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Biomedical subjects

A Grieve

Publications and source records attributed to A Grieve.

At least 19 recordsLinked to original sources
PubMed2001

Rotavirus encephalopathy: pathogenesis reviewed.

Two cases of rotavirus gastroenteritis associated with neurological involvement, one with encephalitis (defined by abnormal neurological signs, cerebrospinal fluid (CSF) pleocytosis and detection of rotavirus genomic nucleic acid in the CSF) and one with a non-inflammatory encephalopathy (defined by abnormal neurological signs, an entirely normal CSF and detection of rotavirus genomic nucleic acid in the CSF), are presented and used as a basis to review and explore potential pathogenetic mechanisms, including direct viral replication within neurons and indirect effects of the newly described rotavirus 'enterotoxin'.

Acyclovir↗
PubMed2000

Preliminary evaluation of an in vitro test for assessment of excitotoxicity by measurement of early gene (c-fos mRNA) levels.

Using primary cultures of mouse cerebellar granule cells as an in vitro model system, it has been demonstrated that different profiles of temporal expression of the c-fos proto-oncogene are observed under non-excitotoxic and excitotoxic conditions. A ratio has been derived previously for the steady-state level of c-fos mRNA after 30 min and 240 min which suggests that a 240 min/30 min ratio of greater than 1 correlates with excitotoxicity, whereas a ratio of less than 1 correlates with a non-excitotoxic outcome. Moreover, a positive correlation is seen with abrogation of excitotoxicity in response to selective excitatory amino acid receptor antagonists. This test, proposed as a specific biomarker for excitotoxicity is undergoing prevalidation. Excitotoxicity is defined as neuronal cell death mediated by hyperactivation of glutamate receptor subtypes and therefore might be expected to be prevented by selective glutamate receptor antagonists. In preliminary evaluation studies, we have conducted work under the direction of the European Center for Validation of Alternate Methods (ECVAM) using compounds specified by ECVAM that have been subdivided into four groups based on known or presumed actions. These groups comprise: Group 1-endogenous/synthetic excitotoxins; Group 2-environmental, putative excitotoxins; Group 3-neurotoxic but non-excitotoxic compounds, and Group 4-non-toxic compounds. The results of this study support the proposal that the c-fos mRNA time-ratio test is a specific biomarker of excitotoxicity. Just as importantly, this test has the potential for application in screening newly-designed EAA receptor antagonists in the search for clinically relevant drugs to treat a variety of neuropathologies.

Animals↗
PubMed1998

Calcium influx via L-type voltage-gated channels mediates the delayed, elevated increases in steady-state c-fos mRNA levels in cerebellar granule cells exposed to excitotoxic levels of glutamate.

The altered kinetics of steady-state c-fos mRNA production in cultured cerebellar granule cells under excitotoxic conditions was investigated in neurons subjected to depolarising stimuli, namely, high KCl and L-glutamate (Glu), in which Ca2+ influx occurs by differing routes. Increases in intracellular-free calcium levels ([Ca2+]i) stimulated by nontoxic or toxic levels of Glu were blocked by selective N-methyl-D-aspartate (NMDA) receptor antagonism; were blocked only partially by the L-type channel blocker, nifedipine; and were unaffected by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate receptor antagonists. Glu-induced cell death was prevented only by NMDA receptor blockade. Exposure of cells to nontoxic levels of Glu resulted in a transient increase in c-fos mRNA levels, whereas an excitotoxic dose produced a delay in the appearance of c-fos mRNA but a subsequent, progressive, and sustained (>4 hr) increase. An excitotoxic dose of Glu in combination with either nifedipine or selective NMDA receptor antagonists resulted in the normal, transient increase of c-fos mRNA levels. Chronic exposure to 55 mM KCl caused no cytotoxicity, although it resulted in a delayed, elevated increase in c-fos mRNA levels that was unaffected by NMDA receptor blockade but reverted to the normal, transient profile of c-fos mRNA formation when it was coadministered with nifedipine. The KCl-induced increase in [Ca2+]i levels was inhibited dramatically by nifedipine but was unaffected by any of the ionotropic Glu receptor antagonists. The results support the notion that the appearance of a delayed but elevated increase in steady-state c-fos mRNA levels following exposure to excitotoxic doses of Glu is mediated specifically by calcium influx via L-type voltage-gated channels.

