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E J Simon

Publications and source records attributed to E J Simon.

At least 37 records · Page 2Linked to original sources
PubMed1995

Structural model for the beta-amyloid fibril based on interstrand alignment of an antiparallel-sheet comprising a C-terminal peptide.

Amyloids are a class of noncrystalline, yet ordered, protein aggregates. A new approach was used to provide the initial structural data on an amyloid fibril--comprising a peptide (beta 34-42) from the C-terminus of the beta-amyloid protein--based on measurement of intramolecular 13C-13C distances and 13C chemical shifts by solid-state 13C NMR and individual amide absorption frequencies by isotope-edited infrared spectroscopy. Intermolecular orientation and alignment within the amyloid sheet was determined by fitting models to observed intermolecular 13C-13C couplings. Although the structural model we present is defined to relatively low resolution, it nevertheless shows a pleated antiparallel beta-sheet characterized by a specific intermolecular alignment.

Alzheimer Disease↗
PubMed1995

Functional reconstitution of a highly purified mu-opioid receptor protein with purified G proteins in liposomes.

A mu-opioid receptor protein (mu-ORP) purified to homogeneity from bovine striatal membranes has been functionally reconstituted in liposomes with highly purified heterotrimeric guanine nucleotide regulatory proteins (G proteins). A mixture of bovine brain G proteins, predominantly GoA, was used for most of the experiments, but some experiments were performed with individual pure G proteins, GoA, GoB, Gi1, and Gi2. Low Km GTPase was stimulated up to 150% by mu-opioid receptor agonists when both mu-ORP and a G protein (either the brain G protein mixture or a single heterotrimeric G protein) were present in the liposomes. Stimulation by a selective mu-agonist was concentration dependent and was reversed by the antagonist (-)-naloxone, but not by its inactive enantiomer, (+)-naloxone. The mu selectivity of mu-ORP was demonstrated by the inability of delta and kappa agonists to stimulate GTPase in this system. High-affinity mu-agonist binding was also restored by reconstitution with the brain G protein mixture and with each of the four pure Gi and G(o) proteins studied. The binding of mu agonists is sensitive to inhibition by GTP gamma S and by sodium.

Animals↗
PubMed1994

Effects of chronic food restriction on mu and kappa opioid binding in rat forebrain: a quantitative autoradiographic study.

It was previously observed that chronic food restriction lowers the threshold for lateral hypothalamic self-stimulation in a manner that is reversible by mu- and kappa-selective opioid antagonists. The present quantitative autoradiographic study was designed to investigate whether chronic food restriction alters regional mu and kappa opioid binding in brain. [3H]DAGO (mu) and mu/delta blocked [3H]BMZ (kappa) binding were analyzed in 34 brain regions from the medial prefrontal cortex to posterior hypothalamus. Significant reductions in mu binding were observed in caudal portions of the medial and lateral habenula, and the basolateral and basomedial nuclei of the amygdala. kappa binding was similarly reduced in medial habenula. Large increases in kappa binding were observed in the bed nucleus of the stria terminalis, ventral pallidum, and medial preoptic area. The possible involvement of these changes in the sensitization of reward by food restriction is discussed.

Analgesics↗
PubMed1994

Immunohistochemical localization of mu-opioid receptors in rat brain using antibodies generated against a peptide sequence present in a purified mu-opioid binding protein.

Light-microscope visualization in rat brain of a pattern of distribution of immunoreactivity, which included immunolabeled perikarya and beaded processes, was achieved using an immunoaffinity purified polyclonal antibody, Ab165, which recognizes the amino acid sequence, IRNLRQDRSKYY, found in the mu-opioid binding protein purified in our laboratory. Immunohistochemical staining with Ab165 was carried out by the avidin-biotin procedure. Antibody, preabsorbed with antigen, served as control. Extensive immunoreactivity was seen in the hippocampal formation, the amygdaloid complex, the striatal complex, cortical regions, select areas of the thalamus and hypothalamus and in laminae I and II of the dorsal horn in spinal cord. The distribution of immunoreactivity in the rat brain of antibody 165, which recognizes a purified mu-opioid binding protein, is concordant with the distribution of mu-opioid binding sites as determined by other laboratories in autoradiographic, electrophysiological and immunocytochemical studies. These findings have enabled us to distinguish areas possessing large fields of mu-opioid receptor containing cell bodies from areas possessing dense networks of immunolabeled neuronal processes or mixtures of both.

