Effects of naltrexone are influenced by childhood adversity during negative emotional processing in addiction recovery.

Naltrexone is an opioid receptor antagonist used in the management of alcohol dependence. Although the endogenous opioid system has been implicated in emotion regulation, the effects of mu-opioid receptor blockade on brain systems underlying negative emotional processing are not clear in addiction. Individuals meeting criteria for alcohol dependence alone (n=18, alcohol) and in combination with cocaine and/or opioid dependence (n=21, alcohol/drugs) and healthy individuals without a history of alcohol or drug dependence (n=21) were recruited. Participants were alcohol and drug abstinent before entered into this double-blind, placebo-controlled, randomized, crossover study. Functional magnetic resonance imaging was used to investigate brain response while viewing aversive and neutral images relative to baseline on 50 mg of naltrexone and placebo. We found that naltrexone modulated task-related activation in the medial prefrontal cortex and functional connectivity between the anterior cingulate cortex and the hippocampus as a function of childhood adversity (for aversive versus neutral images) in all groups. Furthermore, there was a group-by-treatment-by-condition interaction in the right amygdala, which was mainly driven by a normalization of response for aversive relative to neutral images under naltrexone in the alcohol/drugs group. We conclude that early childhood adversity is one environmental factor that influences pharmacological response to naltrexone. Pharmacotherapy with naltrexone may also have some ameliorative effects on negative emotional processing in combined alcohol and drug dependence, possibly due to alterations in endogenous opioid transmission or the kappa-opioid receptor antagonist actions of naltrexone.

Ageing diminishes the modulation of human brain responses to visual food cues by meal ingestion.

BACKGROUND/OBJECTIVES: Rates of obesity are greatest in middle age. Obesity is associated with altered activity of brain networks sensing food-related stimuli and internal signals of energy balance, which modulate eating behaviour. The impact of healthy mid-life ageing on these processes has not been characterised. We therefore aimed to investigate changes in brain responses to food cues, and the modulatory effect of meal ingestion on such evoked neural activity, from young adulthood to middle age. SUBJECTS/METHODS: Twenty-four healthy, right-handed subjects, aged 19.5-52.6 years, were studied on separate days after an overnight fast, randomly receiving 50 ml water or 554 kcal mixed meal before functional brain magnetic resonance imaging while viewing visual food cues. RESULTS: Across the group, meal ingestion reduced food cue-evoked activity of amygdala, putamen, insula and thalamus, and increased activity in precuneus and bilateral parietal cortex. Corrected for body mass index, ageing was associated with decreasing food cue-evoked activation of right dorsolateral prefrontal cortex (DLPFC) and precuneus, and increasing activation of left ventrolateral prefrontal cortex (VLPFC), bilateral temporal lobe and posterior cingulate in the fasted state. Ageing was also positively associated with the difference in food cue-evoked activation between fed and fasted states in the right DLPFC, bilateral amygdala and striatum, and negatively associated with that of the left orbitofrontal cortex and VLPFC, superior frontal gyrus, left middle and temporal gyri, posterior cingulate and precuneus. There was an overall tendency towards decreasing modulatory effects of prior meal ingestion on food cue-evoked regional brain activity with increasing age. CONCLUSIONS: Healthy ageing to middle age is associated with diminishing sensitivity to meal ingestion of visual food cue-evoked activity in brain regions that represent the salience of food and direct food-associated behaviour. Reduced satiety sensing may have a role in the greater risk of obesity in middle age.

Changes in neuropsychological functioning during alcohol detoxification.

This study investigates changes in neuropsychological functioning during early abstinence from alcohol. 30 alcohol-dependent inpatients were tested at intake (day 4 of admission) and post detoxification (day 26), using a test-retest design. The neuropsychological battery included measures of pre-morbid IQ, full-scale IQ, verbal and non-verbal measures of memory and executive function. IQ was within the normal range at intake and comparable with age-adjusted normative values and there were some impairments in memory and executive function. There were significant increases in performance scores post detoxification in working memory, verbal fluency and verbal inhibition but not in non-verbal executive function tasks (mental flexibility and planning ability). Despite increased scores on tests of verbal and memory skills after 3 weeks of abstinence, complex executive abilities showed little change. These may have a negative impact on engagement and response to treatment and compromise clinical outcomes, heightening the risk of relapse.

Induction of patients with moderately severe methadone dependence onto buprenorphine.

