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S Rees

Publications and source records attributed to S Rees.

At least 19 recordsLinked to original sources
PubMed2000

The generation and characterization of antagonist RNA aptamers to human oncostatin M.

Oncostatin M (OSM) is a multifunctional member of the interleukin-6 cytokine family. OSM has been implicated as a powerful proinflammatory mediator and may represent a potentially important, novel therapeutic opportunity for treatment of established rheumatoid arthritis. To further investigate the role of OSM in inflammatory disorders, we have isolated a series of RNA aptamers that bind specifically to human OSM. The highest affinity aptamer, designated ADR58, has been characterized in a series of in vitro and cell based assays. ADR58 has an affinity of 7 nm for human OSM, and it can antagonize OSM binding to the gp130 receptor and specifically antagonize OSM mediated signaling. The aptamer has been truncated in length to 33 bases, all pyrimidine positions are substituted with 2' fluorine, and 14 of 18 purine positions have been substituted with 2' O-methyl to increase stability toward nucleases. This truncated, modified form of ADR58 retains complete affinity and functional activity for OSM. This aptamer may be used as a tool to further investigate the role of OSM in inflammatory disorders and may also have role as a therapeutic agent.

Animals↗
PubMed2000

Use of a dual firefly and Renilla luciferase reporter gene assay to simultaneously determine drug selectivity at human corticotrophin releasing hormone 1 and 2 receptors.

The aim of this study was to investigate and validate the use of a dual glow-signal luciferase reporter gene assay to simultaneously evaluate drug activity at two different seven-transmembrane receptor subtypes. Stable cell lines (CHO) transfected with either human corticotrophin releasing hormone 1 (hCRH1) receptors and a firefly luciferase reporter gene or hCRH2 and a Renilla luciferase reporter gene were created to provide different luciferase readouts for CRH1 and CRH2 receptors, respectively. Cells were combined for stimulation and measurement of luciferase luminescence in a 96-well plate format assay. The nonselective CRH agonists rat/human CRH and sauvagine caused concentration-dependent increases in luminescence via activation of CRH1 (firefly luciferase; pEC50 = 8.40 +/- 0.06 and 8.39 +/- 0.08, respectively, n = 8) and CRH2 (Renilla luciferase; pEC50 = 8.89 +/- 0.14 and 8.92 +/- 0.13, respectively, n = 8) receptors. The nonselective CRH antagonist astressin blocked these agonist-induced increases in luciferase at both CRH1 and CRH2 receptors. The selective CRH1 antagonist CP154,526 blocked r/hCRH- and sauvagine-induced increases in luciferase at CRH1 receptors only. These data report the expected pharmacology for CRH1 and CRH2 receptors. This assay enabled two receptor subtypes to be studied simultaneously in the same 96-well plate and generated robust data with low variability. It has the potential advantage of significant time and cost savings, with application to both basic research and compound screening.

Animals↗
PubMed2000

Exposure to prenatal carbon monoxide and postnatal hyperthermia: short and long-term effects on neurochemicals and neuroglia in the developing brain.

The effects of prenatal exposure to carbon monoxide (CO), a major component of cigarette smoke, was studied alone or in combination with postnatal hyperthermia, on the structural and neurochemical development of the postnatal brain at 1 and 8 weeks. Pregnant guinea pigs (n = 11) were exposed to 200 p.p.m CO for 10 h/day from midgestation until term (68 days), whereas control mothers (n = 10) breathed room air. On postnatal day 4, neonates from the control and CO-exposed pregnancies were exposed to hyperthermia (35 degrees C) for 75 min or remained at ambient (23 degrees C) temperature. Using semiquantitative immunohistochemical techniques the following neurotransmitter alterations were found in the medulla at 1 week: a decrease in met-enkephalin-immunoreactivity (IR) following postnatal hyperthermia and an increase in 5-hydroxytryptamine-IR following a combination of CO and hyperthermia. No alterations were observed in substance P- or tyrosine-hydroxylase-IR in any paradigm. At 8 weeks of age the combination of prenatal CO exposure followed by a brief hyperthermic stress postnatally resulted in lesions throughout the brain and an increase in glial fibrillary acidic protein-IR in the medulla. Such effects on brain development could be of relevance in cardiorespiratory control in the neonate and could have implications for the etiology of Sudden Infant Death Syndrome, where smoking and hyperthermia are major risk factors.