Amino Acid Sequence↗
PubMed1993

Reconstitution of a purified mu-opioid binding protein in liposomes: selective, high affinity, GTP gamma S-sensitive mu-opioid agonist binding is restored.

An opioid binding protein (OBP) purified to homogeneity from bovine striatal membranes has been reconstituted in liposomes. The liposomes were produced by PEG-precipitation of OBP in the presence of a CHAPS extract of bovine striatum, devoid of opioid binding. High affinity mu-agonist binding was restored. The binding was selective for mu-agonists, stereospecific and inhibited by GTP gamma S. These results demonstrate that there is recoupling of OBP with G-protein and confirm our earlier evidence that the purified OBP is a mu-opioid binding site.

Animals↗
PubMed1993

Alterations in delta opioid receptor levels in discrete areas of the neocortex and in the globus pallidus of the aging guinea pig: a quantitative autoradiographic study.

The effect of aging on delta opioid receptors was examined in the brains of guinea pigs aged 1, 6, 24 and 36 months. Quantitative autoradiography was used to monitor the concentrations of delta receptors in various anatomical regions at five rostro-caudal levels. delta opioid receptor populations were found to be remarkably stable throughout the life span of this species. We have, however, discovered anatomical areas which offer striking exceptions. In the globus pallidus, progressive age-related losses of delta receptors reached 50% in the senescent animal. In contrast, laminae I, II of the lateral agranular frontal cortex and laminae I, II and III, IV of the primary somatosensory cortex demonstrated age-related increases in the concentrations of delta receptors ranging from 30 to 45%. These changes are discussed with the view to their being functionally related components of motor circuitry involving pyramidal and extrapyramidal elements.

Aging↗
PubMed1993

Photoaffinity ligands for the mu opioid receptor.

Two affinity ligands, 6 beta-(5-Azido-2-nitrophenacetamido) 14 beta-hydroxy-7,8-dihydromorphinone (4) and 6 beta-(5-azido-2-nitrophenacetamido) 14 beta-hydroxy-7,8-dihydro-N- cyclopropylmethylnormorphinone (5) bind reversibly to opioid receptors present in bovine caudate membranes and photolyse in a range of wavelengths centered about 366 nm to produce wash-resistant binding to the mu receptor. At these wavelengths very little if any photodestruction of the mu receptor occurs over the 20 minute period of irradiation at 0 degree C.

Affinity Labels↗
PubMed1993

Evaluation of three portable suction devices.

Three portable suction devices were evaluated and compared with a wall-mounted vacuum driven suction unit. The Repro-med Res-Q-Vac, the Dräger Sujector 2000 and the Laerdal suction unit were assessed by measuring the time taken to aspirate 140 ml of mock gastric contents. The respective times for each device, expressed as mean and (range) were 7.39 (4.3-10.4), 8.6 (7.8-9.4) and 11.4 (9.4-12.6) s. These compare favourable with the Ohmeda suction unit (7.27 (6.2-8.9)). Each type of device has advantages and disadvantages when factors such as size, power supply and portability are considered, and each will be the most suitable for a particular situation.

Equipment Design↗
PubMed1993

Antisera against peptides derived from a purified mu-opioid binding protein recognize the protein as well as mu-opioid receptors in brain regions and a cell line.

Two peptides, which have no significant homology with known protein structures, were obtained by microsequencing of a mu-opioid binding protein purified to homogeneity from bovine striatal membranes. Polyclonal antibodies generated against portions of these peptides immunoprecipitated up to 65% of radiolabeled purified opioid binding protein. Sequential immunoprecipitations, using antibodies directed against portions of the two different peptides, confirmed that the peptides are derived from the same protein. Immunoblots of the protein with antipeptide antibodies revealed a protein band corresponding to the molecular weight of denatured reduced mu-opioid binding protein. The immunoresponse was blocked by the appropriate peptide and was not observed with irrelevant antisera. The antipeptide antibodies were used for immunoblots of sodium dodecyl sulfate extracts of tissues from bovine brain regions and of the mu receptor-containing cell line SK-N-SH. Affinity-purified antipeptide antibody detected an immunoreactive protein of molecular weight 65,000 in brain regions containing high levels of mu-opioid receptors (striatum, thalamus, hippocampus, and frontal cortex) and in the cell line SK-N-SH. Pons, which contains low levels of receptors, produced a a barely detectable signal, whereas white matter, HeLa cells, and C6 glioma cells, devoid of opioid binding activity, produced no detectable signal. The correlation between immunoreactivity and the presence of mu-opioid binding in brain regions and cell lines and the correspondence of the molecular weight of the immunoreactive protein to that of mu-opioid receptors provide strong evidence that the peptide antisera recognize mu receptors.

