Biomedical subjects

R J Clarke

Publications and source records attributed to R J Clarke.

At least 73 records · Page 4Linked to original sources

PubMed1992

Spectroscopic investigations of the potential-sensitive membrane probe RH421.

The absorbance spectra, fluorescence emission and excitation spectra, and fluorescence anisotropy of the potential-sensitive styryl dye RH421 have been investigated in aqueous solution and bound to the lipid membrane. The potential-sensitive response of the dye has been studied using a preparation of membrane fragments containing a high density of Na+, K(+)-ATPase molecules. In aqueous solution the dye is sensitive both to changes in pH and ionic strength. Evidence has been found that the dye readily aggregates in aqueous solution. Aggregation is enhanced by an increase in ionic strength. The aggregates formed display a low fluorescence intensity. At high pH values (above approx. 8) changes in the dye's fluorescence spectra are observed, which may be due to a reaction of the dye with hydroxide ions. When bound to the membrane the dye also exhibits concentration-dependent fluorescence changes. The potential-sensitive response of the dye in Na(+),K(+)-ATPase membrane fragments after addition of MgATP in the presence of Na+ ions cannot be explained by a purely electrochromic mechanism. The results are consistent with either a potential-dependent equilibrium between membrane-bound dye monomers and membrane-bound dimers, similar to that previously proposed for the dye merocyanine 540, or with a field-induced structural change of the membrane.

Fluorescence Polarization↗

PubMed1992

GABAergic and non-GABAergic projections of accessory optic nuclei, including the visual tegmental relay zone, to the nucleus of the optic tract and dorsal terminal accessory optic nucleus in rat.

This study examines the non-gamma-amino butyric acid (GABA)ergic (group I neurons) and GABAergic neurons (group II neurons) of the accessory optic system projecting to the nucleus of the optic tract (NOT)/dorsal terminal nucleus (DTN) of the accessory optic system in rat. These nuclei include the dorsal (MTNd) and ventral (MTNv) divisions of the medial terminal nucleus, the lateral terminal nucleus, the interstitial nucleus of the superior fasciculus, the posterior fibers, and the visual tegmental relay zone. GABAergic neurons of these nuclei that do not target the NOT/DTN (group III neurons) have also been observed. The fluorescent retrograde tracer fluoro-gold was injected into the pretectum, targeting the NOT/DTN and the tissue prepared immunocytochemically to reveal neurons containing the neurotransmitter GABA. Three groups of neurons (groups I, II, and III neurons) were examined in terms of their distribution, density, and percentage present. Group I neurons are single-labeled with fluoro-gold and represent non-GABAergic neurons projecting to the NOT/DTN. These neurons are of the highest density in the lateral terminal nucleus (204 neurons/mm2). Their densities are also substantial in the MTNv (120 neurons/mm2), interstitial nucleus of the superior fasciculus, posterior fibers (96 neurons/mm2), and visual tegmental relay zone (93 neurons/mm2). Group II neurons are double-labeled with fluoro-gold and GABA. They form a system of GABAergic neurons projecting to the NOT/DTN, which are exceedingly dense in the MTNd (78 neurons/mm2) but are also dense in both the visual tegmental relay zone (49 neurons/mm2) and MTNv (33 neurons/mm2). Group III neurons are GABAergic neurons that do not target the NOT/DTN but must project to other brain nuclei and/or be interneurons. These are of extremely high concentration in the visual tegmental relay zone (316 neurons/mm2) and are also of substantial densities in the MTNd (77 neurons/mm2), lateral terminal nucleus (72 neurons/mm2), and MTNv (44 neurons/mm2). The MTNd has the highest percentage of GABAergic neurons projecting to the NOT/DTN (72%). GABAergic neurons also form significant percentages of the projections to the NOT/DTN from the visual tegmental relay zone (34%) and MTNv (21%). The percentage of the total GABAergic neurons that project to the NOT/DTN is the highest in the MTNd (50%) and MTNv (42%). The described GABAergic afferents to the NOT/DTN may function to process information concerned with the compensation for retinal slip.

Animals↗

PubMed1992

Transcallosally evoked responses in the visual cortex of normal and monocularly enucleated rabbits.