Animals↗
PubMed2000

Neurobiological findings in early phase schizophrenia.

This paper summarises the available information on MRI-determined hippocampal morphometry in first-episode patients as an illustration of the value and interpretation of findings in the neurobiology of early phase schizophrenia. We report a thin slice (1.5 mm) study of 32 first episode and 39 high risk patients which demonstrated significantly smaller hippocampi (right -9%, left -11%) in first episode patients that were of a similar magnitude to those found in chronic patients (right -10%, left -11%) but non-significant volume reductions in high risk individuals, including the 15 subjects who subsequently developed psychoses. Consideration is given to the implications of these findings, including the possible role of early and later neurodevelopmental influences. We present animal data showing that chronic placental insufficiency, as elicited by uterine artery ligation can give rise to substantial reduction (31%) in hippocampal volumes and reflect on other potentially relevant pathophysiological mechanisms, including those that may occur during the early phases of psychotic illnesses, including their prodromes. Greater attention needs to be paid to the study of early phase psychosis in order to obtain a clearer understanding of the nature and time course of neurobiological changes associated with it. Although there is a growing literature on first episode psychosis, there is a striking dearth of information on the neurobiology of the prodrome.

Animals↗
PubMed2000

Reduced number of neurons in the hippocampus and the cerebellum in the postnatal guinea-pig following intrauterine growth-restriction.

Intrauterine growth restriction is a risk factor for neurological and behavioural deficits in children although the precise underlying biological correlate for this is unclear. The present study shows that animals with intrauterine growth restriction, induced by a period of reduced placental blood flow during the second half of pregnancy, demonstrate reduced numbers of neurons in the hippocampus and the cerebellum in conjunction with retarded dendritic and axonal growth within these structures. Intrauterine growth restriction was induced at 30 days gestational age by unilateral uterine artery ligation in pregnant guinea-pigs. At one week of age, the total number of CA1 pyramidal neurons in the hippocampus and the Purkinje neurons in the cerebellum were determined using the combined fractionator/optical disector technique. The Cavalieri Principle was used to determine the volume of specific regions within the hippocampus and cerebellum. The body weight of animals that were classified as intrauterine growth-restricted was reduced by 42% (n=8) compared with control animals (n=8, P<0.001), while there was a smaller effect on brain weight (16% reduction, P<0.01). Estimates of the total number of neurons showed a reduction in CA1 pyramidal neurons in growth-restricted animals (4.19+/-0.43x10(5)) compared with control (5.20+/-0.44x10(5), P<0.01), and the volume of the stratum oriens layer above the CA1 region, which contains the apical dendrites of the CA1 pyramidal neurons, was reduced by 21% (P<0.01) in growth-restricted animals. In the cerebellum there was a reduction in the number of Purkinje neurons in growth-restricted animals (3.97+/-0.50x10(5)) compared with control (5.13+/-0.52x10(5), P<0.01), and in the volume of the molecular layer (17%, P<0.05), the internal granular layer (22%, P<0.01) and in the volume of the cerebellar white matter (23%, P<0.01). These results show that a period of placental insufficiency during the second half of pregnancy can effect brain development in a way which could lead to neurological and behavioural deficits in the postnatal animal.

Animals↗
PubMed2000

Localization of metabotropic glutamate receptor type 2 in the human brain.

