Biomedical subjects

C J Evans

Publications and source records attributed to C J Evans.

At least 55 records · Page 3Linked to original sources

PubMed1998

Phosphorylation is not required for dynamin-dependent endocytosis of a truncated mutant opioid receptor.

Opioid receptors are regulated within minutes after activation by G protein-coupled receptor kinase-mediated phosphorylation and dynamin-dependent endocytosis. We addressed the question of whether phosphorylation is required for opioid receptor endocytosis by examining a functional, truncated mutant delta opioid receptor (DOR344T), which is missing phosphorylation sites located in the carboxyl-terminal cytoplasmic domain. DOR344T receptors expressed in Chinese hamster ovary cells remained predominantly in the plasma membrane, even in the presence of saturating concentrations of agonist, consistent with previous studies demonstrating strongly inhibited endocytosis of truncated receptors in this cell type. In marked contrast, DOR344T receptors expressed at similar levels in human embryonal kidney (HEK) 293 cells exhibited rapid, ligand-induced internalization either in the presence of peptide (DADLE) or alkaloid (etorphine) agonist. Quantitative assays using ELISA and flow cytometric techniques indicated that DOR344T receptors were endocytosed in HEK293 cells with similarly rapid kinetics as full-length DOR (t1/2 < 10 min), and both full-length DOR and DOR344T mutant receptors were endocytosed by a dynamin-dependent mechanism involving clathrin-coated pits. Nevertheless, DOR344T receptors failed to undergo any detectable constitutive or agonist-induced phosphorylation in the same cells in which dynamin-dependent endocytosis was observed. These findings establish the first example of a G protein-coupled receptor that does not require phosphorylation to undergo dynamin-dependent endocytosis, and they suggest that significant cell type-specific differences exist in the biochemical requirements for ligand-induced concentration of opioid receptors in clathrin-coated pits.

Animals↗

PubMed1998

Collaboration between a state alliance for the mentally ill and a state mental health authority in monitoring the consequences of downsizing.

As consumers are released from hospitalization as a result of the downsizing of inpatient psychiatric facilities, their ability to sustain themselves in the community becomes a concern to a number of constituencies. This article describes the development of a program for monitoring persons who were released from hospitals into the community as a result of the downsizing of state psychiatric hospitals. The program's uniqueness was the collaboration between the state mental health authority and the state Alliance for the Mentally Ill in developing and implementing the monitoring system.

Case Management↗

PubMed1998

Alterations in K+ evoked profiles of neurotransmitter and neuromodulator amino acids after focal ischemia-reperfusion.

Secondary elevations in extracellular amino acids occur during reperfusion after transient cerebral ischemia. The delayed accumulation of excitatory amino acids may contribute to the progressive development of neuronal injury. In this study, we explored the mechanisms that may be involved in this phenomenon. Microdialysis samples from probes located in rabbit cortex were analysed with a chiral amino acid procedure. Concentrations of neurotransmitters (L-Glu, GABA), N-methyl-D-aspartate receptor modulators (D-Ser, Gly), an inhibitory neuromodulator (Tau), the lipid component phosphoethanolamine, and L-Gln, L-Ser and L-Ala were measured. Depolarization via perfusion with potassium was used to assess the status of release/reuptake systems at 2 and 4 h reperfusion after 2 h transient focal ischemia. Background experiments classified potassium evoked responses as calcium dependent or calcium-independent by inclusion of 30 microM omega-conopeptide MVIIC or by inclusion of 20 mM magnesium and ommision of calcium. During ischemia, large elevations of almost all amino acids occurred. During reperfusion, secondary elevations in transmitter amino acids (L-Glu, GABA) and N-methyl-D-aspartate receptor modulators (D-Ser, Gly) occurred. Tau remained slightly elevated whereas the lipid component phosphoethanolamine remained high and stable during reperfusion. Reperfusion significantly potentiated the potassium response for amino acids with calcium-dependent responses (L-Glu and GABA). In contrast, calcium-independent responses (Tau, phosphoethanolamine, L-Gln) were significantly attenuated. Intermediate behavior was observed with Gly, while no potassium responses were observed for D-Ser, L-Ser or L-Ala. These data demonstrate that perturbations in evoked amino acid profiles after ischemia-reperfusion are selective. Reduction of calcium-independent responses implicate a general decline in efficacy of transporter mechanisms that restore transmembrane gradients of ions and transmitters. Decreased efficacy of transporter systems may reduce transmitter reuptake and account for the amplified release of L-Glu and GABA, thus contributing to progressive neural dysfunction after cerebral ischemia.