Amino Acid Sequence↗
PubMed1992

Lesioning of the nucleus basalis of Meynert has differential effects on mu, delta and kappa opioid receptor binding in rat brain: a quantitative autoradiographic study.

Opioid receptor binding was investigated in rat brain following lesioning of the nucleus basalis of Meynert (nbM). The nbM, which provides cholinergic input to the cortex, was lesioned unilaterally using ibotenic acid. The efficacy of lesioning was confirmed by the observation of a significant decrease in choline acetyltransferase (ChAT) activity in the ipsilateral prefrontal cortex. The specific laminar and regional distribution of mu, delta and kappa opioid receptor binding was quantitated in various cortical and limbic structures in the rat using autoradiography. Distinct medial to lateral gradients of mu and kappa opioid binding were observed in regions of the cerebral cortex. In the lesioned hemisphere the levels of mu, delta and kappa opioid binding were altered in localized areas of the cerebral cortex and the hippocampus. The direction of these binding changes varied with the opioid receptor type being assessed. Delta opioid binding was increased in the lateral portions of the frontal, occipital, perirhinal and retrosplenial granular cortices. Kappa opioid binding was increased in the lateral portion of the occipital cortex and in the CA3 region of the hippocampus. In contrast, mu opioid binding was decreased in the lateral portions of the frontal, entorhinal and forelimb cortices. These opioid receptor changes are discussed with respect to the interactions between the cholinergic and opioid systems, and relevance of the nbM lesion model to Alzheimer's disease.

Animals↗
PubMed1992

Immunoblots with rhodopsin antisera suggest that a purified mu opioid binding protein has structural characteristics of a G-protein-coupled receptor.

A mu opioid binding protein (OBP), previously purified to homogeneity from bovine striatal membranes, was examined by immunoblotting with six antisera against bovine rhodopsin. An antibody against the carboxyl-terminal tail of rhodopsin and one against membrane-associated rhodopsin gave strong signals at the appropriate molecular mass (65 kDa). An antibody directed against the first cytoplasmic loop of rhodopsin was weakly reactive. Three other antibodies did not recognize OBP. This pattern of crossreactivity was identical to that previously seen with beta-adrenergic receptors. The existence of domains in the OBP, which are antigenically similar to those in two other guanine nucleotide regulatory protein-coupled receptors, supports the hypothesis that mu opioid receptors have the structure characteristic of this receptor family.

Animals↗
PubMed1992

Effects of stress on opioid receptor binding in the rat central nervous system.

The endogenous opioid peptides are known to play a significant role in the modulation and/or mediation of numerous environmental or experimental stressors. However, the specific opioid peptide(s) and receptor type(s) involved, under what physiologic conditions they are engaged and within which regions of the CNS is not well understood. We therefore examined the effects of both a chronic and an acute stressor-90-h water deprivation and a single 20-min foot shock on opioid receptor binding in 17 specific rat brain nuclei. [3H]DSTLE (Tyr-D-Ser-Gly-Phe-Leu-Thr) and [3H]DAGO(Tyr-D-Gly-Phe-NMe-Phe-Gly-ol) were used to label delta and mu receptors, respectively. Foot shock induced profound antinociception as measured by tail-flick latency which outlasted the stressor by several minutes. However, only the septum responded with a decrease in [3H]DAGO binding to this type of stress-induced analgesia. No other alterations in either [3H]DAGO or [3H]DSTLE binding were seen in response to foot shock. In contrast, water deprivation induced increases in [3H-DAGO] binding in the septum as well as increases in [3H]DSTLE binding in the caudate and accumbens nuclei. Moreover, the presumptive mild stress of handling in the foot shock control group was sufficient to decrease mu or delta receptor binding in seven out of 17 brain regions investigated (including the frontal cortex and olfactory tubercle where both mu and delta binding were increased) when compared to unhandled deprivation control animals. These changes in opioid receptor binding may have been the result of alterations in treatment-induced peptide release, receptor regulation, or interactions with other released neurotransmitter ligand/receptor complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Age-related changes in kappa opioid receptors in the guinea-pig brain: a quantitative autoradiographic study.