Metabotropic glutamate receptors are a heterogeneous family of G-protein-coupled receptors that are linked to multiple second messenger systems to regulate neuronal excitability and synaptic transmission. To elucidate the physiological role of these receptors in human central nervous system function and dysfunction at the receptor protein level requires the use of selective antibodies to determine the phenotype of cells expressing particular receptor subtypes. To this end the present study has examined the regional and cellular localization of the metabotropic glutamate type 2 receptor protein in selected human brain regions. After epitope prediction, antibodies have been generated against a short synthetic peptide corresponding to amino acid residues located in the putative intracellular carboxy-terminus and subsequently applied to an immunohistochemical investigation. Antibodies specifically detected the type 2 receptor in transfected mammalian cells and also recognized a major band of 98,000 mol. wt in western blots of human brain tissue membranes. At the light microscope level immunohistochemical studies have demonstrated that type 2-like immunoreactivity was widely distributed in the human brain, being characterized by the presence of a strong immunoreaction in multiple cortical regions, and in structures comprising the basal ganglia, to include the caudate nucleus, putamen, globus pallidus, substantia nigra and subthalamic nucleus. In the hippocampal formation, immunoreactivity was predominant in selective cell layers of both the dentate gyrus and cornu ammonis, the subicular complex and entorhinal cortex. In the thalamus, multiple subnuclei showed reaction product. In the cerebellar cortex, immunoreactivity was expressed in a number of cell layers and cell types. Furthermore, using double immunofluorescence we confirmed that the type 2 receptor is a product of normal resting astrocytes in the cerebral cortex in particular. This antibody provides a new immunological tool with the potential to evaluate the distribution of human metabotropic glutamate receptor 2 protein in other brain regions and in human central nervous system diseases.

Animals↗
PubMed2000

Chronic prenatal exposure to carbon monoxide results in a reduction in tyrosine hydroxylase-immunoreactivity and an increase in choline acetyltransferase-immunoreactivity in the fetal medulla: implications for Sudden Infant Death Syndrome.

Maternal cigarette smoking during pregnancy is associated with a significantly increased risk of Sudden Infant Death Syndrome (SIDS). This study investigated the effects of prenatal exposure to carbon monoxide (CO), a major component of cigarette smoke, on the neuroglial and neurochemical development of the medulla in the fetal guinea pig. Pregnant guinea pigs were exposed to 200 p.p.m CO for 10 h per day from day 23-25 of gestation (term = 68 days) until day 61-63, at which time fetuses were removed and brains collected for analysis. Using immunohistochemistry and quantitative image analysis, examination of the medulla of CO-exposed fetuses revealed a significant decrease in tyrosine hydroxylase-immunoreactivity (TH-IR) in the nucleus tractus solitarius, dorsal motor nucleus of the vagus (DMV), area postrema, intermediate reticular nucleus, and the ventrolateral medulla (VLM), and a significant increase in choline acetyltransferase-immunoreactivity (ChAT-IR) in the DMV and hypoglossal nucleus compared with controls. There was no difference between groups in immunoreactivity for the m2 muscarinic acetylcholine receptor, substance P- or met-enkephalin in any of the medullary nuclei examined, nor was there evidence of reactive astrogliosis. The results show that prenatal exposure to CO affects cholinergic and catecholaminergic pathways in the medulla of the guinea pig fetus, particularly in cardiorespiratory centers, regions thought to be compromised in SIDS.

Animals↗
PubMed2000

Tracing cranial nerve pathways (glossopharyngeal, vagus, and hypoglossal) in SIDS and control infants: a DiI study.

It has been proposed that Sudden Infant Death Syndrome (SIDS) might occur as a consequence of a developmental deficit associated with the cardiorespiratory and arousal control centers located within the brainstem. In this study 1.1' dioctadecyl-3,3,3',3-tetramethylindocarbocyanine perchlorate (DiI) was used to investigate the trajectories of the glossopharyngeal and vagus nerves which carry essential afferent and efferent fiber tracts associated with cardiac and respiratory control and of the hypoglossal nerve which innervates the tongue, in SIDS (n = 14) and control (n = 7) infants. The postnatal development of the trajectories of these nerves was examined in non-SIDS brains and comparisons were then made with age-matched SIDS brains. The mean profile area of hypoglossal and dorsal motor neurons were also assessed. In controls, no major alterations were observed in the trajectories of axon bundles with increasing age (7 wk to 2 yr) in each of the nerves investigated although axon bundles appeared to increase in thickness with age. In SIDS cases (2 wk to 44 wk), the trajectories of the cranial nerves were not different from those seen in age-matched control cases. The mean profile area of hypoglossal and dorsal motor neurons was not significantly different between control and SIDS infants. We conclude that the DiI tracing technique can be used successfully to trace the pathways of cranial nerves in human infant fixed-tissue. Furthermore, if functional differences exist between SIDS and non-SIDS brains in the control of respiration, circulation, or arousal they do not appear to be related to markedly reduced or aberrant projections of the glossopharyngeal, vagus, or hypoglossal nerves.