Animals↗
PubMed1998

Estimating treatment effects in clinical crossover trials.

Some current approaches to modeling crossover trials in two treatments are critically reviewed from the perspective of the practical requirements of the drug developer. Particular attention is paid to the AB/BA design, and the inadequacies of the once popular two-stage procedure are discussed in detail. The use of baseline data is also examined. Both frequentist and Bayesian alternatives to approaches currently advocated are considered and critically compared. It is concluded that it is crucial for the applied statistician working in this field to have an appreciation of the practical medical and pharmacological background.

Algorithms↗
PubMed1997

Caring amidst technology.

Nurses have sometimes allowed the medical model and its associated technology to dominate their practice at the expense of patient care. But change is possible and patients can again become the central focus of care.

Empathy↗
PubMed1996

NMDA receptor-mediated cGMP synthesis in primary cultures of mouse cerebellar granule cells appears to involve neuron-astrocyte communication with NO operating as the intercellular messenger.

The possibility that neuron-astrocyte communication may be responsible for glutamate (Glu)-stimulated cGMP formation even in relatively homogeneous primary cultures of mouse cerebellar granule cells (7 days in vitro) was investigated. Pharmacological analysis using selective excitatory amino acid (EAA) receptor antagonists showed that cGMP production, stimulated in these cultures by Glu and a variety of endogenous EAAs structurally-related to Glu (namely, L-aspartate, L-cysteine sulphinate, L-homocysteate, S-sulpho-L-cysteine), was mediated wholly by N-methyl-D-aspartate (NMDA) receptor activation. Moreover, EAA-induced responses were dependent on the presence of extracellular calcium but unaffected by addition of the L-type voltage-sensitive calcium channel blockers nifedipine (10 microM) or verapamil (5 microM). The mode of calcium entry was also shown to be important since the calcium ionophore, A23187 (10 microM), was unable to stimulate cGMP levels above basal. cGMP formation was blocked by the competitive nitric oxide synthase inhibitor, L-NG-nitroarginine (100 microM), consistent with a role of nitric oxide (NO) in this signalling pathway. In the presence of added haemoglobin (1 microM), acting as a membrane-impermeable NO scavenger, Glu-stimulated cGMP formation was abolished implying that NO must act as an intercellular messenger. When the neuronal population was destroyed following a 24 hr exposure to the excitotoxin, S-sulpho-L-cysteine (200 microM), Glu-stimulated cGMP formation was abolished; whereas responses to the NO donor, sodium nitroprusside (SNP), although markedly reduced were still double that stimulated by Glu in the absence of the excitotoxin, suggesting the presence of non-neuronal cells that can generate cGMP if supplied directly with NO. Consistent with this suggestion, low levels of the glial specific enzyme, glutamine synthetase, were detected in granule cell cultures. Furthermore, omission or delayed addition of the antimitotic agent, cytosine arabinoside (20 microM), to the growth medium caused a significant increase in the level of Glu-stimulated cGMP formation.

Animals↗
PubMed1996

Glutamate toxicity in primary cerebellar cultures from mouse brain is unaffected by changes in cGMP levels.

During evaluation of potential end-points for in vitro neurotoxicity screening we investigated what influence changes in cyclic GMP (cGMP) levels might exert on the degree of glutamate (Glu)-induced neurotoxicity in primary cultures of mouse cerebellar granule cells. Depletion of Glu-stimulated cGMP levels by N-methyl-D-aspartate receptor antagonists fully protected against Glu-induced toxicity. However, when Glu-stimulated cGMP levels were either depleted or elevated by the use of a variety of pharmacological agents acting intracellularly at various points of the NO/cGMP signalling pathway the degree of cytotoxicity exerted by Glu was unaltered. These results imply that cGMP and NO do not modulate the toxic effects of Glu and are therefore unsuitable as biomarkers of excitotoxicity in these cells.

Animals↗
PubMed1996

A prototypic intracellular calcium antagonist, TMB-8, protects cultured cerebellar granule cells against the delayed, calcium-dependent component of glutamate neurotoxicity.

