Biomedical subjects

P R Dodd

Publications and source records attributed to P R Dodd.

At least 19 recordsLinked to original sources

PubMed2005

NMDA receptor subunit-dependent modulation by conantokin-G and Ala7-conantokin-G.

The modulation of recombinant NMDA receptors by conantokin-G (con-G) and Ala7-conantokin-G (Ala7-Con-G) was investigated in Xenopus oocytes injected with capped RNA coding for NR1 splice variants and NR2 subunits using the two-electrode voltage clamp technique. Glutamate exhibited a marginally higher apparent affinity for NR2A-containing receptors than NR2B-containing receptors, regardless of the NR1 subunit present. Conantokins were bath applied to give cumulative concentration responses in the presence of 3 and 30 mum glutamate. Both contantokins exhibited biphasic concentration-response relationships at NR2A-containing NMDA receptors, producing potentiation at low conantokin concentrations and inhibition at high concentrations. These effects were stronger with glutamate concentrations near its EC50, and less marked at saturating concentrations. In contrast, the conantokin concentration-response relation was monophasic and inhibitory at NR2B-containing receptors. We conclude that the combinations of subunits that comprise the NMDA receptor complex influence conantokin and glutamate affinities and the nature of the responses to conantokins.

Alanine↗

PubMed2005

The effects of alanine-substituted conantokin-G and ifenprodil on the human spermine-activated N-methyl-D-aspartate receptor.

We evaluated the effects of Ala-7-conantokin-G (Con-G(A7)) and ifenprodil on the modulation by spermine of [(3)H]MK801 binding to human cortical membranes. Human cortical tissue was obtained at autopsy and stored at -80 degrees C until assay. Both Con-G(A7) and ifenprodil inhibited [(3)H]MK801 binding, but spermine affected these inhibitions differently. Con-G(A7) IC(50) changed little with spermine concentration, indicative of a non-competitive interaction, whereas the rightward shift in ifenprodil IC(50) with increasing spermine concentration suggested partial competition. When the two agents were tested against the biphasic activation of [(3)H]MK801 binding by spermine, they again differed in their effects. In the activation phase Con-G(A7) was a non-competitive inhibitor of spermine activation, and may even enhance the spermine EC(50), while the ifenprodil data indicated a partially competitive interaction. Both agents were non-competitive in the inhibitory phase. Overall, the data suggest that Con-G(A7) and ifenprodil interact differently with the polyamine modulation of the glutamate-N-methyl-D-aspartate receptor.

Alanine↗

PubMed2004

Association studies of neurotransmitter gene polymorphisms in alcoholic Caucasians.

Ethanol enhances mesolimbic/cortical dopamine activity in reward and reinforcement circuits. We investigated the hypothesis that risk for alcoholism may be mediated by genes for neurotransmitters associated with the dopamine reward system as well as genes for enzymes involved in ethanol metabolism. DNA was extracted from brain tissue collected at autopsy from pathologically characterized alcoholics and controls. PCR-based assays showed that alcoholism was associated with polymorphisms of the dopamine D2 receptor (DRD2) TaqI B (P = .029) and the GABAA-beta2 subunit C1412T (P = .012) genes, but not with the glutamate receptor subunit gene NMDAR2B (366C/G), the serotonin transporter gene (5HTTL-PR), the dopamine transporter gene DAT1(SLC6A3), the dopamine D2 receptor gene DRD2 TaqI A, or the GABAA alpha1(A15G), alpha6(T1519C), and gamma2(G3145A) subunit genes. The glial glutamate transporter gene EAAT2 polymorphism G603A was associated with alcoholic cirrhosis (P = .048). The genotype for the most active alcohol dehydrogenase enzyme ADH1C was associated with a lower risk of alcoholism (P = .026) and was less prevalent in alcoholics with DRD2TaqIA2/A2 (P = .047), GABAA-beta2 1412C/C (P = .01), or EAAT2 603G/A (P = .022) genotypes. Combined DRD2TaqI A or B with GABAA-beta2 or EAAT2 G603A genotypes may have a concerted influence in the predisposition to alcoholism.