Brain Stem↗
PubMed2000

Resolution of inverse agonist-induced up-regulation from constitutive activity of mutants of the alpha(1b)-adrenoceptor.

Constitutively active forms of the hamster alpha(1b)-adrenoceptor can be produced from the point mutations Asp(142)Ala or Ala(293)Glu or exchange of a small segment of the third intracellular loop with the equivalent region of the beta(2)-adrenoceptor. Green fluorescent protein (GFP)-tagged forms of each of these mutants and of the wild type alpha(1b)-adrenoceptor were expressed stably in HEK293 cells. The wild type alpha(1b)-adrenoceptor-GFP was expressed both at the plasma membrane and with a distinctly perinuclear punctate pattern. Sustained treatment with a range of antagonist/inverse agonist ligands failed to modulate the cellular distribution or levels of expression of this construct. The form of the alpha(1b)-adrenoceptor containing the beta(2)-adrenoceptor sequence substitution was predominantly located in punctate intracellular vesicles and sustained challenge with the same series of antagonists/inverse agonists produced a 5-fold up-regulation of protein levels with elevation of both plasma membrane and intracellular receptor. Quantification of these effects could be produced by spectrofluorometric analysis of cells grown in a 96-well microtiter plate. In contrast, both the Asp(142)Ala and Ala(293)Glu forms of the alpha(1b)-adrenoceptor-GFP were located predominantly at the plasma membrane. Levels of these two point mutants were not increased by any of the antagonist/inverse agonist ligands tested, although the sequence substitution mutation encompasses codon 293. Resolution of constitutive activity and ligand-induced up-regulation was further exemplified by a mutant lacking eight serine residues in the C-terminal tail that displayed little constitutive activity but was up-regulated by sustained ligand challenge. These results demonstrate the nonequivalence of mutations in their regulation by antagonist/inverse agonist ligands.

Adrenergic alpha-Antagonists↗
PubMed1999

Functional analysis of a human A(1) adenosine receptor/green fluorescent protein/G(i1)alpha fusion protein following stable expression in CHO cells.

Fusion proteins between the human A(1) adenosine receptor and the pertussis toxin resistant (Cys351Gly) mutant of the G-protein alpha subunit G(i1)alpha (A1/Gi), and between the human A(1) adenosine receptor, the Aequorea victoria green fluorescent protein (GFP) and Cys351Gly G(i1)alpha (A1/GFP/Gi), were expressed in CHO cells. The agonist NECA caused a stimulation of [(35)S]GTPgammaS binding at both fusion proteins with similar concentration dependence as at the native receptor. However in the presence of pertussis toxin NECA stimulation of [(35)S]GTPgammaS binding was only seen at the A1/GFP/Gi fusion protein. The regulation of the adenylyl cyclase and MAP kinase effector systems by both fusion proteins was attenuated following pertussis toxin treatment. These studies demonstrate for the first time the characterisation of a fusion protein between a G-protein coupled receptor, GFP and a G-protein alpha subunit.

Animals↗
PubMed1999

Ventriculomegaly and reduced hippocampal volume following intrauterine growth-restriction: implications for the aetiology of schizophrenia.

Structural alterations in the brains of some schizophrenic patients suggest an impairment of brain development, possibly as a result of intrauterine compromise. In this study we have tested the hypothesis that placental insufficiency during the second half of pregnancy in the guinea pig results in structural alterations similar to those seen in some schizophrenic patients. Placental insufficiency was induced in pregnant guinea pigs via uterine artery ligation at midgestation. At 60 days gestation (term: 68 days gestation) the fetal brains were prepared for quantitative histological and immunohistochemical analysis and compared with controls. Placental insufficiency resulted in growth-restricted animals with significantly larger cerebral ventricles, reduced cross-sectional area of the cerebral cortex and the striatum and reduced hippocampal volume compared with controls. There were fewer neuronal nitric oxide synthase (nNOS)-positive cells in layers 5-6 of the cingulate cortex, and in layer 1 of the frontal and temporal cortices. In contrast, there were no significant alterations in the optical density of tyrosine hydroxylase (TH), a rate-limiting enzyme in the biosynthesis of catecholamines and the dopamine transporter (DAT) in the striatum in growth-restricted animals compared with controls. These findings indicate that developmental disturbances can produce anatomical changes that resemble those found in some individuals with schizophrenia.

