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R H Edwards

Publications and source records attributed to R H Edwards.

At least 37 records · Page 2Linked to original sources
PubMed2000

Synaptic vesicle transporter expression regulates vesicle phenotype and quantal size.

While the transporters that accumulate classical neurotransmitters in synaptic vesicles have been identified, little is known about how their expression regulates synaptic transmission. We have used adenoviral-mediated transfection to increase expression of the brain vesicular monoamine transporter VMAT2 and presynaptic amperometric recordings to characterize the effects on quantal release. In presynaptic axonal varicosities of ventral midbrain neurons in postnatal culture, VMAT2 overexpression in small synaptic vesicles increased both quantal size and frequency, consistent with the recruitment of synaptic vesicles that do not normally release dopamine. This was confirmed using noncatecholaminergic AtT-20 cells, in which VMAT2 expression induced the quantal release of dopamine. The ability to increase quantal size in vesicles that were already competent for dopamine release was shown in PC12 cells, in which VMAT2 expression increased the quantal size but not the number of release events. These results demonstrate that vesicle transporters limit the rate of transmitter accumulation and can alter synaptic strength through two distinct mechanisms.

Adenoviridae↗
PubMed2000

Uptake of glutamate into synaptic vesicles by an inorganic phosphate transporter.

Previous work has identified two families of proteins that transport classical neurotransmitters into synaptic vesicles, but the protein responsible for vesicular transport of the principal excitatory transmitter glutamate has remained unknown. We demonstrate that a protein that is unrelated to any known neurotransmitter transporters and that was previously suggested to mediate the Na(+)-dependent uptake of inorganic phosphate across the plasma membrane transports glutamate into synaptic vesicles. In addition, we show that this vesicular glutamate transporter, VGLUT1, exhibits a conductance for chloride that is blocked by glutamate.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2000

Amino acid transport system A resembles system N in sequence but differs in mechanism.

Classical amino acid transport System A accounts for most of the Na(+)-dependent neutral amino acid uptake by mammalian cells. System A has also provided a paradigm for short- and long-term regulation by physiological stimuli. We now report the isolation of a cDNA encoding System A that shows close similarity to the recently identified System N transporter (SN1). The System A transporter (SA1) and SN1 share many functional characteristics, including a marked sensitivity to low pH, but, unlike SN1, SA1 does not mediate proton exchange. Transport mediated by SA1 is also electrogenic. Amino acid transport Systems A and N thus appear closely related in function as well as structure, but exhibit important differences in ionic coupling.

Amino Acid Sequence↗
PubMed2000

A phosphorylation site regulates sorting of the vesicular acetylcholine transporter to dense core vesicles.

Vesicular transport proteins package classical neurotransmitters for regulated exocytotic release, and localize to at least two distinct types of secretory vesicles. In PC12 cells, the vesicular acetylcholine transporter (VAChT) localizes preferentially to synaptic-like microvesicles (SLMVs), whereas the closely related vesicular monoamine transporters (VMATs) localize preferentially to large dense core vesicles (LDCVs). VAChT and the VMATs contain COOH-terminal, cytoplasmic dileucine motifs required for internalization from the plasma membrane. We now show that VAChT undergoes regulated phosphorylation by protein kinase C on a serine (Ser-480) five residues upstream of the dileucine motif. Replacement of Ser-480 by glutamate, to mimic the phosphorylation event, increases the localization of VAChT to LDCVs. Conversely, the VMATs contain two glutamates upstream of their dileucine-like motif, and replacement of these residues by alanine conversely reduces sorting to LDCVs. The results provide some of the first information about sequences involved in sorting to LDCVs. Since the location of the transporters determines which vesicles store classical neurotransmitters, a change in VAChT trafficking due to phosphorylation may also influence the mode of transmitter release.

Amino Acid Substitution↗
PubMed2000

Estimation of body composition in muscular dystrophy by MRI and stereology.

We have applied the Cavalieri method of modern design stereology with magnetic resonance imaging for estimating the volume of whole-body muscle and fat compartments in four patients with muscular dystrophy, a patient with myopathy, five controls, an anorexic subject, and a body builder. Detailed systematic series (ie, 50) of axial MR images (T1-weighted, TR/TE 400/10 msec) were obtained throughout the whole body of each subject. The results showed that 15, 20, and 35 axial sections through the body are sufficient to secure coefficients of error (CEs) on the estimates of total muscle and fat volume of around 10%, 5%, and 3% respectively in muscular dystrophy patients and controls. The mean normalized volumes of muscle in four muscular dystrophy patients were decreased by 27% (t-test: P < 0.05), and those of total fat were increased by 12% (t-test: P > 0.05) relative to controls. The Cavalieri method provides a direct, efficient, and mathematically unbiased approach for studying human body compartments and may have application in assessing treatment efficacy in patients with muscular dystrophy. J. Magn. Reson. Imaging 2000;12:467-475.