In visual cortex of normal adult rabbits, callosal projections are restricted to a 2 mm wide band at the area 17/18 border. In adult rabbits which are monocularly enucleated (ME) on the day of birth, the callosal zone extends 4 mm into the medial region of area 17 in the cortex ipsilateral to the remaining eye. In this study, the function of these anomalous callosal projections in ME rabbits was investigated using electrophysiological techniques. A microelectrode was placed in the visual cortex ipsilateral to the enucleated eye at the 17/18 border, bipolar stimulating electrodes were placed in a homotopic location in the contralateral cortex, and averaged evoked responses (AERs) to stimulation were recorded. The stimulating electrodes were then moved mediolaterally in 1 mm steps, and the AERs were recorded for each location of the stimulating electrodes. In the normal rabbit, a maximal short latency evoked response was recorded when the stimulating electrodes were at a location homotopic to the recording electrode. When the stimulating electrodes were moved a distance of 1 mm or more from this optimal position, this short latency response was either absent or dramatically decreased in amplitude, reflecting the precise topographic pattern of the normal callosal projection. In contrast, in ME rabbits, a consistent response was evoked at the 17/18 border when the stimulating electrodes were moved as much as 3 mm medial to the homotopic position. Since antidromically activated responses and both pre- and postsynaptic orthodromically activated responses contribute to the AER, recordings were also made from single cells in some animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

The effects of monocular enucleation on visual topography in area 17 in the rabbit.

The effects of neonatal monocular enucleation on the topographic representation of the ipsilateral visual field in the visual cortex of the rabbit were investigated, using electrophysiological recordings of multi-unit activity in area 17. Topography of receptive fields was determined in normal adult rabbits, adult rabbits monocularly enucleated on the day of birth and adult rabbits monocularly enucleated as adults. In normal rabbits and in adult enucleates, the projection from the ipsilateral eye is represented by a strip of cortex extending approximately 2 mm from the 17/18 border. In neonatal enucleates, the width of the area of cortex in which the projection from the ipsilateral eye is represented was approximately twice as large as normal. Visual topography was normal in the superior-inferior axis but was distorted in the nasotemporal axis. Our data suggest that the abnormal topography observed in the visual cortex of neonatally enucleated rabbits may play a major role in shaping the abnormal visual callosal projections observed in these animals. In addition, our data indicate that, following neonatal monocular enucleation, developmental abnormalities in the topography of geniculocortical projections can occur independently of any alteration in the retinogeniculate projection patterns.

Animals↗

PubMed1992

An adsorption isotherm for the interaction of membrane-permeable hydrophobic ions with lipid vesicles.

An adsorption isotherm is presented which describes the association of membrane-permeable hydrophobic ions with lipid vesicles. The theory is based upon the Langmuir adsorption isotherm, but is has been significantly extended to take into account diffusion of ions across the membrane and dissociation into the intravesicular space as well as electrical effects due to the build up of boundary potentials within the membrane. The boundary potentials are calculated according to the three-capacitor model of the lipid membrane. In contrast to the Gouy-Chapman theory, which is only applicable when the charged group is located directly at the membrane-solution interface, the theory presented here allows the charge to be located at any position within the membrane.

Journal Article↗

PubMed1992

Biological fate of sulphur mustard, 1,1'-thiobis(2-chloroethane): isolation and identification of urinary metabolites following intraperitoneal administration to rat.

1. The metabolism of sulphur mustard, 1,1'-thiobis(2-chloroethane), in vivo was investigated following i.p. administration to rat. 2. Approx. 60% of dose was excreted in the 24 h urine. Many metabolites were present; nine have been isolated by h.p.l.c. and characterized by mass spectrometry. Structural assignments were confirmed by comparison with authentic synthetic standards. 3. Some metabolites result from initial hydrolysis of the sulphur mustard, but the majority are formed by conjugation with glutathione. These are further metabolized to N-acetylcysteine conjugates, or to methylthio/methylysulphinyl derivatives by a pathway probably involving beta-lyase, accompanied by oxidation of the mustard sulphur atom to sulphoxide or sulphone. 4. Thiodiglycol sulphoxide, 1,1'-sulphonylbis[2-S(N-acetylcysteinyl)ethane] and 1,1'-sulphonylbis[2-methylsulphinyl)ethane] or 1-methylsulphinyl-2-[2-(methylthio ethylsulphonyl]ethane were the most prevalent metabolites resulting from the three major pathways. Metabolic pathways for the formation of the excretion products are proposed.