The effect(s) of a prototypic intracellular Ca2+ antagonist, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), on glutamate-induced neurotoxicity was investigated in primary cultures of mouse cerebellar granule cells. Glutamate evoked an increase in cytosolic free-Ca2+ levels ([Ca2+]i) that was dependent on the extracellular concentration of Ca2+ ([Ca2+]o). In addition, this increase in [Ca2+]i correlated with a decrease in cell viability that was also dependent on [Ca2+]o. Glutamate-induced toxicity, quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining, was shown to comprise two distinct components, an "early" Na+/Cl(-)-dependent component observed within minutes of glutamate exposure, and a "delayed" Ca(2+)-dependent component (ED50 approximately 50 microM) that coincided with progressive degeneration of granule cells 4-24 h after a brief (5-15 min) exposure to 100 microM glutamate. Quantitative analysis of cell viability and morphological observations identify a "window" in which TMB-8 (at > 100 microM) protects granule cells from the Ca(2+)-dependent, but not the Na+/Cl(-) -dependent, component of glutamate-induced neurotoxic damage, and furthermore, where TMB-8 inhibits glutamate-evoked increases in [Ca2+]i. These findings suggest that Ca2+ release from a TMB-8-sensitive intracellular store may be a necessary step in the onset of glutamate-induced excitotoxicity in granule cells. However, these conclusions are compromised by additional observations that show that TMB-8 (1) exhibits intrinsic toxicity and (2) is able to reverse its initial inhibitory action on glutamate-evoked increases in [Ca2+]i and subsequently effect a pronounced time-dependent potentiation of glutamate responses. Dantrolene, another putative intracellular Ca2+ antagonist, was completely without effect in this system with regard to both glutamate-evoked increases in [Ca2+]i and glutamate-induced neurotoxicity.

Animals↗
PubMed1995

Modulation by ionotropic excitatory amino acids and potassium of (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid-stimulated phosphoinositide hydrolysis in mouse cerebellar granule cells.

The effect of ionotropic excitatory amino acids and potassium on the formation of inositol phosphates elicited by the metabotropic glutamate receptor agonist (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) was studied in mouse cerebellar granule cells. In Mg(2+)-containing buffers, NMDA (50-100 microM), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA; 10-1,000 microM), and high potassium (10-30 mM) enhanced synergistically the response to a maximally effective concentration of 500 microM trans-ACPD. Potentiation of the trans-ACPD response was blocked by higher concentrations of NMDA (> 500 microM) and potassium (> 35 mM) but not by AMPA (up to 1 mM). The potentiation by NMDA of the trans-ACPD-stimulated phosphoinositide hydrolysis was blocked by D,L-2-amino-5-phosphonopentanoic acid (APV), a competitive NMDA-receptor antagonist. Under Mg(2+)-free conditions, the accumulation of inositol phosphates in the presence of trans-ACPD alone was equal to that attained by trans-ACPD in Mg(2+)-containing buffers when costimulated with maximally enhancing concentrations of NMDA (50 microM). trans-ACPD potentiated synergistically the NMDA-evoked increases in cytosolic free-Ca2+ levels in Mg(2+)-containing but not in Mg(2+)-free solutions, and moreover did not enhance the AMPA-evoked increases in cytosolic free-Ca2+ levels. The calcium ionophore A23187 caused a dose-dependent increase in inositol phosphate accumulation but did not enhance the response stimulated by trans-ACPD alone. These results demonstrate the existence of cross talk between metabotropic and ionotropic glutamate receptors in cerebellar granule cells. The exact mechanism remains unclear but appears to involve interplay of G protein-coupled phospholipase C activation and regulated elevation of cytosolic free-Ca2+ levels. This study may provide a framework for future investigations at the cellular and molecular level that clarify the functional relevance and molecular mechanisms that are described.

Animals↗
PubMed1994

Mixed actions of TMB-8 as a Ca2+ antagonist in cultured mouse cortical neurones.

The action of TMB-8 [8-N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate] on glutamate (Glu)- and 55 mM KCl (K+)-induced increases in intracellular free calcium levels ([Ca2+]i) was studied in cultured mouse cerebral cortical neurones. Glu-induced responses were mediated by two mechanisms, one independent of and the other dependent on extracellular Ca2+, both being inhibited by TMB-8 in a dose-dependent manner. TMB-8 had no effect on [3H]dizocilpine binding kinetics in cortical membrane preparations. The transient and sustained phases of K(+)-induced increases in [Ca2+]i were also inhibited by TMB-8 in a dose-dependent manner. The Ca2+ channel antagonist, verapamil, blocked K(+)-induced responses but had no effect on Glu-induced responses. Although TMB-8 displays multiple effects, its action at voltage-gated Ca2+ channels does not appear to contribute significantly to its inhibition of the Glu-induced increase in [Ca2+]i.