Alcoholism↗

PubMed2003

Increased TUNEL positive cells in human alcoholic brains.

Alcohol-sensitive neuronal cell loss, which has been reported in the superior frontal cortex and hippocampus, may underlie the pathogenesis of subsequent cognitive deficits. In the present study, we have used the TUNEL labeling to detect the DNA damage in human alcoholic brains. Seven out of eleven alcoholics exhibited TUNEL-positive cells in both superior frontal cortex and hippocampus, which were co-localized with GFAP immunoreactivity. In contrast, almost no positive cells were detected in the non-alcoholic controls. None of the TUNEL-positive cells showed any typical morphological features of apoptosis or necrosis. TUNEL-positive cells observed in the present study may indicate DNA damage induced by ethanol-related overproduction of reactive oxygen species.

Adult↗

PubMed2003

Synaptic vesicle transport and synaptic membrane transporter sites in excitatory amino acid nerve terminals in Alzheimer disease.

The apparent l-[3H]glutamate uptake rate (v') was measured in synaptic vesicles isolated from cerebral cortex synaptosomes prepared from autopsied Alzheimer and non-Alzheimer dementia cases, and age-matched controls. The initial synaptosome preparations exhibited similar densities of d-[3H]aspartate membrane binding sites (BMAX values) in the three groups. In control brain the temporal cortex d-[3H]aspartate BMAX was 132% of that in motor cortex, parallel with the l-[3H]glutamate v' values (temporal=139% of motor; NS). Unlike d-[3H]aspartate BMAX values, l-[3H]glutamate v' values were markedly and selectively lower in Alzheimer brain preparations than in controls, particularly in temporal cortex. The difference could not be attributed to differential effects of autopsy interval or age at death. Non-Alzheimer dementia cases resembled controls. The selective loss of vesicular glutamate transport is consistent with a dysfunction in the recycling of transmitter glutamate.

Aged↗

PubMed2001

Molecular cloning and characterization of hNP22: a gene up-regulated in human alcoholic brain.

An improved differential display technique was used to search for changes in gene expression in the superior frontal cortex of alcoholics. A cDNA fragment was retrieved and cloned. Further sequence of the cDNA was determined from 5' RACE and screening of a human brain cDNA library. The gene was named hNP22 (human neuronal protein 22). The deduced protein sequence of hNP22 has an estimated molecular mass of 22.4 kDa with a putative calcium-binding site, and phosphorylation sites for casein kinase II and protein kinase C. The deduced amino acid sequence of hNP22 shares homology (from 67% to 42%) with four other proteins, SM22alpha, calponin, myophilin and mp20. Sequence homology suggests a potential interaction of hNP22 with cytoskeletal elements. hNP22 mRNA was expressed in various brain regions but in alcoholics, greater mRNA expression occurred in the superior frontal cortex, but not in the primary motor cortex or cerebellum. The results suggest that hNP22 may have a role in alcohol-related adaptations and may mediate regulatory signal transduction pathways in neurones.

Adult↗

PubMed2001

Glutamate(NMDA) receptor NR1 subunit mRNA expression in Alzheimer's disease.

We analyzed the expression profile of two NMDAR1 mRNA isoform subsets, NR1(0XX) and NR1(1XX), in discrete regions of human cerebral cortex. The subsets are characterized by the absence or presence of a 21-amino acid N-terminal cassette. Reverse transcription polymerase chain reaction for NR1 isoforms was performed on total RNA preparations from spared and susceptible regions from 10 pathologically confirmed Alzheimer's disease (AD) cases and 10 matched controls. Primers spanning the splice insert yielded two bands, 342 bp (NR1(0XX)) and 405 bp (NR1(1XX)), on agarose gel electrophoresis. The bands were visualized with ethidium and quantified by densitometry. NR1(1XX) transcript expression was calculated as a proportion of the NR1(1XX) + NR1(0XX) total. Values were significantly lower in AD cases than in controls in mid-cingulate cortex, p < 0.01, superior temporal cortex, p < 0.01 and hippocampus, p approximately 0.05. Cortical proportionate NR1(1XX) transcript expression was invariant over the range of ages and areas of controls tested, at approximately 50%. This was also true for AD motor and occipital cortex. Proportionate NR1(1XX) expression in AD cingulate and temporal cortex was lower at younger ages and increased with age: this regression was significantly different from that in the homotropic areas of controls. Variations in NR1 N-terminal cassette expression may underlie the local vulnerability to excitotoxic damage of some areas in the AD brain. Alternatively, changes in NR1 mRNA expression may arise as a consequence of the AD disease process.