Animals↗

PubMed1991

Suppression of thromboxane A2 but not of systemic prostacyclin by controlled-release aspirin.

BACKGROUND: The antithrombotic efficacy of aspirin is attributed to its inhibition of the enzyme prostaglandin G/H synthase, which is necessary for the formation of thromboxane A2 in platelets. Thromboxane A2 is a potent vasoconstrictor and platelet agonist. However, the formation of prostacyclin by vascular endothelium also requires prostaglandin G/H synthase, and prostacyclin exerts opposite effects on platelet function and vascular tone. We wanted to see whether controlled-release aspirin would affect the formation of thromboxane A2 but not prostacyclin by reducing the aspirin concentration that reaches the posthepatic circulation. METHODS: A controlled-release formulation containing 75 mg of aspirin, designed to release 10 mg per hour, was developed to inhibit prostaglandin G/H synthase in platelets in the prehepatic circulation. The effects of the controlled-release preparation on plasma levels of aspirin and salicylate, serum levels of thromboxane B2, and urinary dinor metabolites of prostacyclin and thromboxane B2 (measured by gas chromatography-mass spectrometry) were compared with those of conventional immediate-release aspirin in normal volunteers. Prostacyclin release was stimulated by intravenous bradykinin. RESULTS: Steady-state inhibition of serum thromboxane B2 required two to four days and appeared slower with 75 mg of controlled-release than with the same amount of immediate-release aspirin. Maximal inhibition was achieved rapidly by adding a single loading dose of 162.5 mg of immediate-release aspirin to the regimen. Over a 28-day period, suppression of thromboxane A2 with this regimen was comparable to that with immediate-release aspirin taken either as 162.5 mg daily or as 325 mg on alternate days, despite the minimal systemic bioavailability of controlled-release aspirin. Bleeding time was prolonged to a similar degree with each of the three regimens. The five- to sixfold increase in the prostacyclin metabolite induced by bradykinin was depressed by pretreatment for four days with 75 mg of immediate-release aspirin, but not by 75 mg of controlled-release aspirin. CONCLUSIONS: Maximal inhibition of platelet thromboxane A2 production was sustained during long-term dosing with controlled-release aspirin, whereas basal prostacyclin biosynthesis fell only slightly and systemic synthesis of prostacyclin stimulated by bradykinin was preserved. Controlled-release aspirin may facilitate determination of the clinical importance of preserving prostacyclin during platelet inhibition in humans.

Adolescent↗

PubMed1991

Analysis of 1,1'-sulphonylbis[2-(methylsulphinyl)ethane] and 1-methylsulphinyl-2-[2-(methylthio)ethylsulphonyl]ethane, metabolites of sulphur mustard, in urine using gas chromatography-mass spectrometry.

A method has been developed for the detection of 1,1'-sulphonylbis[2-(methylsulphinyl)ethane] and 1-methylsulphinyl-2-[2-(methylthio)ethylsulphonyl]ethane, which have been identified as urinary metabolites of sulphur mustard in the rat. The two metabolites were reduced to the single analyte 1,1'-sulphonylbis[2-(methylthio)ethane] by treatment of urine with acidic titanium trichloride. 1,1'-Sulphonylbis[2-(methylthio)ethane] was readily extracted from urine by passing through a C8 reversed-phase extraction column, or by solvent extraction from a solid absorbent tube, and detected by gas chromatography-mass spectrometry using ammonia positive ion chemical ionisation. The limit of detection was 2 ng/ml for 1-ml samples of urine. There were no background levels of analyte in human or rat urine. If man metabolises sulphur mustard by a similar pathway, the detection of these metabolites should constitute firm evidence of an exposure to sulphur mustard.