Animals↗
PubMed1994

L-trans-pyrrolidine-2,4-dicarboxylate and cis-1-aminocyclobutane-1,3-dicarboxylate behave as transportable, competitive inhibitors of the high-affinity glutamate transporters.

The ability of two conformationally restricted analogues of L-glutamate to function as non-transportable inhibitors of plasma membrane L-glutamate transport was investigated in primary cultures of cerebellar granule cells and cortical astrocytes. L-trans-Pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC) and cis-1-aminocyclobutane-1,3-dicarboxylic acid (cis-ACBD) behaved as linear competitive inhibitors of the uptake of D-[3H]aspartate (used as a non-metabolizable analogue of L-glutamate) exhibiting Ki values between 40 and 145 microM; L-trans-PDC being the more potent inhibitor in each preparation. However, both L-trans-PDC and cis-ACBD, over a concentration range of 1 microM-5 mM, dose-dependently stimulated the release of exogenously supplied D-[3H]aspartate from granule cells maintained in a continuous superfusion system. The stimulated release was independent of extracellular calcium ions; essentially superimposable dose-response profiles being obtained in the absence and presence of 1.3 mM CaCl2 and yielding EC50 values of 16-25 microM and 180-220 microM for L-trans-PDC and cis-ACBD, respectively. Stimulated release of D-[3H]aspartate was unaffected by either 300 microM D-(-)-2-amino-5-phosphonopentanoic acid [D-APV; a selective antagonist of the N-methyl-D-aspartate (NMDA) receptor] or by 25 microM 6-cyano-7-nitroquinoxaline-2,3-dione [CNQX; a selective antagonist of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor]. The release of D-[3H]-aspartate following stimulation by either L-trans-PDC or cis-ACBD was however markedly attenuated following substitution in the superfusion medium of sodium ions by choline ions. Taken together, these results support an action of L-trans-PDC and cis-ACBD consistent with that of being competitive substrates rather than non-transportable blockers of the plasma membrane L-glutamate uptake system.

Amino Acid Transport System X-AG↗
PubMed1992

Neuronal and glial plasma membrane carrier-mediated uptake of L-homocysteate is not selectively blocked by beta-p-chlorophenylglutamate.

The proposed action of beta-p-chlorophenylglutamate (chlorpheg) as a selective blocker of L-homocysteate uptake was studied in primary cultures of mouse brain neurons and astrocytes and in rat brain synaptosomes. The preparations were incubated with 1 microM to 10 mM L-homocysteate and D-aspartate in the absence and presence of 7.5 mM chlorpheg. In each preparation uptake of the two amino acids comprised a saturable uptake and a non-saturable (passive diffusion) component. L-Homocysteate was of at least 100-fold lower affinity than D-aspartate as a substrate for the amino acid transporter. Chlorpheg was shown to be essentially equieffective as a weak competitive inhibitor of only the saturable uptake of L-homocysteate and D-aspartate. It is concluded that chlorpheg is not a selective blocker of L-homocysteate uptake.

Amino Acid Transport Systems↗
PubMed1992

Simultaneous measurement by HPLC of the excitatory amino acid transmitter candidates homocysteate and homocysteine sulphinate supports a predominant astrocytic localisation.

Primary cultures of mouse cerebral cortex neurons, cerebellar granule cells and cortical astrocytes were maintained in vitro for respectively 8-10, 7-10 and 21-24 days. Following these times, amino acids were extracted from the cells by use of ice-cold 70% (v/v) ethanol and the extracts lyophilised. The lyophilised extracts when resuspended were subjected to reverse-phase high performance liquid chromatographic (HPLC) analysis for detection of free amino acids. Samples of cell culture growth medium and water blanks were treated in a similar manner. Identification of L-homocysteate (HCA) and L-homocysteine sulphinate (HCSA) was undertaken by matching retention times with regard to external standards and by 'spiking' cell extracts with authentic compounds. On this basis, HCA and HCSA were consistently detectable in astrocytes at levels of, respectively, 72.3 +/- 33.7 pmol/mg protein (n = 24) and 49.4 +/- 28.7 pmol/mg protein (n = 24). However, in neurons, a peak corresponding to HCSA could not be detected above the background noise, while the area of the peak corresponding to HCA was always greater than, but not significantly different from, that of the background noise present in water blanks. HCA and HCSA were not detectable in the serum used for preparation of the cell culture growth medium. Taken together, these findings indicate a predominant localisation of HCA and HCSA in astrocytes which, at least in culture, appear to possess the metabolic machinery necessary for synthesising and storing these amino acids without any neuronal influence.