Adipose Tissue↗
PubMed2000

The effect of motor learning in facioscapulohumeral muscular dystrophy patients.

Two models of motor learning (ML) practice (complex and simple) were used to investigate upper limb function in individuals with facioscapulohumeral dystrophy (FSHD). The effect of ML practice was studied by examining changes in the ability to undertake a simulated drinking task. In the complex model, seven FSHD patients and seven age- and gender-matched healthy volunteers were studied. Performance was assessed by measurement of the electromyographic (EMG) activity of the biceps brachii and brachioradialis, elbow joint flexion, shoulder joint flexion and abduction, maximum acceleration at the onset of movement, movement time and reaction time, both before and after a 30-min complex ML task. In the simple model, a second group of six FSHD patients and six age- and gender-matched healthy subjects were studied. The same parameters were measured as for the complex ML task, except that the EMG activity of the triceps and deltoid muscles (anterior part) were measured instead of that of the brachioradialis. In both studies, the FSHD patients showed significantly larger values for all parameters except the flexion of shoulder joint and reaction time, compared with controls before the ML task. In the FSHD group, while the complex ML resulted in decreases in the brachioradialis EMG activity (P<0.005) and reaction time (P<0.0001), the simple ML model resulted in significant changes towards the normal value in all parameters measured except shoulder flexion. The change in the measured variables towards normal values indicates that ML may help to improve performance in FSHD.

Adult↗
PubMed1999

Molecular analysis of system N suggests novel physiological roles in nitrogen metabolism and synaptic transmission.

The amino acid glutamine has a central role in nitrogen metabolism. Although the molecular mechanisms responsible for its transport across cell membranes remain poorly understood, classical amino acid transport system N appears particularly important. Using intracellular pH measurements, we have now identified an orphan protein related to a vesicular neurotransmitter transporter as system N. Functional analysis shows that this protein (SN1) involves H+ exchange as well as Na+ cotransport and, under physiological conditions, mediates glutamine efflux as well as uptake. Together with the pattern of SN1 expression, these unusual properties suggest novel physiological roles for system N in nitrogen metabolism and synaptic transmission.

Amino Acid Sequence↗
PubMed1999

Signature amino acid changes in latent membrane protein 1 distinguish Epstein-Barr virus strains.

Sequence variations in the Epstein-Barr virus (EBV) latent membrane protein 1 gene have been described in numerous EBV-associated tumors with some of these variations, most notably a 30-base pair deletion in the cytoplasmic carboxyl-terminal domain, suggested as associated with an increase in tumorigenicity. In this study, EBV DNA sequence was determined from 92 tissue specimens or cell lines, including nasopharyngeal carcinoma, oral hairy leukoplakia, post-transplant lymphoma, post-transplant without pathology, mononucleosis, Burkitt's lymphoma, parotid tumor, and normal from distinct geographical regions. The amino- and carboxyl-terminal sequences and, in some cases, the full-length sequences of latent membrane protein 1 were determined. Characteristic sequence patterns distinguished strains, with the carboxyl-terminal sequence being the most informative in distinguishing among the strains. Phylogenetic relationships between strains were determined, as were signature amino acid changes that discriminate between them. A correlation between strain and disease or strain and geographic location was not detected. The sequence variation and signature sequences identified at least seven distinct strains, as well as hybrid strains that apparently result from recombination.

Amino Acid Sequence↗
PubMed1999

In vivo microdialysis-A technique for analysis of chemical activators of muscle pain.

The accurate measurement of the chemical activators of pain in skeletal muscle has proved to be a major challenge. This study examined the applicability of microdialysis to the measurement of pain-producing substances in skeletal muscle using a defined model of ischemia and reperfusion in the rat. Microdialysis probes were placed into muscle of anesthetized rats. Ischemia was induced for 4 h, followed by reperfusion for 1 h. Perfusates were analyzed for hypoxanthine, potassium, prostaglandin (PG) E(2) and histamine. A 20-fold increase in perfusate hypoxanthine concentration was seen prior to reperfusion (70.1 +/- 27.1 microM for ischemic versus 3.7 +/- 1.9 microM for control; P < 0.05). An initial increase in PGE(2) concentration was seen during ischemia (7.4 +/- 2.0 nM versus 3.4 +/- 1.4 nM; P < 0.05) and immediately post-reperfusion (17.9 +/- 5.2 nM versus 4.0 +/- 1.1 nM; P < 0.05). Potassium concentration was significantly increased following occlusion and reperfusion. This indicates the applicability of microdialysis to the measurement of pain-producing substances in muscle during ischemia and reperfusion. Further use will provide novel information on muscle pain both in defined model systems and in clinical situations in humans.