Aged↗

PubMed2001

GABA(A) receptor alpha-subunit proteins in human chronic alcoholics.

Antibodies were raised against specific peptides from N-terminal regions of the alpha1 and alpha3 isoforms of the GABA(A) receptor, and used to assess the relative expression of these proteins in the superior frontal and primary motor cortices of 10 control, nine uncomplicated alcoholic and six cirrhotic alcoholic cases were matched for age and post-mortem delay. The regression of expression on post-mortem delay was not statistically significant for either isoform in either region. In both cortical areas, the regression of alpha1 expression on age differed significantly between alcoholic cases, which showed a decrease, and normal controls, which did not. Age had no effect on alpha3 expression. The alpha1 and alpha3 isoforms were found to be expressed differentially across cortical regions and showed a tendency to be expressed differentially across case groups. In cirrhotic alcoholics, alpha1 expression was greater in superior frontal than in motor cortex, whereas this regional difference was not significant in controls or uncomplicated alcoholics. In uncomplicated alcoholics, alpha3 expression was significantly lower in superior frontal than in motor cortex. Expression of alpha1 was significantly different from that of alpha3 in the superior frontal cortex of alcoholics, but not in controls. In motor cortex, there were no significant differences in expression between the isoforms in any case group.

Adult↗

PubMed2000

Localisation of GABA(A) receptor subunits in the CNS using RT-PCR.

This paper presents an RT-PCR in situ histochemistry (RT-ISH) method for the detection and localisation of isoforms of the GABA(A) receptor in formalin-fixed, paraffin-embedded brain sections. RT-ISH was performed using PCR conditions already established in our laboratory for the amplification of the alpha(1-3) and beta(1-3) subunits of the GABA(A) receptor [2,5]. Initial experiments determined whether mRNA isolated from such sections was suitable for use in RT-PCR. Transcripts encoding both the alpha- and beta-subunits of the GABA(A) receptor were successfully amplified. RT-ISH, a one-step RT-PCR method, was used to amplify the transcripts and digoxigenin-labeled nucleotides were directly incorporated into the amplified products during cycling. RT-PCR products were detected using anti-digoxigenin antibody conjugated to alkaline-phosphatase and signal was visualised using light microscopy. This protocol may be used to study the expression of GABA(A) receptor isoforms in vivo and examine alterations in receptor composition during development and disease states.

Brain Stem↗

PubMed2000

Expression and distribution of GABAA receptor subtypes in human alcoholic cerebral cortex.

Long-term alcohol abuse is known to target specific areas of the brain such as the superior frontal cortex (SFC), resulting in neuronal cell loss. Abnormal transmission of the inhibitory neurotransmitter GABA may contribute to this damage. Previous work in our laboratory has found differential expression and distribution of certain a subunit genes of the GABAA receptor in the SFC of human alcoholic brain, suggesting that differences in GABAA receptor subunit expression could give rise to the locally altered GABAA pharmacology which is associated with alcohol abuse. A competitive RT-PCR assay has been developed to study the expression of the GABAA receptor beta-subunit genes beta1, beta2, and beta3. A single set of primers homologous to all three beta isoform sequences has been shown to amplify each of the beta isoforms from mRNA isolated from human brain tissue obtained at autopsy. An internal standard has been designed which is identical to the target except for a 61-bp deletion and a unique restriction enzyme (RE) site. This is co-amplified with the target sequences to allow amplification efficiency to be assessed and thus enable the quantitation of gene expression. A range of GABAA receptor ligands were used to look at differential distribution of receptor subtypes in the cortical laminae by autoradiography. Differences in distribution of the ligands were demonstrated, consistent with a hypothesis of alcohol-induced variations in the expression of receptor subunits.