Animals↗

PubMed1998

Quantitative immunolocalization of mu opioid receptors: regulation by naltrexone.

The present study utilized a newly developed quantitative immunohistochemical assay to measure changes in mu opioid receptor abundance following chronic administration of the opioid receptor antagonist naltrexone. These data were compared with those obtained from mu receptor radioligand binding on adjacent tissue sections, in order to determine whether the characteristic antagonist-induced increase in radioligand binding is due to an increase in the total number of mu receptors and/or to an increase in the proportion of receptors that are in an active binding conformation in the absence of a change in the total number of receptors. Adult male Sprague-Dawley rats were administered naltrexone, 7-8 mg/kg per day, or saline continuously for seven days by osmotic minipumps, after which time their brains were processed for immunohistochemistry and receptor autoradiography on adjacent fresh frozen tissue sections. Semiquantitative immunohistochemistry was performed using a radiolabelled secondary antibody for autoradiographic determination and a set of radioactive standards. Results demonstrate an overall concordance between the distribution of mu opioid receptors as measured by the two different methods with a few exceptions. Following naltrexone administration, mu receptor immunoreactivity was significantly higher in the amygdala, thalamus, hippocampus, and interpeduncular nucleus as compared with the saline-treated control animals. [3H]D-Ala2,N-Me-Phe4,Gly-ol5-enkephalin binding to mu opioid receptors was significantly higher in the globus pallidus, amygdala, thalamus, hypothalamus, hippocampus, substantia nigra, ventral tegmental area, central gray, and interpeduncular nucleus of the naltrexone-treated rats. These findings indicate that in some brain regions chronic naltrexone exposure increases the total number of mu opioid receptors, while in other regions there is an increase in the percent of active receptors without an observable change in the total number of receptors. Quantitative receptor immunodetection together with ligand autoradiography provides a new approach for investigating the regulation of mu opioid receptors on tissue sections.

Analgesics, Opioid↗

PubMed1998

Prevalence of venous reflux in the general population on duplex scanning: the Edinburgh vein study.

PURPOSE: The prevalence of reflux in the deep and superficial venous systems in the Edinburgh population and the relationship between patterns of reflux and the presence of venous disease on clinical examination were studied. METHODS: A cross-sectional survey was done on men and women ranging in age from 18 to 64 years, randomly selected from 12 general practices. The presence of varicose veins and chronic venous insufficiency was noted on clinical examination, as was the duration of venous reflux by means of duplex scanning in 8 vein segments on each leg. Results were compared using cut-off points for reflux duration (RD) of 0.5 seconds or more (RD >/= 0.5) and more than 1.0 second (RD > 1.0) to define reflux. RESULTS: There were 1566 study participants, 867 women and 699 men. The prevalence of reflux was similar in the right and left legs. The proportion of participants with reflux was highest in the lower thigh long saphenous vein (LSV) segment (18.6% in the right leg and 17.5% in the left leg for RD >/= 0.5), followed by the above knee popliteal segments (12.3% in the right leg and 11.0% in the left leg for RD >/= 0.5), the below knee popliteal (11.3% in the right leg and 9.5% in the left leg for RD >/= 0.5), upper LSV (10.0% in the right leg and 10.8% in the left leg for RD >/= 0.5) segments, the common femoral vein segments (7.8% in the right leg and 8.0% in the left leg for RD >/= 0.5), the lower superficial femoral vein (SFV) segments (6.6% in the right leg and 6.4% in the left leg for RD >/= 0.5), and the upper SFV (5.2% in the right leg and 4.7% in the left leg for RD >/= 0.5) and short saphenous vein (SSV) (4.6% in the right leg and 5.6% in the left leg for an RD >/= 0.5) segments. In the superficial vein segments, there was little difference in the occurrence of reflux whether RD >/= 0.5 or RD > 1.0 was used; but in the different deep vein segments, the prevalence of reflux was 2 to 4 times greater for RD >/= 0.5 rather than RD > 1.0. Men had a higher prevalence of reflux in the deep vein segments than women, reaching statistical significance (P </=.01) in 4 of 5 segments for RD >/= 0.5. In general, the prevalence of reflux increased with age. Those with "venous disease" had a significantly higher prevalence of reflux in all vein segments than those with "no disease" (P </=.001). CONCLUSION: The prevalence of venous reflux in the general population was related to the presence of "venous disease," although it was also present in those without clinically apparent disease. There was a higher prevalence of reflux in the deep veins in men than the deep veins in women. Follow-up study of the population will determine the extent to which reflux is a predictor of future disease and complications.

