mean SBP in the lisinopril treatment groups compared with the nifedipine group at the end of the placebo run-in period. Both treatments lowered lying and standing diastolic blood pressures (DBP) to the same extent and the response rates to the two treatments were the same. The effects of the two drugs on heart rate were indistinguishable from each other. There were six lisinopril and 12 nifedipine-treated patients withdrawn during randomized treatment (P = 0.22). Nineteen per cent of lisinopril patients reported an adverse event compared with 36% of nifedipine patients. The relative risk of an adverse event on lisinopril compared with nifedipine was 0.42 (confidence limits 1.027-0.172) a difference which approached statistical significance (P = 0.0573). Lisinopril produced a greater reduction in both lying and standing SBP than nifedipine and both were associated with equivalent reductions in DBP. Lisinopril may be better tolerate than nifedipine.

Adolescent↗

PubMed1993

Metabolism of thiodiglycol (2,2'-thiobis-ethanol): isolation and identification of urinary metabolites following intraperitoneal administration to rat.

1. The metabolism of thiodiglycol, 2,2'-thiobis-ethanol, was investigated following i.p. administration to rat. 2. Approximately 90% of the administered dose was excreted in the 0-24-h urine. Four metabolites were isolated by h.p.l.c. and identified by mass spectrometry. Structural assignments were confirmed by comparison with authentic synthetic standards. 3. Thiodiglycol sulphoxide was the major metabolite accounting for approximately > or = 90% of the excreted radioactivity following i.p. injection of 13C4, 35S-thiodiglycol. Thiodiglycol sulphone, S-(2-hydroxyethylthio)acetic acid and S-(2-hydroxy-ethylsulphinyl)acetic acid were identified as minor metabolites. 4. Analysis for thiodiglycol by GC-MS indicated approximately 0.5-1% of the applied dose was excreted unmetabolized.

Animals↗