Animals↗
PubMed1999

Membrane trafficking of neurotransmitter transporters in the regulation of synaptic transmission.

Many psychoactive drugs influence the transport of neurotransmitters across biological membranes, suggesting that the physiological regulation of neurotransmitter transport might contribute to normal and perhaps abnormal behaviour. Over the past few years, molecular characterization of the neurotransmitter transporters has enabled investigation of their subcellular location and regulation. The analysis of location suggests that membrane trafficking has an important role in the normal function of these proteins. One of the major regulatory mechanisms also involves changes in localization that might contribute to synaptic plasticity. This article discusses recent work on the membrane trafficking of neurotransmitter transporters and its role in regulating their activity.

Affect↗
PubMed1999

Evidence for early hematopoietic progenitor cell involvement in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) represents a subtype of acute myeloid leukemia with characteristic morphologic, molecular, and immunophenotypic features. Previous immunophenotypic analyses have shown that leukemic cells in APL typically express the myeloid markers CD33 and CD13 but lack expression of the early hematopoietic progenitor cell antigens CD34 and HLA-DR. We analyzed selected immunophenotypic features of APL by flow cytometry and showed that 7 (41%) of 17 cases contained significant subsets of CD34+ leukemic cells: CD34+ myeloid cells predominated in 2 APL cases. By using a fluorescence-activated cell sorter-fluorescence in situ hybridization approach, we confirmed that the CD34+ cells harbored the t(15;17) translocation characteristic of APL. By using the same experimental approach, CD34+ populations were stratified into primitive CD34+ CD38- and committed CD34+ CD38+ progenitor cell subpopulations; cells in both subsets contained the t(15;17) translocation. The knowledge that APL may be partly or largely CD34+ is important for proper diagnosis. Furthermore, identification of the t(15;17) translocation in CD34+ CD38- blasts indicates that, in at least some cases, the leukemogenic mutation in APL occurs within primitive hematopoietic progenitor cells.

Antigens, CD34↗
PubMed1999

Nocturnal oxygenation and prognosis in Duchenne muscular dystrophy.

REM-related oxygen desaturation occurs in advanced Duchenne muscular dystrophy (DMD) and might be an independent predictor of disease progression. We have followed 18 patients for 10 yr after an initial respiratory sleep study or until death or onset of nasal ventilation. We measured baseline spirometry, blood gas tensions, maximal respiratory pressures, and body mass index. In 11 cases, VC was recorded serially. Median survival was 50 (range, 13 to 89) mo from initial study and unrelated to age at time of study, BMI, or mouth pressures but correlated with PaCO2 (r = -0.72, p < 0.005, n = 17), minimal nocturnal SaO2 (r = 0.62, p < 0.007, n = 18) and VC (r = 0. 65, p < 0.005, n = 17). Cox regression analysis showed a VC of less than 1 L at the time of study to be the best single predictor of subsequent survival. The only measure associated with age of death was the age at which the VC fell below 1 L (r = 0.79, p < 0.004). These data suggest measurement of PaCO2 or serial assessment of VC should be studied further as valid methods of assessing prognosis in DMD.

Adolescent↗
PubMed1998

Neurotransmitter release: variations on a theme.

Similarities between the ways that synaptic vesicles and large dense-core vesicles release their contents have been emphasized, but recent studies have revealed important mechanistic differences between these two exocytotic processes.

Animals↗
PubMed1998

The vesicular GABA transporter, VGAT, localizes to synaptic vesicles in sets of glycinergic as well as GABAergic neurons.

A transporter thought to mediate accumulation of GABA into synaptic vesicles has recently been cloned (McIntire et al., 1997). This vesicular GABA transporter (VGAT), the first vesicular amino acid transporter to be molecularly identified, differs in structure from previously cloned vesicular neurotransmitter transporters and defines a novel gene family. Here we use antibodies specific for N- and C-terminal epitopes of VGAT to localize the protein in the rat CNS. VGAT is highly concentrated in the nerve endings of GABAergic neurons in the brain and spinal cord but also in glycinergic nerve endings. In contrast, hippocampal mossy fiber boutons, which although glutamatergic are known to contain GABA, lack VGAT immunoreactivity. Post-embedding immunogold quantification shows that the protein specifically associates with synaptic vesicles. Triple labeling for VGAT, GABA, and glycine in the lateral oliva superior revealed a higher expression of VGAT in nerve endings rich in GABA, with or without glycine, than in others rich in glycine only. Although the great majority of nerve terminals containing GABA or glycine are immunopositive for VGAT, subpopulations of nerve endings rich in GABA or glycine appear to lack the protein. Additional vesicular transporters or alternative modes of release may therefore contribute to the inhibitory neurotransmission mediated by these two amino acids.