Adolescent↗

PubMed1998

A survey of mental health consumers' and family members' involvement in advocacy.

As the service delivery system is redesigned, the involvement of consumers and family members in mental health policy-making is essential. Advocacy is a way for them to have an impact on policy or decision-making. To determine the extent of their participation in advocacy efforts, consumers and family members were surveyed by other consumers and family members about their involvement in advocacy and mental healthcare reform. Although more family than consumers reported receiving mental healthcare reform information, both groups had difficulty understanding this information. Consumers were more likely to have received information from a mental health professional while family were more likely to have received information from an advocacy group. Lack of economic resources and communication problems were the most often cited barriers to advocacy for consumers. Strategies are suggested for how to increase involvement of consumers and family in advocacy efforts. The limitations of conducting a state-wide study with consumers and family as data collectors is discussed.

Adult↗

PubMed1998

mu-Opioid receptor internalization: opiate drugs have differential effects on a conserved endocytic mechanism in vitro and in the mammalian brain.

mu-Opioid receptors are the pharmacological targets of endogenous opioid peptides and morphine-like alkaloid drugs. Previous studies of transfected cells and peripheral neurons indicate that opioid receptors are rapidly internalized after activation by the alkaloid agonist etorphine but not after activation by morphine. To determine whether opioid receptors in the central nervous system are regulated by a similar process of agonist-selective internalization, mu-opioid receptors were examined in rat brain neurons after treatment of animals with opioid drugs. Internalized mu receptors were observed within 30 min after intraperitoneal injection of the alkaloid agonist etorphine, and this process was blocked by the antagonist naloxone. Colocalization of internalized opioid receptors with transferrin receptors in confocal optical sections indicated that receptor internalization observed in vivo is mediated by a membrane trafficking pathway similar to that observed previously in vitro using transfected human embryonic kidney 293 cells. Morphine failed to induce detectable rapid internalization of receptors, even when administered to animals at doses far in excess of those required to induce analgesia. To quantify these agonist-selective differences and to analyze an array of opioid ligands for their ability to trigger internalization, we used flow cytometry on stably transfected 293 cells. These studies indicated that the different effects of individual agonists are not correlated with their potencies for receptor activation and that a variety of clinically important agonists differ significantly in their relative abilities to stimulate the rapid internalization of opioid receptors.

Animals↗

PubMed1997

Presynaptic versus postsynaptic localization of mu and delta opioid receptors in dorsal and ventral striatopallidal pathways.

Parallel studies have demonstrated that enkephalin release from nerve terminals in the pallidum (globus pallidus and ventral pallidum) can be modulated by locally applied opioid drugs. To investigate further the mechanisms underlying these opioid effects, the present study examined the presynaptic and postsynaptic localization of delta (DOR1) and mu (MOR1) opioid receptors in the dorsal and ventral striatopallidal enkephalinergic system using fluorescence immunohistochemistry combined with anterograde and retrograde neuronal tracing techniques. DOR1 immunostaining patterns revealed primarily a postsynaptic localization of the receptor in pallidal cell bodies adjacent to enkephalin- or synaptophysin-positive fiber terminals. MOR1 immunostaining in the pallidum revealed both a presynaptic localization, as evidenced by punctate staining that co-localized with enkephalin and synaptophysin, and a postsynaptic localization, as evidenced by cytoplasmic staining of cells that were adjacent to enkephalin and synaptophysin immunoreactivities. Injections of the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) or the retrograde tracer Texas Red-conjugated dextran amine (TRD) into the dorsal and ventral striatum resulted in labeling of striatopallidal fibers and pallidostriatal cell bodies, respectively. DOR1 immunostaining in the pallidum co-localized only with TRD and not PHA-L, whereas pallidal MOR1 immunostaining co-localized with PHA-L and not TRD. These results suggest that pallidal enkephalin release may be modulated by mu opioid receptors located presynaptically on striatopallidal enkephalinergic neurons and by delta opioid receptors located postsynaptically on pallidostriatal feedback neurons.

