PubMed Health⌕ Search

Biomedical subjects

P Emson

Publications and source records attributed to P Emson.

At least 19 recordsLinked to original sources

AXOR12, a novel human G protein-coupled receptor, activated by the peptide KiSS-1.

A novel human G protein-coupled receptor named AXOR12, exhibiting 81% homology to the rat orphan receptor GPR54, was cloned from a human brain cDNA library. Heterologous expression of AXOR12 in mammalian cells permitted the identification of three surrogate agonist peptides, all with a common C-terminal amidated motif. High potency agonism, indicative of a cognate ligand, was evident from peptides derived from the gene KiSS-1, the expression of which prevents metastasis in melanoma cells. Quantitative reverse transcriptase-polymerase chain reaction was used to study the expression of AXOR12 and KiSS-1 in a variety of tissues. The highest levels of expression of AXOR12 mRNA were observed in brain, pituitary gland, and placenta. The highest levels of KiSS-1 gene expression were observed in placenta and brain. A polyclonal antibody raised to the C terminus of AXOR12 was generated and used to show localization of the receptor to neurons in the cerebellum, cerebral cortex, and brainstem. The biological significance of these expression patterns and the nature of the putative cognate ligand for AXOR12 are discussed.

Amino Acid Sequence↗

Indexing-based differential display--studies on post-mortem Alzheimer's brains.

In this study we demonstrate for the first time that a novel indexing-based differential display technique generates valid and reproducible results when applied to human post-mortem tissue. We studied expression profiles in prefrontal cortex tissue derived from Alzheimer's disease (AD) and control brains, respectively, and found robust changes in several expressed genes, some of which have a known association with the disease process in AD. These included the dramatic reduction of calcineurin (known to be involved in tau phosphorylation) and GAP-43 (associated with synapse remodelling). Differential display results were confirmed by semi-quantitative RT-PCR on a larger number of brains.

Aged↗

Immunohistochemical localization of the P2Y1 purinergic receptor in Alzheimer's disease.

The biological actions of extracellular nucleotides are mediated by two distinct classes of P2 receptor, P2X and P2Y. The G protein-coupled P2Y receptors comprise five mammalian subtypes, P2Y(1-11). The P2Y1 subtype is expressed abundantly throughout the human brain and is specifically localized to neuronal structures. In the present study, the distribution of the P2Y1 receptor was investigated in Alzheimer's disease (AD) brains. In contrast to control human brain, the P2Y1 receptor was localized to a number of characteristic AD structures such as neurofibrillary tangles, neuritic plaques and neuropil threads. Immunoblot analysis showed that this specific immunostaining observed over tangles was not a result of cross-reactivity between the anti-P2Y1 antiserum and abnormal tau protein, the major constituent of tangles. The significance of this altered P2Y1 cellular distribution in AD brains is at present unclear.

Adenosine Diphosphate↗

Regional and cellular distribution of the P2Y(1) purinergic receptor in the human brain: striking neuronal localisation.

The biological actions of extracellular nucleotides are exerted via two families of P2 receptors, P2X and P2Y. The metabotropic P2Y receptors comprise at least 7 distinct subtypes, which have been cloned from a number of species. However, none of the P2Y receptor proteins have been visualised yet in human brain. In the present study, the regional and cellular distribution of the P2Y(1) receptor was investigated in the human brain by using immunohistochemistry. Polyclonal antibodies were raised against a synthetic peptide from the C-terminus of the P2Y(1) protein. Immunoblot analysis demonstrated that P2Y(1) antiserum specifically recognised a 63-kDa band in human and rat brain membranes. Similarly, the antiserum specifically detected the human P2Y(1) receptor in transfected 1321N1 cells. Immunohistochemical analysis on perfusion-fixed human brain tissue showed a widespread distribution for this receptor throughout the brain. At the cellular level, the P2Y(1) receptor was strikingly localised to neuronal structures of the cerebral cortex, cerebellar cortex, hippocampus, caudate nucleus, putamen, globus pallidus, subthalamic nucleus, red nucleus, and midbrain. Expression of the P2Y(1) receptor was not detected in other non-neuronal cell types. These results provide the first characterisation of the cellular distribution of a P2Y receptor in the human brain. The widespread and abundant distribution of the P2Y(1) receptor suggests its involvement in a number of important functions within the human brain. The neuronal localisation of this receptor points towards a possible role in neurotransmission, and also highlights a major role for extracellular nucleotides as signaling molecules within the brain.