Current clinical practice allows patients with low levels of physiological dependence on opioids (equivalent to methadone doses of 30 mg/d or less) to be transferred to buprenorphine. This study investigated the response of opioid-dependent patients receiving doses of methadone between 30-70 mg/d when transferred to buprenorphine at doses between 12-16 mg/d. Twenty-three patients receiving inpatient opioid detoxification agreed to take part in a trial of facilitated transfer to buprenorphine. Following the last morning dose of methadone, buprenorphine was substituted in doses increasing from 4 mg to a maximum of 16 mg, with adjunctive lofexidine (maximum of 2.4 mg/d). All except two patients successfully completed transfer to buprenorphine. To investigate the effect of initial methadone dose, the group was split into intermediate dose (ID; 30 - 49 mg/d; n = 10) and high dose (HD; 50-70 mg/d; n = 11) groups. Average stabilisation dose of buprenorphine for the sample who completed transfer was 14.0 mg/d (SD 2.3) and average daily lofexidine dose during transfer was 0.57 mg (SD 0.39). The HD group used significantly more lofexidine to complete transfer compared to the ID group. Increased opioid withdrawal symptoms, of mild severity as measured by the Short Opiate Withdrawal Scale (SOWS), were found in the HD group compared with the ID group during the first and last day of buprenorphine stabilisation. However, average SOWS scores for the whole of the period of transfer were not significantly different from those during the period of stabilisation on buprenorphine in either the ID or HD groups. This study suggests that transfer to buprenorphine is relatively uncomplicated from daily methadone doses of 30-70 mg in an inpatient setting and may be facilitated by use of lofexidine. This procedure may allow a larger proportion of opioid-dependent patients access to buprenorphine treatment.

Differential changes in brain glucose metabolism during hypoglycaemia accompany loss of hypoglycaemia awareness in men with type 1 diabetes mellitus. An [11C]-3-O-methyl-D-glucose PET study.

AIMS/HYPOTHESIS: Hypoglycaemia unawareness in type 1 diabetes increases the risk of severe hypoglycaemia and impairs quality of life for people with diabetes. To explore the central mechanisms of hypoglycaemia awareness, we used [11C]-3-O-methyl-D-glucose (CMG) positron emission tomography (PET) to measure changes in global and regional brain glucose metabolism between euglycaemia and hypoglycaemia in aware and unaware diabetic subjects. MATERIALS AND METHODS: Twelve men with type 1 diabetes, of whom six were characterised as aware and six as unaware of hypoglycaemia, underwent two CMG-PET brain scans while plasma glucose was controlled by insulin and glucose infusion either at euglycaemia (5 mmol/l) or at hypoglycaemia (2.6 mmol/l) in random order. RESULTS: With hypoglycaemia, symptoms and sweating occurred only in the aware group. Brain glucose content fell in both groups (p=0.0002; aware, 1.18+/-0.45 to 0.02+/-0.2 mmol/l; unaware, 1.07+/-0.46 to 0.19+/-0.23 mmol/l), with a relative increase in tracer uptake in prefrontal cortical regions, including the anterior cingulate. No detectable differences were found between groups in global brain glucose transport parameters (K1, k2). The cerebral metabolic rate for glucose (CMRglc) showed a relative rise in the aware subjects (11.839+/-2.432 to 13.958+/-2.372) and a fall in the unaware subjects (from 12.457+/-1.938 to 10.16+/-0.801 micromol 100 g(-1) min(-1), p=0.043). CONCLUSIONS/INTERPRETATION: Hypoglycaemia is associated with reduced brain glucose content in aware and unaware subjects, with a relative preservation of metabolism in areas associated with sympathetic activation. The relative rise in global glucose metabolic rate seen in aware subjects during hypoglycaemia contrasted with the relative fall in the unaware subjects and suggests that cortical neuronal activation is a necessary correlate of the state of hypoglycaemia awareness.

Amnesic syndrome and severe ataxia following the recreational use of 3,4-methylene-dioxymethamphetamine (MDMA, 'ecstasy') and other substances.