Animals↗

PubMed1997

cAMP decreases steady-state levels of delta-opioid receptor mRNA in NG108-15 cells.

We have compared several drug combinations for their ability to increase basal cAMP and to down-regulate delta-opioid receptor mRNA levels. Continuous treatment for up to 48 h with a phosphodiesterase inhibitor in combination with the adenylyl cyclase activator forskolin showed an early peak response, but cAMP levels returned to control after 8 and 24 h. Increases in cAMP level up to 150-fold were observed after treatment for 1 h with a series of drugs (rolipram, IBMX/forskolin, rolipram/forskolin, dibutyryl cAMP, and prostaglandin E2) that increase cAMP by different mechanisms. A significant decrease in DOR mRNA level, to 31% of control, followed the three treatments that produced the largest increases in cAMP level: IBMX/forskolin, rolipram/forskolin, and prostaglandin E2.

1-Methyl-3-isobutylxanthine↗

PubMed1997

Orphanin FQ inhibits synaptic transmission and long-term potentiation in rat hippocampus.

It is known that opioid peptides acting on opioid receptors can modulate hippocampal synaptic functions. Although a novel member of the opioid receptor family, ORL1 receptors, that displays high-sequence homology with classical opioid receptors is abundant in the hippocampus, little is known regarding its role in synaptic function. The present study was designed to investigate whether activation of the ORL1 receptor by its natural ligand, orphanin FQ, could modulate synaptic transmission and synaptic plasticity in the hippocampus. The actions of orphanin FQ in the CA1 and dentate gyrus were examined by field potential recordings in response to stimulation of Schaffer collaterals and perforant path, respectively. Our results showed that orphanin FQ, but not the inactive analog des-Phe1-orphanin FQ, reduced both the slope of the excitatory postsynaptic potentials and population spike amplitude. The inhibitory effect of orphanin FQ is dose dependent and probably involves a presynaptic mechanism, as suggested by the significantly increased paired-pulse facilitation evoked in the presence of orphanin FQ. In addition, orphanin FQ was found to inhibit the induction of long-term potentiation at the Schaffer collateral-CA1 synapse. These results demonstrate that orphanin FQ can function as an inhibitory modulator regulating synaptic transmission and synaptic plasticity in the hippocampus, suggesting that activation of ORL1 receptors may play an important role in synaptic plasticity involved in learning and memory.

Amino Acid Sequence↗

PubMed1997

The use of associated particle timing based on the D + D reaction for elemental analysis of bulk samples such as the human body.

The use of associated particle timing based on the D + D reaction has been demonstrated for elemental analysis of bulk samples such as the human body. The neutron energy of 2.8 MeV eliminates the background from organic matrices. The nanosecond timing of a HPGe detector renders it possible to identify the spatial origin of the measured gamma radiation limiting the sensitive area to a single pixel. By this technique the background could be reduced by a factor of > or = 1000, but the present set-up has achieved an effective factor only in the range 20-100, due to losses in the generation of timing signals. The very clean gamma-spectra obtained permit the use of high efficiency scintillation detectors. Sensitivities for measuring Al, Ti, and Fe are presented at an extrapolated dose of 10 mSv.

Aluminum↗

PubMed1997

Amygdala kindling modifies extracellular opioid peptide content in rat hippocampus measured by microdialysis.