Alcoholism↗

PubMed2000

Gene expression in human alcoholism: microarray analysis of frontal cortex.

BACKGROUND: Changes in brain gene expression are thought to be responsible for the tolerance, dependence, and neurotoxicity produced by chronic alcohol abuse, but there has been no large scale study of gene expression in human alcoholism. METHODS: RNA was extracted from postmortem samples of superior frontal cortex of alcoholics and nonalcoholics. Relative levels of RNA were determined by array techniques. We used both cDNA and oligonucleotide microarrays to provide coverage of a large number of genes and to allow cross-validation for those genes represented on both types of arrays. RESULTS: Expression levels were determined for over 4000 genes and 163 of these were found to differ by 40% or more between alcoholics and nonalcoholics. Analysis of these changes revealed a selective reprogramming of gene expression in this brain region, particularly for myelin-related genes which were down-regulated in the alcoholic samples. In addition, cell cycle genes and several neuronal genes were changed in expression. CONCLUSIONS: These gene expression changes suggest a mechanism for the loss of cerebral white matter in alcoholics as well as alterations that may lead to the neurotoxic actions of ethanol.

Aged↗

PubMed1999

Structure-activity studies of conantokins as human N-methyl-D-aspartate receptor modulators.

The activities of conantokin-G (con-G), conantokin-T (con-T), and several novel analogues have been studied using polyamine enhancement of [3H]MK-801 binding to human glutamate-N-methyl-D-aspartate (NMDA) receptors, and their structures have been examined using CD and 1H NMR spectroscopy. The potencies of con-G[A7], con-G, and con-T as noncompetitive inhibitors of spermine-enhanced [3H]MK-801 binding to NMDA receptor obtained from human brain tissue are similar to those obtained using rat brain tissue. The secondary structure and activity of con-G are found to be highly sensitive to amino acid substitution and modification. NMR chemical shift data indicate that con-G, con-G[D8, D17], and con-G[A7] have similar conformations in the presence of Ca2+. This consists of a helix for residues 2-16, which is kinked in the vicinity of Gla10. This is confirmed by 3D structure calculations on con-G[A7]. Restraining this helix in a linear form (i.e., con-G[A7,E10-K13]) results in a minor reduction in potency. Incorporation of a 7-10 salt-bridge replacement (con-G[K7-E10]) prevents helix formation in aqueous solution and produces a peptide with low potency. Peptides with the Leu5-Tyr5 substitution also have low potencies (con-G[Y5,A7] and con-G[Y5,K7]) indicating that Leu5 in con-G is important for full antagonist behavior. We have also shown that the Gla-Ala7 substitution increases potency, whereas the Gla-Lys7 substitution has no effect. Con-G and con-G[K7] both exhibit selectivity between NMDA subtypes from mid-frontal and superior temporal gyri, but not between sensorimotor and mid-frontal gyri. Asn8 and/or Asn17 appear to be important for the ability of con-G to function as an inhibitor of polyamine-stimulated [3H]MK-801 binding, but not in maintaining secondary structure. The presence of Ca2+ does not increase the potencies of con-G and con-T for NMDA receptors but does stabilize the helical structures of con-G, con-G[D8,D17], and, to a lesser extent, con-G[A7]. The NMR data support the existence of at least two independent Ca2+-chelating sites in con-G, one involving Gla7 and possibly Gla3 and the other likely to involve Gla10 and/or Gla14.

Amino Acid Sequence↗

PubMed1999

The modulatory effect of spermine on the glutamate-NMDA receptor is regionally variable in normal human adult cerebral cortex.