Animals↗
PubMed1998

Transient developmental expression of monoamine transporters in the rodent forebrain.

Neurons in first-order sensory thalamic nuclei have been shown to express functional plasma membrane serotonin (SERT) and vesicular monoamine (VMAT2) transporters during early postnatal development. In the present study, we provide an extensive description of the spatial and the temporal patterns of VMAT2 and SERT expression, during early embryonic development and postnatal life, by using in situ hybridization and immunocytochemistry. VMAT2 and SERT genes are transiently expressed in a wide population of non-monoaminergic neurons in the central and peripheral nervous system with a large overlap in the temporal and spatial pattern of expression of both genes. A selective pattern of expression of both genes was observed in the thalamus with expression limited to the dorsal thalamus and more particularly to primary sensory relay nuclei that convey point to point projection maps. Transient expression of the transporters was also observed in sensory cranial nerves, in the hippocampus, cerebral cortex, septum, and amygdala. VMAT2 and SERT gene expression was not necessarily linked, as some neural populations expressed only VMAT2, while others only contained SERT. Since VMAT2 serves to transport catecholamines besides serotonin, we examined the developmental expression of the plasma membrane dopamine and norepinephrine transporters but found no transient expression of these genes. Despite minor temporal disparities, VMAT2 and SERT extinguished almost simultaneously during the second and third weeks of post-natal life. These expressions did not seem to be dependent on peripheral neural inputs, since monocular enucleations and infraorbital nerve cuts effected on the day of birth, did not modify the period of transporter expression or of extinction.

Animals↗
PubMed1998

The localization of the brain-specific inorganic phosphate transporter suggests a specific presynaptic role in glutamatergic transmission.

Molecular cloning has recently identified a vertebrate brain-specific Na+-dependent inorganic phosphate transporter (BNPI). BNPI has strong sequence similarity to EAT-4, a Caenorhabditis elegans protein implicated in glutamatergic transmission. To characterize the physiological role of BNPI, we have generated an antibody to the protein. Immunocytochemistry of rat brain sections shows a light microscopic pattern that is suggestive of reactivity in nerve terminals. Excitatory projections are labeled prominently, and ultrastructural analysis confirms that BNPI localizes almost exclusively to terminals forming asymmetric excitatory-type synapses. Although BNPI depends on a Na+ gradient and presumably functions at the plasma membrane, both electron microscopy and biochemical fractionation show that BNPI associates preferentially with the membranes of small synaptic vesicles. The results provide anatomic evidence of a specific presynaptic role for BNPI in glutamatergic neurotransmission, consistent with the phenotype of eat-4 mutants. Because an enzyme known as the phosphate-activated glutaminase produces glutamate for release as a neurotransmitter, BNPI may augment excitatory transmission by increasing cytoplasmic phosphate concentrations within the nerve terminal and hence increasing glutamate synthesis. Expression of BNPI on synaptic vesicles suggests a mechanism for neural activity to regulate the function of BNPI.

Animals↗
PubMed1998

Expression of the Epstein-Barr virus latent membrane protein 1 induces B cell lymphoma in transgenic mice.

The latent membrane protein 1 (LMP1) of the Epstein-Barr virus has transforming properties in rodent fibroblasts and is expressed in most of the cancers associated with Epstein-Barr virus (EBV) infection including posttransplant lymphomas, Hodgkin's disease, nasopharyngeal carcinoma, and AIDS-related lymphomas. In this study, three lineages of LMP1 transgenic mice were established with LMP1 expressed under the control of the Ig heavy chain promoter and enhancer. Lymphoma developed in all three lineages, and the incidence of lymphoma increased significantly with age with lymphomas developing in 42% of transgenic mice over 18 months. The expression of LMP1 was detected at high levels in the lymphoma tissues but only at trace levels in normal lymphoid tissues. Gene rearrangement of the Ig heavy chain indicated monoclonality or oligoclonality in all lymphomas, some of the lymphoid hyperplastic spleens, and some histologically normal spleens. These data reveal that LMP1, without the expression of other EBV genes, is oncogenic in vivo and indicate that LMP1 is a major contributing factor to the development of EBV-associated lymphomas.

Animals↗