Animals↗

The CCTTT polymorphism in the NOS2A gene is associated with dementia with Lewy bodies.

We report the analysis of the allele distribution of a (CCTTT)n pentanucleotide repeat within the promoter region of the NOS2A gene in DNA samples from patients with autopsy confirmed Alzheimer's disease (AD) and dementia with Lewy bodies (DLB) type. A significant difference was observed in the allelic distribution between the control group and the DLB group (chi2 = 15.175, df = 5; p<0.01), with an increased occurrence of the eight and nine repeat alleles, and a marked under representation of the 11 repeat allele. Genotype frequencies in the DLB group also differed significantly from controls (p<0.012). These results suggest that variations in the NOS2A gene may predispose to the development of DLB.

Alleles↗

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↗

Temporal lobe epilepsy associated up-regulation of metabotropic glutamate receptors: correlated changes in mGluR1 mRNA and protein expression in experimental animals and human patients.

Aberrant axonal reorganization and altered distribution of neurotransmitter receptor subtypes have been proposed as major pathogenic mechanisms for hippocampal hyperexcitability in chronic temporal lobe epilepsies (TLE). Recent data point to excitatory class I metabotropic glutamate receptors (mGluR1 and mGluR5) as interesting candidates. Here, we have analyzed the hippocampal distribution and mRNA expression of mGluR1 and mGluR5 in two rat models of limbic seizures, i.e. electrical kindling and intraperitoneal kainate injections, as well as in human TLE. Quantitative RT-PCR analysis detected a significant increase of hippocampal mGluR1 gene transcript levels in kainate treated and kindled rats. In addition, microdissected hippocampal tissue samples localized this increase to the dentate gyrus. Using immunohistochemistry with mGluR1alpha subtype specific antibodies, increased labeling was observed within the dentate gyrus molecular layer (DG-ML). A similar pattern of increased mGluR1alpha neuropil staining was found within the DG-ML of epilepsy patients (n = 42) compared with peritumoral hippocampus specimens obtained from nonepileptic patients (biopsy controls, n = 3). This increase was detected in TLE patients with segmental hippocampal cell loss, as well as in TLE patients with focal lesions but no histopathological alterations of the hippocampus. In contrast, mGluR5 immunoreactivity and mRNA expression were not significantly altered in the DG-ML. Our data demonstrate a striking regional induction of mGluR1alpha in the hippocampal dentate gyrus of experimental animals with limbic seizures as well as in human patients with chronic, intractable TLE. This increase corresponds to functional alterations of class I mGluRs observed in seizure models and may significantly contribute to hippocampal hyperexcitability in focal human epilepsies.

Adult↗

Localization of the type VI voltage-gated sodium channel protein in human CNS.

The cellular distribution of the type VI human voltage-gated sodium channel (Type VI) was examined in selected human brain regions. Antibodies designed to be specific to rat and human Type VI were raised against a synthetic peptide from the predicted NH2-terminal of the protein, and used for an immunohistochemical investigation. Immunoblot experiments showed that purified antibodies specifically detected the presence of Type VI in transfected cells and human brain membrane preparations. Immunohistochemistry on perfusion fixed human tissue revealed a predominantly somato-dendritic distribution of Type VI in major output neurons of the cerebellum, cerebral cortex and hippocampus. The observed localisation of this channel may reflect an important role in the integration of synaptic input in the human CNS.

Animals↗

Heterodimerization is required for the formation of a functional GABA(B) receptor.