Animals↗

PubMed1991

Binding and diffusion kinetics of the interaction of a hydrophobic potential-sensitive dye with lipid vesicles.

The interaction of the dye oxonol V with unilamellar dioleoylphosphatidylcholine vesicles has previously been investigated using a fluorescence stopped-flow technique. It has been found that the most suitable mathematical description of the equilibrium and kinetic data is obtained by assuming the presence of saturable dye binding sites in both monolayers of the vesicle membrane and a potential-dependent diffusion across the membrane interior between these two classes of sites. A kinetic model is presented which takes into account the degree of saturation of the binding sites, the degree of fluorescence quenching within the membrane, and the production of an electrical potential gradient across the membrane interior by the binding of the negatively charged dye. The model successfully predicts the time course of the fluorescence change due to binding and diffusion over the complete range of dye and vesicle concentrations as well as the fluorescence response of the dye to changing membrane potential.

Computer Simulation↗

PubMed1991

Indapamide: a diuretic of choice for the treatment of hypertension?

In recent years, the therapeutic approach to mild hypertension has evolved from a stepped care approach to one including varied, initial monotherapies, with the selection of drug based on factors present in an individual patient, followed by combination therapy. The greatest cumulative experience of the value of antihypertensive therapy has been obtained with diuretics. The reduction in risk of cardiovascular disease with successful antihypertensive therapy using conventional agents, including diuretics, is not as great as might have been anticipated by the magnitude of change in blood pressure. This could be due to antecedent cardiovascular injury prior to therapy or to risk factors induced by therapy. The recent introduction of a new generation of drugs with combined diuretic and hypotensive effects that reduce blood pressure without inducing the biochemical changes associated with thiazides, offers an opportunity to evaluate this question. Indapamide is the first of this new generation to be released. Its pharmacologic characteristics show that it has the prerequisites to be considered a first-line drug for use in hypertension. It meets the requirements of a Phase II or second generation drug in that it has similar efficacy, but less short term toxicity than first generation drugs. It has been introduced at a cost that is competitive with thiazides and potassium-sparing combination drugs and is cheaper than many other classes of antihypertensive drugs. The major caution in promoting its use is that its long term effect on morbidity and mortality in hypertensive disease has not yet been evaluated. In summary, this is a promising new drug that has the potential to replace first generation thiazides in the routine management of hypertension.

Blood Glucose↗

PubMed1991

Combined administration of aspirin and a specific thrombin inhibitor in man.

BACKGROUND: Heparin is of limited value as an antithrombotic drug in the presence of platelet activation and residual thrombus. Greater anticoagulant activity can be achieved in vivo with more specific thrombin inhibitors. Heparin may also increase the risk of bleeding by an effect on platelets that is independent of its thrombin inhibitory activity. METHODS AND RESULTS: The pharmacodynamic and pharmacokinetic effects of a novel thrombin inhibitor, argatroban, were examined alone and in combination with aspirin in normal male volunteers. Argatroban induced a dose-dependent prolongation of the thrombin time and the activated partial thromboplastin time (aPTT). aPTT had returned to its pretreatment value 1 hour after stopping the infusion of argatroban. Six male subjects received an infusion of 1 micrograms/kg/min argatroban after the administration of two doses of 162.5 mg aspirin or a matching placebo. At this dose, aspirin decreased serum thromboxane B2 by a mean of 99% and prolonged the bleeding time (230 +/- 52 versus 320 +/- 113 seconds, p less than 0.01). Argatroban given alone increased thrombin time by 454 +/- 18% and aPTT by 160 +/- 3%. Steady-state plasma concentrations were achieved at 1 hour and declined exponentially with an elimination half-life of 24 +/- 4 minutes. Neither the anticoagulant effects nor the plasma concentrations of argatroban were altered by aspirin. Furthermore, argatroban did not increase the bleeding time when given alone and did not further prolong the bleeding time when combined with aspirin. CONCLUSION: The combination of aspirin and argatroban may prove to be an effective therapeutic strategy in the prevention of coronary thrombosis.

Adult↗

PubMed1990

Opioid receptors in the accessory optic system of the rat: effects of monocular enucleation.