Animals↗
PubMed1999

Comparative analysis of the efficacy of A1 adenosine receptor activation of Gi/o alpha G proteins following coexpression of receptor and G protein and expression of A1 adenosine receptor-Gi/o alpha fusion proteins.

HEK293T cells were transiently transfected to express either the human A1 adenosine receptor together with pertussis toxin-resistant cysteine-to-glycine forms of the alpha subunits of Gi1 (C351G), Gi2 (C352G), and Gi3 (C351G) and wild-type Go1alpha or fusion proteins comprising the A1 adenosine receptor and these Gi/o G proteins to compare A1 adenosine receptor agonist-mediated activation of these Gi family G proteins upon coexpression of individual Gi/o G proteins and receptor versus expression as receptor-G protein fusion proteins. Addition of the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) to membranes of pertussis toxin-treated cells resulted in a concentration-dependent stimulation of [35S]GTPgammaS binding with comparable amounts of NECA required to produce half-maximal stimulation following transfection of A1 adenosine receptor and Gi/o G proteins either as fusion proteins or as separate polypeptides. However, the magnitude of agonist-mediated activation of GTPgammaS binding was greatly enhanced by expressing the A1 adenosine receptor and Gi family G proteins from chimaeric open reading frames. This observation was consistent following the study of more than 40 agonists. No preferential activation of any G protein was observed with more than 40 A1 receptor agonists following cotransfection of receptor with G protein or transfection of receptor-G protein fusion proteins. These studies demonstrate the utility of using fusion proteins to study receptor-G protein interaction, show that the A1 adenosine receptor couples equally well to the Gi/o G proteins Gi1alpha, G i2alpha, Gi3alpha, and Go1alpha, and demonstrate that for a range of agonists there is no selectivity for activation of any particular A1 adenosine receptor-Gi/o G protein combination.

Adenosine-5'-(N-ethylcarboxamide)↗
PubMed1999

Chimaeric G alpha proteins: their potential use in drug discovery.

Approaches that allow ligand occupancy of a wide range of G protein-coupled receptors to be converted into robust assays amenable to relatively high-throughput analysis are ideal for screening for novel ligands at this class of receptor. Many attempts have been made to design universal ligand-screening systems such that any GPCR can be screened using a common assay end-point. Manipulation of the G protein within the assay system offers the possibility of achieving this. To better understand the domains involved in the interactions between G protein-coupled receptors, G proteins and effector polypeptides and the fine details of these contacts, a wide range of chimaeric G protein alpha subunits have been produced. Graeme Milligan and Stephen Rees discuss the information generated by such studies and the ways in which such chimaeric G proteins can be integrated into assay systems for drug discovery.

Drug Evaluation, Preclinical↗
PubMed1999

Corrigendum

Explore the source record for details and available documents.

Journal Article↗
PubMed1999

Effects of a range of beta2 adrenoceptor agonists on changes in intracellular cyclic AMP and on cyclic AMP driven gene expression in cultured human airway smooth muscle cells.

1. The effects of the selective beta2 adrenoceptor agonists salbutamol, terbutaline and salmeterol and the non-selective beta adrenoceptor agonist isoprenaline on [3H]-cyclic AMP formation and cyclic AMP response element (CRE) driven luciferase expression, assessed using the construct p6CRE/luc, were studied in primary cultures of human airway smooth muscle (HASM) cells. 2. Optimal transfection conditions for transient expression of pGL3 Control were 4 microg DNA/well71 in a 6 well plate and 1.8 microl Transfectam/microg DNA. Expression was maximal at 48 - 72 h. 3. Salbutamol (maximum response 19%, EC50 0.6 microM), terbutaline (maximum response 38%, EC50 2.3 microM) and salmeterol (maximum response 18%, EC50 0.0012 microM) were all partial agonists for cyclic AMP formation compared with isoprenaline (EC50 0.08 microM). However, all of the beta2 adrenoceptor agonists produced increases in CRE-driven luciferase activity, in cultured HASM transfected with the vector p6CRE/luc, which were equivalent or greater (salmeterol) than those seen with isoprenaline. 4. Both salbutamol and salmeterol were more potent at increasing luciferase expression than in elevating cyclic AMP levels in these cells. The potency ratios (EC50 (cyclic AMP)/EC50 (LUC)) for the agents studied were isoprenaline: 0. 2 fold, terbutaline: 3 fold, salbutamol: 24 fold, salmeterol: 38 fold. 5. These data suggest that important quantitative differences exist in the ability of beta2 adrenoceptor agonists to increase whole cell cyclic AMP levels in airway smooth muscle and to drive gene expression via a CRE-driven mechanism.