GABA (gamma-aminobutyric acid) is the main inhibitory neurotransmitter in the mammalian central nervous system, where it exerts its effects through ionotropic (GABA(A/C)) receptors to produce fast synaptic inhibition and metabotropic (GABA(B)) receptors to produce slow, prolonged inhibitory signals. The gene encoding a GABA(B) receptor (GABA(B)R1) has been cloned; however, when expressed in mammalian cells this receptor is retained as an immature glycoprotein on intracellular membranes and exhibits low affinity for agonists compared with the endogenous receptor on brain membranes. Here we report the cloning of a complementary DNA encoding a new subtype of the GABAB receptor (GABA(B)R2), which we identified by mining expressed-sequence-tag databases. Yeast two-hybrid screening showed that this new GABA(B)R2-receptor subtype forms heterodimers with GABA(B)R1 through an interaction at their intracellular carboxy-terminal tails. Upon expression with GABA(B)R2 in HEK293T cells, GABA(B)R1 is terminally glycosylated and expressed at the cell surface. Co-expression of the two receptors produces a fully functional GABA(B) receptor at the cell surface; this receptor binds GABA with a high affinity equivalent to that of the endogenous brain receptor. These results indicate that, in vivo, functional brain GABA(B) receptors may be heterodimers composed of GABA(B)R1 and GABA(B)R2.

Amino Acid Sequence↗

Human metabotropic glutamate receptor 2 couples to the MAP kinase cascade in chinese hamster ovary cells.

We have examined the functional coupling of the human metabotropic glutamate receptor type 2 (mGluR2) with the regulation of the mitogen activated protein kinase (MAP kinase) signal transduction cascade. We demonstrated that L-glutamate stimulation of the human mGluR2 receptor transiently expressed in chinese hamster ovary (CHO) cells leads to a rapid increase in the activity of p42/p44 MAP kinase (also known as the extracellular signal regulated kinases, ERK1 and ERK2). Activation of p42/p44 MAP kinase has been demonstrated in a peptide phosphorylation assay and through the demonstration of a shift in electrophoretic mobility of p42 MAP kinase following activation. In both assay systems L-glutamate stimulation of MAP kinase was inhibited by pertussis toxin and by the MEK (MAP/ERK activating kinase) inhibitor PD 98059. We conclude that L-glutamate stimulation of the mGluR2 receptor in CHO cells mediated regulation of p42/p44 MAP kinase following the activation of pertussis toxin-sensitive G alpha(i) G-proteins via a distinct protein kinase signalling pathway that utilizes MEK.

Animals↗

Immunohistochemical localisation of mGluR7 protein in the rodent and human cerebellar cortex using subtype specific antibodies.

Metabotropic glutamate receptors (mGluRs) 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 characterise the protein expression of the two mGluR7 receptor splice variants in human and rat cerebellar cortex, antibodies specific to mGluR7 were generated. Antibodies were raised against a glutathione-S-transferase fusion protein containing amino acid residues located in the extracellular domain common to both the human and rat mGluR7 splice variants. These antibodies specifically detected human mGluR7a in mammalian cells transfected with this receptor. In agreement with mGluR7 in situ hybridisation studies, immunohistochemistry performed at the light microscope level revealed that mGluR7 protein expression occurred most prominently in a particular population of nerve cells common to both the human and rat, located within the cerebellar cortex of gray matter contained within transverse folia. Moreover, strong mGluR7-like immunoreactivity was seen in Purkinje cell body cytoplasm of the Purkinje cell layer. In the most superficial cerebellar cortical layer, the molecular layer, immunostaining was observed in Purkinje cell associated proximal and distal dendritic trees. No detectable labelling was evident in intrinsic deep cerebellar nuclei known to contain GABAergic terminals of projecting Purkinje cell axons. These data are suggestive of a post-synaptic location of mGluR7 in this central nervous system structure. In the rodent, additional non-Purkinje cells thought to represent inhibitory interneurones were labelled at all levels in the molecular layer. mGluR7-like immunoreactivity was not associated with glial cells.

Animals↗

Localization of mGluR4 protein in the rat cerebral cortex and hippocampus.

The cellular distribution of the rat metabotropic glutamate receptor type 4 (mGluR4) was examined in the adult rat cerebral cortex and hippocampus. Antibodies were raised against amino acid residues located in the extracellular amino terminal domain that is common to both the mGluR4a and mGluR4b splice variants, and used for an immunohistochemical investigation. The affinity-purified antibodies on immunoblot analysis specifically detected mGluR4 protein in transfected mammalian cells, showing no cross-reactivity with other members of the mGluR family. At the light microscope level intense mGluR4-like immunoreactivity was detected in the CA2 region of Ammon's horn in the hippocampus. In the cerebral cortex numerous non-pyramidal cells were strongly immunolabelled.