The MK-801, glutamate and polyamine binding sites on the N-methyl-D-aspartate class of glutamate receptors labelled with [3H]MK-801 were characterized in four cortical areas (sensorimotor, superior temporal, mid-frontal and occipital) from seven human adult control cases. Age, post-mortem delay, tissue storage time and sex had no significant effects on any of the parameters measured. Dissociation constants (K(D) values) for MK-801 showed similar mean values in the four cortical areas, whereas receptor densities (B(max) values) showed significant differences between sensorimotor or occipital and superior temporal or mid-frontal cortex. There were marked regional differences in the profiles of the spermine- and glutamate-incremented enhancement of specific [3H]MK-801 binding. The EC(50) for the glutamate enhancement was significantly higher in the occipital than in the mid-frontal and sensorimotor cortex, whereas maximal glutamate-enhanced binding values did not differ. The maximal enhancement of [3H]MK-801 binding by spermine and glutamate varied between the cases, ranging from zero to 40.4+/-9.3 fmol x mg protein(-1) for spermine, and from 85+/-5 to 111+/-10 fmol x mg protein(-1) for glutamate. Maximal spermine enhancement of [3H]MK-801 binding was significantly more variable in superior temporal or mid-frontal than in sensorimotor or occipital cortex. The results suggest that N-methyl-D-aspartate receptor sites, especially the polyamine site, are heterogeneous in human cerebral cortex, and show a high degree of regional and individual variability.

Age Factors↗

PubMed1999

Increased expression of mitochondrial genes in human alcoholic brain revealed by differential display.

Polymerase chain reaction (PCR)-based differential display was used to screen for alterations in gene expression in the mesolimbic system of the human alcoholic brain. Total RNA was extracted from the nucleus accumbens of five alcoholic and five control brains. A selected subpopulation of mRNA was reverse-transcribed to cDNA and amplified by PCR. A differentially expressed cDNA fragment was recovered, cloned, and sequenced. Full sequence analysis of this 467 bp fragment revealed 98.2% homology with the human mitochondrial 12S rRNA gene. Dot-blot analysis showed increased expression of this gene in nucleus accumbens and hippocampus, but not in the superior frontal cortex, primary motor cortex, caudate, and pallidus/putamen in a total of eight human alcoholic brains, compared with seven control brains. A similar increased expression was observed by dot-blot analysis, using RNA from the cerebral cortex of rats chronically treated with alcohol vapor. Hybridization of a 16S rRNA oligonucleotide probe indicated that the expression of both rRNAs genes was significantly increased in nucleus accumbens. These results indicate that chronic alcohol consumption induces alteration in expression of mitochondrial genes in selected brain regions. The altered gene expression may reflect mitochondrial dysfunction in the alcohol-affected brain.

Adult↗

PubMed1998

Association of missense and 5'-splice-site mutations in tau with the inherited dementia FTDP-17.

Thirteen families have been described with an autosomal dominantly inherited dementia named frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), historically termed Pick's disease. Most FTDP-17 cases show neuronal and/or glial inclusions that stain positively with antibodies raised against the microtubule-associated protein Tau, although the Tau pathology varies considerably in both its quantity (or severity) and characteristics. Previous studies have mapped the FTDP-17 locus to a 2-centimorgan region on chromosome 17q21.11; the tau gene also lies within this region. We have now sequenced tau in FTDP-17 families and identified three missense mutations (G272V, P301L and R406W) and three mutations in the 5' splice site of exon 10. The splice-site mutations all destabilize a potential stem-loop structure which is probably involved in regulating the alternative splicing of exon10. This causes more frequent usage of the 5' splice site and an increased proportion of tau transcripts that include exon 10. The increase in exon 10+ messenger RNA will increase the proportion of Tau containing four microtubule-binding repeats, which is consistent with the neuropathology described in several families with FTDP-17.

Alternative Splicing↗

PubMed1998

Cell death mediated by amino acid transmitter receptors in human alcoholic brain damage: conflicts in the evidence.