Investigation into the effect of aging on kappa opioid receptors in the brains of guinea-pigs was carried out in animals aged one, six, 24 and 36 months. Quantitative autoradiography was used to monitor the concentration of kappa receptors in various anatomic regions at five rostrocaudal levels in each age-group. Areas of high binding were found in the deep layers (laminae V, VI) of the neocortex and in the internal band of the periallocortical, dorsal agranular insular cortex. Among non-cortical areas examined, the nucleus accumbens and the substantia nigra possessed kappa binding levels equal to those seen in the deep neocortical layers. In all cases where an age-related change in the level of kappa receptors was detected, the direction of the change was one of decreased binding with advancing age. Statistical analysis of the binding data revealed that the one-month-old animal possessed the highest levels of kappa binding among all age groups examined. The vast majority of age-related changes in kappa binding levels occurred in laminae V and VI of neocortical regions. The per cent decreases (18-42%), as well as their age of onset (six to 36 months) varied in different anatomical regions. Possible mechanisms to explain the age-related decreases in kappa opioid binding are presented. The majority of the age-related decreases in kappa opioid binding are found in areas of the neocortex which are characterized by their motor, sensory and associative functions. It is within these three areas of function that diminutions in performance are most apparent in senescence.

Aging↗
PubMed1992

Characterization of solubilized opioid receptors: reconstitution and uncoupling of guanine nucleotide-sensitive agonist binding.

Opioid receptors were solubilized from bovine striatal membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate-(CHAPS). High concentrations of NaCl (0.5-1.0 M) were necessary to ensure optimal yields, which ranged from 40 to 50% of membrane-bound receptors. This requirement was found to be specific for sodium, with only lithium able to substitute partially, as previously reported for solubilization with digitonin. Opioid antagonists, but not agonists, were able to bind to soluble receptors with high affinity. High-affinity binding of mu, delta, and kappa agonists was reconstituted following polyethylene glycol precipitation and resuspension of CHAPS extract. Evidence is presented suggesting that this is the result of inclusion of receptors in liposomes. Competition and saturation studies indicate that the three opioid receptor types retain their selectivity and that they exist in the reconstituted CHAPS extract in a ratio (50:15:35) identical to that in the membranes. In reconstituted CHAPS extract, as in membranes, mu-agonist binding was found to be coupled to a guanine nucleotide binding protein (G protein), as demonstrated by the sensitivity of [3H][D-Ala2,N-methyl-Phe4,Gly5-ol]-enkephalin ([3H]DAGO) binding to guanosine 5'-O-(thiotriphosphate) (GTP gamma S). In the reconstituted CHAPS extract, complete and irreversible uncoupling by GTP gamma S was observed, whereas membrane-bound receptors were uncoupled only partially. Treatment with GTP gamma S, at concentrations that uncoupled the mu receptors almost completely, resulted in a fourfold decrease in the Bmax of [3H]DAGO binding with a relatively small change in the KD. Competition experiments showed that the Ki of DAGO against [3H]bremazocine was increased 200-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Lumbar subarachnoid ethylenediaminetetraacetate induces hindlimb tetanic contractions in rats: prevention by CaCl2 pretreatment; observation of spinal nerve root degeneration.

Disodium ethylenediaminetetraacetate (Na2EDTA) has replaced sodium bisulfite as the antioxidant in 2-chloroprocaine, Nesacaine CE. This study was undertaken to determine whether this new formulation has neurotoxic effects when administered in the subarachnoid space. Sprague-Dawley rats receiving subarachnoid injections of 1.5 mM or higher concentrations of Na2EDTA immediately initiated a circling behavior that was followed by the development of tetanic contractions of the hindlimbs lasting for 15-20 min. The tetanic contractions were followed by a brief period of hindlimb paralysis. Pretreatment of rats by subarachnoid injections of 1 mM CaCl2 prevented the development of tetanic contractions and paralysis of the hindlimb. Histologic examination of animals receiving Na2EDTA revealed moderate to severe focal degenerative changes in spinal nerve roots. Control rats receiving subarachnoid injections of normal saline solution did not develop tetanic contraction nor pathological changes on light microscopy. These results suggest that the preservative used in Nesacaine-MPF may be neurotoxic.

Animals↗