Animals↗

Expression of a novel splice variant of human mGluR1 in the cerebellum.

We have isolated clones of a novel splice variant of metabotropic glutamate receptor type 1 (mGluR1) from a human cerebellum cDNA library. Translation of this variant, mGluR1g would result in the addition of just one amino acid after the exon/intron boundary where the other splice variants diverge. RNA dot blot analysis using an mGluR1g-specific probe demonstrated expression in the cerebellum and also high levels in the kidney. Northern blotting using the same probe showed expression of a 4 kb transcript in the cerebellum. In situ hybridization studies in the cerebellum showed that mGluR1g mRNA is only expressed in granule cells, compared with mGluR1a/b mRNA which is also found in Purkinje cells and basket cells. Transcripts of the analogous splice variant are also present in rat brain mRNA.

Alternative Splicing↗

Inducible nitric oxide synthase gene transcription and protein activity in the rat heart during endotoxaemia.

Septicaemia leads to an impairment of myocardial contractility in animals and humans. Cytokines released during endotoxaemia are capable of increasing inducible nitric oxide synthase (iNOS) expression in vitro in myocytes, endothelial cells and macrophages. The aim of this study was to assess whether iNOS gene transcription occurs in the myocyte in vivo. Rats were injected with intraperitoneal endotoxin. Myocardial sections obtained 4, 6 and 8 hours after infection were hybridised with oligonucleotides complementary to iNOS cDNA. Myocardial homogenates were used to measure NOS enzyme activity and to detect iNOS mRNA. Uninfected control animals did not demonstrate myocardial iNOS expression. Myocardium from endotoxaemic animals contained iNOS mRNA and high calcium-independent NOS enzyme activity. In situ hybridisation did not localise iNOS to myocytes but to cells located between myocytes. Endotoxaemia leads to iNOS gene transcription and calcium-independent NOS enzyme activity in the rat myocardium. In situ hybridisation demonstrates that iNOS is not transcribed by the myocyte in vivo.

Animals↗

Effects of combined ventral forebrain grafts to neocortex and amygdala on behavior of rats with damage to the nucleus basalis magnocellularis.

In rats with damage to the nucleus basalis magnocellularis, transplantation of the embryonic ventral forebrain to the neocortex improves behavioral performance in some behavioral tasks. The present investigation focuses on improvement of behavioral performance by combined graft placement to both neocortex and amygdala. Male rats received unilateral microinjections of quisqualate to the nucleus basalis magnocellularis to produce cell damage. Embryonic ventral forebrain cell suspensions were placed in one group of rats in the frontal and parietal neocortex, in a second group in the amygdala, and in a third group in the frontal and parietal neocortex and in the amygdala. These groups were compared to a group of nonoperated rats and a group of rats with damage but with no grafts. Quisqualate-induced damage to the nucleus basalis magnocellularis reduced cholinergic innervation in the ipsilateral cortical hemisphere, impaired performance in the one-trial training version of passive avoidance, an increased motility and time spent in the open arms of the elevated plus maze. Combined graft placement to neocortex and amygdala normalized performance of passive avoidance and restored the normal time spent in the open arms of an elevated plus maze. These results suggest that after damage to the nucleus basalis magnocellularis, modulation of function in multiple brain regions may be necessary for optimization of adaptive behavior in situations involving induction of fear.

Amygdala↗

Developmental expression of nitric oxide synthase in the rat diencephalon with special reference to the thalamic paratenial nucleus.

Using immunohistochemistry and in situ hybridization the distribution of nitric oxide synthase (NOS) was investigated in the rat brain during pre- and postnatal development. At E15 weak NOS-like immunoreactivity (NOS-LI) could be seen in the differentiation field of the anterior hypothalamus. At E17 strong NOS-LI was observed in the developing neurons of the hypothalamic paraventricular nucleus, supraoptic nucleus, anterodorsal nucleus and lateral hypothalamic areas. In the thalamic paratenial nucleus a strong NOS-LI was observed in these neurons at E17, E18 and P1 with a weaker intensity at P3, P7, P9 and P15, whereas at P30 and in adult rats no NOS-positive neurons could be detected. NOS expression at E17 and P3 was verified by in situ hybridization. These results suggest that NO may have a developmental role at least in one of the regions studied, the thalamic paratenial nucleus.