Amino Acids↗
PubMed1992

Synaptosomal plasma membrane transport of excitatory sulphur amino acid transmitter candidates: kinetic characterisation and analysis of carrier specificity.

The transport kinetics of the excitatory sulphur-containing amino acid (SAA) transmitter candidates, L-cysteine sulphinate (L-CSA), L-cysteate (L-CA), L-homocysteine sulphinate (L-HCSA), and L-homocysteate (L-HCA), together with their plasma membrane carrier specificity, was studied in cerebrocortical synaptosome fractions by a sensitive high performance liquid chromatographic assay. A high affinity uptake system could be demonstrated for L-CSA (Km = 57 +/- 6 microM; Vmax = 1.2 +/- 0.1 nmol/min/mg protein) and L-CA (Km = 23 +/- 3 microM; Vmax = 3.6 +/- 0.1 nmol/min/mg protein), whereas L-HCSA (Km = 502 +/- 152 microM; Vmax = 6.1 +/- 1.3 nmol/min/mg protein) and L-HCA (Km = 1550 +/- 169 microM; Vmax = 10.3 +/- 1.1 nmol/min/mg protein) exhibited much lower affinity as transport substrates. In all cases, only a single, saturable Na(+)-dependent component of uptake could be identified, co-existing with a non-saturable, Na(+)-independent influx component. Plasma membrane carrier specificity of the SAAs was established following comparison with other high-affinity neurotransmitter systems. High-affinity L-CSA and L-CA transport and low-affinity L-HCSA and L-HCA transport demonstrate strong positive correlations in inhibition profiles when compared against each other or individually against the high-affinity transport of L-[3H]glutamate, L-[3H]aspartate, or D-[3H]aspartate. Moreover, the transport systems for the excitatory SAAs exhibited a negative correlation when compared in inhibition profiles with the high affinity transport of both [3H] gamma-aminobutyric acid (GABA) and [3H]taurine.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1992

Sulphur-containing excitatory amino acid-evoked Ca(2+)-independent release of D-[3H]aspartate from cultured cerebellar granule cells: the role of glutamate receptor activation coupled to reversal of the acidic amino acid plasma membrane carrier.

Sulphur-containing excitatory amino acid transmitter candidates (500 microM) stimulated the Ca(2+)-independent efflux of exogenously-supplied D-[3H]aspartate from primary cultures of cerebellar granule cells superfused continuously with HEPES-buffered saline containing CoCl2 (1 mM) in place of CaCl2. The stimulated release of D-[3H]aspartate was markedly attenuated by 200 microM 6,7-dinitroquinoxalinedione, a concentration at which the antagonist inhibits both non-N-methyl-D-aspartate and N-methyl-D-aspartate ionotropic excitatory amino acid receptors. The Ca(2+)-independent component of evoked release was also markedly attenuated and, in some cases, abolished by removing NaCl from the superfusion medium. Furthermore, when 700 microM dihydrokainate (demonstrated herein as a mixed/non-competitive inhibitor of the high-affinity dicarboxylic amino acid transporter in cultured granule cells) was included in the superfusion medium, stimulated efflux of D-[3H]aspartate was reduced by between 15-78% of the control response; the extent of inhibition varying with the agonist employed. In constrast, agents which act as competitive inhibitors of the plasma membrane carrier in granule cells, e.g. beta-methylene-D,L-aspartate, potentiated the release of D-[3H]aspartate in a synergistic manner. Taken together, these findings are consistent with a mechanism for the Ca(2+)-independent release of D-[3H]aspartate that is mediated predominantly by activation of excitatory amino acid receptors resulting in a reversal of the high-affinity dicarboxylic amino acid transport system. Although the physiological relevance of such non-vesicular release from the cytosol remains obscure and is still a matter of some debate, this mode of release may be of pathological significance.

Animals↗