Opioid peptide release in the hippocampus was shown to be increased immediately following amygdala kindling stimulation in freely moving rats using microdialysis combined with a universal opioid peptide radioimmunoassay (RIA). Extracellular opioid peptide levels were elevated (55% above basal levels) within the first 10 min after electrical stimulation-induced partial seizures in previously nonkindled animals. Fully kindled rats showed lower extracellular opioid peptide levels (40% reduction) during the interictal period [16 +/- 2.1 days (mean +/- SEM) after the last stage V seizure], in comparison with values obtained from the sham-kindled group under basal conditions. However, opioid peptide release in fully kindled rats increased above 152% of interictal levels within the first 20 min after onset of fully kindled seizures, attaining peak levels equal to that of the partial kindled group and returning to prestimulation conditions 40-60 min following the ictal events. The majority of the immunoreactive material recovered from the hippocampus within the first 20 min following partial and generalized kindled seizures coeluted with dynorphin-A (1-6), dynorphin-A (1-8), and Leu-enkephalin by HPLC/RIA analysis. It is proposed that the enhanced opioid peptide release in hippocampus induced by amygdala kindling stimulation might be associated with either enhanced excitability or seizure suppression as seizure susceptibility fluctuates. The reduced interictal opioid peptide levels may also underlie some interictal behavioral disturbances.

Amygdala↗

PubMed1997

Monte Carlo design study of a moderated 252Cf source for in vivo neutron activation analysis of aluminium.

The Monte Carlo computer code MCNP has been used to design a moderated 252Cf neutron source for in vivo neutron activation analysis of aluminium (Al) in the bones of the hand. The clinical motivation is the need to monitor Al body burden in subjects with renal dysfunction, at risk of Al toxicity. The design involves the source positioned on the central axis at one end of a cylindrical deuterium oxide moderator. The moderator is surrounded by a graphite reflector, with the hand inserted at the end of the moderator opposing the source. For a 1 mg 252Cf source, 15 cm long x 20 cm radius moderator and 20 cm thick reflector, the estimated minimum detection limit is 0.5 mg Al for a 20 min irradiation, with an equivalent dose of 16.5 mSv to the hand. Increasing the moderator length and/or introducing a fast neutron filter (for example silicon) further reduces interference from fast-neutron-induced reactions on phosphorus in bone, at the expense of decreased fluence of the thermal neutrons which activate Al. Increased source strengths may be necessary to compensate for this decreased thermal fluence, to allow measurements to be made within an acceptable time limit for the comfort of the patient.

Aluminum↗

PubMed1997

The performance of an infra-red interactance instrument for assessing total body fat.

Infra-red interactance has been evaluated as a technique for measuring total body fat in comparison with a range of alternative methods. The alternative techniques employed were neutron activation analysis, tritiated water dilution, whole-body potassium-40 counting, skinfold anthropometry, bioelectrical impedance analysis and the body mass index. The study group consisted of 43 healthy adults (16 males and 27 females). For 11 women, measurements were obtained before and after 11 weeks on a very low-calorie diet, giving a total of 54 sets of data. Correlation coefficients between infra-red interactance and the other techniques varied between 0.58 (p < 0.0002) and 0.80 (p < 0.0001) for females, and between 0.64 (p < 0.009) and 0.94 (p < 0.0001) for males. The average fat for the study group was underestimated by 15% using infra-red interactance in comparison with the average fat obtained from the other techniques. It was also noted that the infra-red interactance instrument yielded a very narrow range of body fats in females in comparison with the other techniques. It is essential that these differences are reconciled before infra-red interactance takes a significant role in body composition analysis.

Adipose Tissue↗

PubMed1996

Sequestration of the delta opioid receptor. Role of the C terminus in agonist-mediated internalization.