Adrenergic beta-2 Receptor Agonists↗
PubMed1999

Reduction in choline acetyltransferase immunoreactivity but not muscarinic-m2 receptor immunoreactivity in the brainstem of SIDS infants.

The cholinergic neurotransmitter system is vital for several brainstem functions including cardiorespiratory control and central chemosensitivity. This study has examined aspects of the cholinergic neurotransmitter system in the brainstem of sudden infant death syndrome (SIDS) and control infants. The cellular localisation and the optical density of the immunoreactivity of the cholinergic enzyme choline acetyltransferase (CHAT-IR) and the muscarinic acetylcholine receptor m2 (m2-IR) in the medulla was described in 14 SIDS and 9 control cases. There was a reduction in the number of CHAT-IR neurons in the hypoglossal nucleus (control: 71.2+/-8.3% vs SIDS: 46.1+/-5.3%) and the dorsal motor nucleus of the vagus (DMV) (control: 77.2+/-5.0% vs SIDS: 52.5+/-7.4%) and reduced optical density of CHAT-IR in the hypoglossal nucleus (control: 0.20+/-0.01 vs SIDS; 0.14+/-0.02) in SIDS infants. In contrast there were no changes in the optical density of m2-IR in the hypoglossal nucleus, the DMV, or the arcuate nucleus. Hypoplasia of the arcuate nucleus was observed in one SIDS infant. These results suggest that there is a specific defect in some cholinergic motor neurons in the medulla of SIDS infants. This could lead to abnormal control of cardiovascular and respiratory function and airway patency and may be one of the contributing factors in the etiology of SIDS.

Brain Stem↗
PubMed1999

Hypoxemia near mid-gestation has long-term effects on fetal brain development.

We tested the hypotheses that an episode of hypoxemia near mid-gestation in fetal sheep has long-term effects on brain development and that the extent and type of damage is related to the stage of development within a particular brain structure at the time of the hypoxemia. Fetal sheep (n = 8) were made hypoxemic at 90 +/- 2 days (term approximately 147 days) by restricting the maternal blood supply to the placenta for 12 hours (h) using a vascular clamp so as to reduce fetal arterial O2 saturation by 50%-60%. Fetuses were killed 35 days later and the brains analysed histologically and immunohistochemically. Age-matched fetuses (n = 8) were used as controls. Gross brain damage was observed in only 1 fetus, the most acidemic during the period of hypoxemia. There was a reduction of 12% (p < 0.05) in the cross-sectional area of the cerebral cortex in hypoxemic fetuses compared with controls. In lobule 6 of the cerebella of hypoxemic fetuses, significant reductions were seen in (a) the volume density of Purkinje cells (33%), (b) the width of the molecular layer (13%), (c) the area of the inner granule cell layer (13%), (d) the area of the white matter (18%), and (e) the total cross-sectional area (15%). There were also significant reductions in the area of arborization of Purkinje cell dendritic trees (50%), in the branching density (25%), and in the number of dendritic spines (31%). In the ventral hippocampi of hypoxemic fetuses, there was a 36% reduction (p < 0.05) in the volume density of CA1 pyramidal cells and a 50% increase (p < 0.05%) in the number of astrocytes. We conclude that an episode of hypoxemia near mid-gestation reduces neuronal numbers in the hippocampus and cerebellum and probably also in the cerebral cortex. The growth of neural processes in a particular region will be significantly retarded if the hypoxemia occurs at an early stage of the growth of neural processes (e.g. cerebellum) but not if development is well advanced at the time of the insult (e.g. hippocampus). Damage is sustained in the white matter of the cerebral hemispheres if the insult is particularly severe. Together, these deficits could affect neural connectivity and impair postnatal brain function.

Animals↗