The presence and concentrations of each of the three subtypes of opioid receptors (mu, kappa, and delta) has been studied in the accessory optic nuclei (dorsal, lateral, and medial terminal nuclei and the interstitial nucleus of the superior fasciculus, posterior fibers: DTN, LTN, MTN, and inSFp) in normal young rats with radioligands directed towards each opioid receptor subtype. The changes in mu opioid receptors have also been investigated in monocularly enucleated rats in which one eye was removed and the rats sacrificed at postoperative day (PO) 2, 3, 5, 7, 14, and 30. As the MTN is the only accessory optic nucleus of the rat large enough for semiquantitative evaluation, the mu receptor population of the MTN has been subjected to optical microdensitometric analysis. All four of the accessory optic nuclei (AOS nuclei) are found to contain exceedingly high levels of mu opioid receptor binding with the selective radioligand [3H]-[D-Ala,MePhe4, Gly-ol5] (DAGO), low levels of kappa opioid receptor binding using the radioligand [3H]-[ethylketocyclazocine] (EKC) together with the competing agents [D-Pro4]-morphiceptin and [D-Ser2,Thr6]-Leu-enkephalin, and an absence of delta opioid receptor binding with the radioligand [3H]-[D-Ala2,D-Leu5]-enkephalin (DADLE) combined with the competing agent [D-Pro4]-morphiceptin. Monocular enucleation, as studied on the mu opioid receptor population with this experimental approach, results in virtually a complete loss of mu opioid receptors throughout all four of the contralaterally located AOS nuclei, including both dorsal and ventral subdivisions of the medial terminal nucleus (MTNd,v). Kappa and delta receptors are very few (kappa receptors) or are lacking (delta receptors) in the AOS nuclei, and for this reason, the effects of monocular enucleation on these two opioid receptor subtypes have not been investigated. Monocular enucleation also produces a significant lowering in mu receptor binding in other primary optic nuclei (the lateral geniculate nuclei, nucleus of the optic tract, and superficial layers of the superior colliculus) and in the pars principalis of the medial geniculate nucleus (description of changes in mu receptors in non-accessory optic primary optic nuclei will be considered elsewhere). Microdensitometric study of the MTNd,v shows that the decreased binding of mu receptors in this nucleus is barely detectable (about 6%) at PO2 and rises to 6-15% at PO3. At PO5 receptor loss reaches approximately 62%, whereas at PO7 it is about 81% complete. At PO14 and PO30, the mu receptor loss is nearly complete at around 93%.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1990

The development of the retinal projection to the olivary pretectal nucleus in normal and monocularly enucleated hamsters.

The projection to the olivary pretectal nucleus (OPN) from the contralateral eye is observed on the first day after birth and appears adult-like on postnatal day 5. The ipsilateral projection is present at postnatal day 4, and expands to fill the nucleus overlapping the contralateral projection, though never as dense, between days 6 and 8. Then, in normal hamsters, ipsilaterally projecting fibers retract to the ventral side of the OPN by day 10. However, the dense expanded projection in the dorsal OPN ipsilateral to the remaining eye in monocularly enucleated hamsters persists into adulthood.

Animals↗

PubMed1990

The projection of GABA-ergic neurons of the medial terminal accessory optic nucleus to the pretectum in the rat.

Large numbers of neurons were retrogradely labeled in both the dorsal and ventral medial terminal nucleus (MTN) after fluoro-gold injections into the rat pretectal nucleus of the optic tract/dorsal terminal nucleus (NOT/DTN). Fluorescence immunocytochemistry for GABA in the same brains revealed GABA-positive neurons distributed mainly in the dorsal MTN. Approximately half of all the GABAergic neurons in the MTN were double-labeled. Therefore, GABAergic neurons comprise a significant component of the MTN-NOT/DTN projection which most likely inhibits the pretectal pathway mediating horizontal optokinetic nystagmus.

Animals↗

PubMed1989

Pump current and Na+/K+ coupling ratio of Na+/K+-ATPase in reconstituted lipid vesicles.