Aging↗

Nitric oxide synthase, choline acetyltransferase, catecholamine enzymes and neuropeptides and their colocalization in the anterior pelvic ganglion, the inferior mesenteric ganglion and the hypogastric nerve of the male guinea pig.

By the indirect immunofluorescence method, the distribution of nitric oxide synthase (NOS)-like immunoreactivity (LI) and its possible colocalization with neuropeptide immunoreactivities, with two enzymes for the catecholamine synthesis pathway, tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH), as well as the enzyme for the acetylcholine synthesis pathway, choline acetyltransferase (ChAT) were studied in the anterior pelvic ganglion (APG), the inferior mesenteric ganglion (IMG) and the hypogastric nerve in the male guinea pig. The analyses were performed on tissues from intact animals, as well as after compression/ligation or cut of the hypogastric nerve. In some cases the colonic nerves were also cut. Analysis of the APG showed two main neuronal cell populations, one group containing NOS localized in the caudal part of the APG and one TH-positive group lacking NOS in its cranial part. The majority of the NOS-positive neurons contained ChAT-LI. Some NOS-positive cells did not contain detectable ChAT, but all ChAT-positive cells contained NOS. NOS neurons often contained peptides, including vasoactive intestinal peptide (VIP), neuropeptide tyrosine (NPY), somatostatin (SOM) and/or calcitonin gene-related peptide (CGRP). Some NOS cells expressed DBH, but never TH. The second cell group, characterized by absence of NOS, contained TH, mostly DBH and NPY and occasionally SOM and CGRP. Some TH-positive neurons lacked DBH. In the IMG, the NOS-LI was principally in nerve fibers, which were of two types, one consisting of strongly immunoreactive, coarse, varicose fibers with a patchy distribution, the other one forming fine, varicose, weakly immunoreactive fibers with a more general distribution. In the coarse networks, NOS-LI coexisted with VIP- and DYN-LI and the fibers surrounded mainly the SOM-containing noradrenergic principal ganglion cells. A network of ChAT-positive, often NOS-containing nerve fibers, surrounded the principal neurons. Occasional neuronal cell bodies in the IMG contained both NOS- and ChAT-LI. Accumulation of NOS was observed, both caudal and cranial, to a crush of the hypogastric nerve. VIP accumulated mainly on the caudal side and often coexisted with NOS. NPY accumulated on both sides of the crush, but mainly on the cranial side, and ENK was exclusively on the cranial side. Neither peptide coexisted with NOS. Both substance P (SP) and CGRP showed the strongest accumulation on the cranial side, possibly partly colocalized with NOS. It is concluded that the APG in the male guinea-pig consists of two major complementary neuron populations, the cholinergic neurons always containing NOS and the noradrenergic neurons containing TH and DBH. Some NOS neurons lacked ChAT and could represent truly non-adrenergic, non-cholinergic neurons. In addition, there may be a small dopaminergic neuron population, that is containing TH but lacking DBH. The cholinergic NOS neurons contain varying combinations of peptides. The noradrenergic population often contained NPY and occasionally SOM and CGRP. It is suggested that NO may interact with a number of other messenger molecules to play a role both within the APG and IMG and also in the projection areas of the APG.

Animals↗

Molecular cloning and structural organization of the human inducible nitric oxide synthase gene (NOS2).

Previously, we reported the isolation and molecular cloning of human inducible nitric oxide synthase gene (NOS2) sequences from human chromosome 17 cosmid libraries. Here we describe the further characterization and sequencing of the NOS2 gene. The genomic structure of the NOS2 gene was determined from two overlapping cosmid clones, namely, pcos4A and pcos20. the NOS2 open reading frame is encoded by 27 exons, with translation initiation and termination in exon 2 and exon 27, respectively. These results differ from the previously reported organization of the iNOS gene, where 26 exons were reported for the genomic structure of NOS2.

Alternative Splicing↗