The primary structure of the opioid receptors have revealed that many of the structural features that are conserved in other G protein-coupled receptors are also conserved in the opioid receptors. Upon exposure to agonists, some G protein-coupled receptors internalize rapidly, whereas other structurally homologous G protein-coupled receptors do not. It is not known whether opioid receptors are regulated by rapid endocytosis. In transfected Chinese hamster ovary cells expressing the epitope-tagged wild type delta opioid receptor, exposure to 100 nM [D-Ala2,D-Leu5]enkephalin causes internalization of the receptor within 30 min as determined by confocal microscopy. The rate of internalization of the wild type receptor is rapid with a half-maximal reduction by about 10 min, as determined by the reduction in mean surface receptor fluorescence intensity measured using flow cytometry. In contrast, the cells expressing receptors lacking the C-terminal 15 or 37 amino acids exhibit a substantially slower rate of internalization. Furthermore, the cells expressing receptors with point mutations of any of the Ser/Thr between Ser344 and Ser363 in the C-terminal tail exhibit a significant reduction in the rate of receptor internalization. These results suggest that a portion of the C-terminal tail is involved in receptor internalization. Agents that block the formation of clathrin-coated pits considerably reduce the extent of agonist-mediated internalization of the wild type receptor. Taken together, these results suggest that the wild type opioid receptor undergoes rapid agonist-mediated internalization via a classic endocytic pathway and that a portion of the C-terminal tail plays an important role in this internalization process.

Amino Acid Sequence↗

PubMed1996

Multiple opioid receptor-like genes are identified in diverse vertebrate phyla.

Polymerase chain reaction was used to determine whether opioid receptor-like sequences are present in species from the protostome and deuterostome branches of the metazoan kingdom. Multiple opioid receptor-like sequences were found in all vertebrates, but no specific fragments were obtained from any invertebrates. Delta, mu, kappa and ORL-1 receptors were identified from bovine DNA, and three different opioid receptor-like fragments were identified from the other vertebrates analyzed. The data suggest that the opioid receptor gene family has been highly conserved during vertebrate evolution and that, even in the primitive jawless fish, multiple members of the opioid receptor family appear to be present.

Amino Acid Sequence↗

PubMed1996

Morphine activates opioid receptors without causing their rapid internalization.

We have examined the endocytic trafficking of epitope-tagged delta and mu opioid receptors expressed in human embryonic kidney (HEK) 293 cells. These receptors are activated by peptide agonists (enkephalins) as well as by the alkaloid agonist drugs etorphine and morphine. Enkephalins and etorphine cause opioid receptors to internalize rapidly (t1/2 approximately 6 min) by a mechanism similar to that utilized by a number of other classes of receptor, as indicated by localization of internalized opioid receptors in transferrin-containing endosomes and inhibition of opioid receptor internalization by hypertonic media. Remarkably, morphine does not stimulate the rapid internalization of either delta or mu opioid receptors, even at high concentrations that strongly inhibit adenylyl cyclase. These data indicate that agonist ligands, which have similar effects on receptor-mediated signaling, can have dramatically different effects on the intracellular trafficking of a G protein-coupled receptor.

Cell Line↗

PubMed1996

Immunohistochemical localization of ORL-1 in the central nervous system of the rat.

A novel member of the opioid receptor family (ORL-1) has been cloned from a variety of vertebrates. ORL-1 does not bind any of the classical opioids, although a high affinity endogenous agonist with close homology to dynorphin has recently been identified. We have generated a monoclonal antibody to the N-terminus of ORL-1 to map areas of receptor expression in rat central nervous system (CNS). Intense and specific immunolabeling was observed in multiple areas in the diencephalon, mesencephalon, pons/medulla, and spinal cord. In the telencephalon, intense labeling was observed in the neuropil throughout layers II-V in the neocortex, the anterior olfactory nuclear complex, the pyriform cortex, the CA1-CA4 fields and dentate gyrus of the hippocampus, and in many of the septal and basal forebrain areas. In contrast to other members of the opioid receptor family, light labeling for ORL-1 was observed in telencephalic areas such as caudate-putamen. In the cerebellum, ORL-1 immunoreactivity was only observed in the deep nuclei. Throughout the CNS the majority of labelling was localized to fiber processes and fine puncta, although labeled scattered perikarya were observed in a few brain areas such as the hilus dentate in the hippocampus and some nuclei in the brainstem and spinal cord. The present mapping study is consistent with the reported distribution of ORL-1 mRNA and provides the first immunohistochemical report on anatomical and cellular distribution of ORL-1 receptor in the rat CNS.

Animals↗