A method is described for studying the coupling ratio of the Na+/K+ pump, i.e., the ratio of pump-mediated fluxes of Na+ and K+, in a reconstituted system. The method is based on the comparison of the pump-generated current with the rate of K+ transport. Na+/K+-ATPase from kidney is incorporated into the membrane of artificial lipid vesicles; ATPase molecules with outward-oriented ATP-binding site are activated by addition of ATP to the medium. Using oxonol VI as a potential-sensitive dye for measuring transmembrane voltage, the pump current is determined from the change of voltage with time t. In a second set of experiments, the membrane is made selectively K+-permeable by addition of valinomycin, so that the membrane voltage U is equal to the Nernst potential of K+. Under this condition, dU/dt reflects the change of intravesicular K+ concentration and thus the flux of K+. Values of the Na+/K+ coupling ratio determined in this way are close to 1.5 in the experimental range (10-75 mM) of extravesicular (cytoplasmic) Na+ concentrations.

Animals↗

PubMed1989

A stopped-flow kinetic study of the interaction of potential-sensitive oxonol dyes with lipid vesicles.

The interaction of the dyes oxonol V and oxonol VI with unilamellar dioleoylphosphatidylcholine vesicles was investigated using a fluorescence stopped-flow technique. On mixing with the vesicles, both dyes exhibit an increase in their fluorescence, which occurs in two phases. According to the dependence of the reciprocal relaxation time on vesicle concentration, the rapid phase appears to be due to a second-order binding of the dye to the lipid membrane, which is very close to being diffusion-controlled. The slow phase is almost independent of vesicle concentration, and it is suggested that this may be due to a change in dye conformation or position within the membrane, possibly diffusion across the membrane to the internal monolayer. The response times of the dyes to a rapid jump in the membrane potential has also been investigated. Oxonol VI was found to respond to the potential change in less than 1 s, whereas oxonol required several minutes. This has been attributed to lower mobility of oxonol V within the lipid membrane.

Fluorescent Dyes↗

PubMed1989

Neurons of the medial terminal accessory optic nucleus of the rat are poorly collateralized.

The vast majority of neurons of the rat medial terminal nucleus (MTN) project to the nucleus of the optic tract (NOT), but the MTN also projects to a lesser degree upon a number of other brainstem nuclei controlling optokinetic nystagmus. Because of the diversity of targets of the MTN, it is possible that individual neurons have branched axons that project to two or more brainstem nuclei. The possibility that axons of MTN-NOT neurons collateralize to innervate other MTN targets is examined in the rat with the fluorescent, double-labeling, retrograde tracer technique. Fluoro-Gold was injected into the NOT while Fast Blue was simultaneously injected into each of five other known targets of the MTN: the supraoculomotor-periaqueductal gray; the dorsal cap of the inferior olive; the visual tegmental relay zone; the dorsolateral nucleus of the basal pons; and the superior/lateral vestibular nuclei. Brainstem sections were processed for fluorescence microscopy and the MTN was examined for single- and double-labeled neurons. Results show that virtually all neurons of the MTN (greater than 97.5%), together with neurons in the visual tegmental relay zone immediately surrounding the MTNd, are single-labeled in all paired injections involving the NOT and the other target nuclei. It was found that about 69% of MTN neurons project exclusively to the NOT, 5-10% project to each one of the other nuclei, and 3% of MTN neurons project to more than one target. Based upon cell counts from the fluorescent material, and previous analysis of Nissl-stained serial sections, the findings show that virtually all MTN neurons are projection neurons. It was concluded that the MTN is comprised of independent projection systems, possibly involved in different aspects of generating optokinetic nystagmus.

Amidines↗

PubMed1989

Ganglion cell size and distribution in the retina of the two-toed sloth (Choloepus didactylus L.).

The distribution of ganglion cell densities and sizes was studied in Nissl-stained flat-mount retinae of the two-toed sloth. The area centralis, a weak specialization with low ganglion cell density, is located in the temporal retina close to the center of the eye. The presence of a visual streak was noted. The distribution of different ganglion cell sizes was approximately equal throughout the retina. Although the retinal organization differs from that of the closely related three-toed sloth, the presumed function of retinal specializations in both species is to guide limb movements by permitting visualization of the branch along which the animal is climbing.

Adaptation, Physiological↗