Human alcoholics have reduced neuronal counts in certain brain regions, such as superior frontal cortex (SFC), where the form and quantity of synaptic gamma-aminobutyric acid type A (GABAA) receptor sites are atypical. We measured the expression of GABAA receptor isoform mRNA and protein, since GABAA receptor pharmacology is strongly influenced by its subunit composition. Cortex samples were obtained at autopsy; whole-tissue extracts were assayed for mRNA by quantitative reverse transcriptase polymerase chain reaction (RT-PCR), while synaptic membranes were studied for both GABAA receptor pharmacology and subunit protein levels by Western blots with isoform-specific antibodies. Although alpha 1 and alpha 3 mRNA species were strongly expressed in alcoholics irrespective of cirrhosis than in controls, alpha 1 protein differed little between case groups, and alpha 3 protein showed some complex variations. Differences in GABAA pharmacology conformed more closely with differences in protein levels than with altered mRNA expression.

Alcoholism↗

PubMed1998

Expression of GABA(A) receptor isoform genes in the cerebral cortex of cirrhotic and alcoholic cases assessed by S1 nuclease protection assays.

Pathogenic processes underlying the localized reduction in neuronal number in cerebral cortex in human alcoholics have been reported to be associated with selective variations in the parameters of GABA(A) receptor site binding. Since the properties of the receptor complex depend on its isoform composition, we studied how the expression of GABA(A) receptor subunit isoform genes varied with alcoholism. Cerebral cortex tissue was obtained at autopsy from chronic human alcoholics (average ethanol intake > 80 g/day for most of their adult lives; n = 17) and matched controls (< 20 g/day ethanol; n = 15). Eight of the alcoholics and five of the controls had pathologically confirmed cirrhosis of the liver. Expression of alpha1, alpha2, alpha3, alpha5, beta1, beta3, and gamma2 GABA(A) mRNA was assessed by S1 nuclease protection assays. After phosphorimager quantitation and normalization to GAPDH mRNA and 18S rRNA, none of the mRNA species showed significantly different expression in uncomplicated alcoholics. Analysis of differences in the patterns of expression of the various subunits showed the alpha1 signal was strongest in combined cirrhotic motor cortex while the alpha3 and beta3 values were greatest in combined cirrhotic frontal cortex. It appears that only major differences in mRNA expression may be detected by this technique in human brain.

Adult↗

PubMed1998

Regional development of glutamate-N-methyl-D-aspartate receptor sites in asphyxiated newborn infants.

The N-methyl-D-aspartate (NMDA) subclass of glutamate receptors was examined in newborn infants dying between 25 weeks' gestation and term, either from acute cerebral hypoxia, or from other noncerebral conditions incompatible with life. Frontal, occipital, temporal, and motor cortex tissue samples were obtained at autopsy (post mortem delay: median, 45.9 hr; range, 24-96 hr) and frozen for subsequent [3H]MK801 homogenate binding assays. Whereas no significant variation was observed in ligand affinity (KD), in all cases receptor density (BMAX) increased with gestational age, in occipital cortex (27 weeks, BMAX = 222 +/- 44 fmol x mg protein(-1); 39 weeks, 439 +/- 42 fmol x mg protein[-1]), but not in motor or temporal cortex. The gestational-age increase also occurred in control frontal cortex (27 weeks, 284 +/- 80; 39 weeks, 567 +/- 40 fmol x mg protein[-1]), but was significantly less marked in frontal cortex in hypoxia cases (27 weeks, 226 +/- 90; 39 weeks, 326 +/- 47 fmol x mg protein[-1]). In all cortical areas except temporal, the maximal response to glutamate did not vary across case groups. Hypoxia cases showed an increased response to glutamate enhancement selectively in temporal cortex. Binding site density did not correlate with degree of hypoxia as assessed pathologically, suggesting that receptor differences preceded the hypoxic episode. Regional differences in glutamate-NMDA receptor sites may underlie increased vulnerability to hypoxia at birth.

Autopsy↗