A 26-year-old woman suffered disseminated intravascular coagulation (DIC) and a brief respiratory arrest following recreational use of 3,4-methylene-dioxymethamphetamine (MDMA; 'ecstasy'), together with amyl nitrate, lysergic acid (LSD), cannabis and alcohol. She was left with residual cognitive and physical deficits, particularly severe anterograde memory disorder, mental slowness, severe ataxia and dysarthria. Follow-up investigations have shown that these have persisted, although there has been some improvement in verbal recognition memory and in social functioning. Magnetic resonance imaging and quantified positron emission tomography investigations have revealed: (i) severe cerebellar atrophy and hypometabolism accounting for the ataxia and dysarthria; (ii) thalamic, retrosplenial and left medial temporal hypometabolism to which the anterograde amnesia can be attributed; and (iii) some degree of fronto-temporal-parietal hypometabolism, possibly accounting for the cognitive slowness. The putative relationship of these abnormalities to the direct and indirect effects of MDMA toxicity, hypoxia and ischaemia is considered.

The remarkable structural and functional organization of the eukaryotic pyruvate dehydrogenase complexes.

The three-dimensional reconstruction of the bovine kidney pyruvate dehydrogenase complex (M(r) approximately 7.8 x 10(6)) comprising about 22 molecules of pyruvate dehydrogenase (E(1)) and about 6 molecules of dihydrolipoamide dehydrogenase (E(3)) with its binding protein associated with the 60-subunit dihydrolipoamide acetyltransferase (E(2)) core provides considerable insight into the structural and functional organization of the largest multienzyme complex known. The structure shows that potentially 60 centers for acetyl-CoA synthesis are organized in sets of three at each of the 20 vertices of the pentagonal dodecahedral core. These centers consist of three E(1) molecules bound to one E(2) trimer adjacent to an E(3) molecule in each of 12 pentagonal openings. The E(1) components are anchored to the E(1)-binding domain of the E(2) subunits through an approximately 50-A-long linker. Three of these linkers emanate from the outside edges of the triangular base of the E(2) trimer and form a cage around its base that may shelter the lipoyl domains and the E(1) and E(2) active sites. The docking of the atomic structures of E(1) and the E(1) binding and lipoyl domains of E(2) in the electron microscopy map gives a good fit and indicates that the E(1) active site is approximately 95 A above the base of the trimer. We propose that the lipoyl domains and its tether (swinging arm) rotate about the E(1)-binding domain of E(2,) which is centrally located 45-50 A from the E(1), E(2), and E(3) active sites, and that the highly flexible breathing core augments the transfer of intermediates between active sites.

Changes in regional brain (18)F-fluorodeoxyglucose uptake at hypoglycemia in type 1 diabetic men associated with hypoglycemia unawareness and counter-regulatory failure.

We examined the effects of acute moderate hypoglycemia and the condition of hypoglycemia unawareness on regional brain uptake of the labeled glucose analog [(18)F]fluorodeoxyglucose (FDG) using positron emission tomography (PET). FDG-PET was performed in diabetic patients with (n = 6) and without (n = 7) hypoglycemia awareness. Each patient was studied at plasma glucose levels of 5 and 2.6 mmol/l, applied by glucose clamp techniques, in random order. Hypoglycemia-unaware patients were asymptomatic during hypoglycemia, with marked attenuation of their epinephrine responses (mean [+/- SD] peak of 0.77 +/- 0.39 vs. 7.52 +/- 2.9 nmol/l; P < 0.0003) and a reduced global brain FDG uptake ([mean +/- SE] 2.592 +/- 0.188 vs. 2.018 +/- 0.174 at euglycemia; P = 0.027). Using statistical parametric mapping (SPM) to analyze images of FDG uptake, we identified a subthalamic brain region that exhibited significantly different behavior between the aware and unaware groups. In the aware group, there was little change in the normalized FDG uptake in this region in response to hypoglycemia ([mean +/- SE] 0.654 +/- 0.016 to 0.636 +/- 0.013; NS); however, in the unaware group, the uptake in this region fell from 0.715 +/- 0.015 to 0.623 +/- 0.012 (P = 0.001). Our data were consistent with the human hypoglycemia sensor being anatomically located in this brain region, and demonstrated for the first time a change in its metabolic function associated with the failure to trigger a counter-regulatory response.

Direct evidence for the size and conformational variability of the pyruvate dehydrogenase complex revealed by three-dimensional electron microscopy. The "breathing" core and its functional relationship to protein dynamics.

Structural studies by three-dimensional electron microscopy of the Saccharomyces cerevisiae truncated dihydrolipoamide acetyltransferase (tE(2)) component of the pyruvate dehydrogenase complex reveal an extraordinary example of protein dynamics. The tE(2) forms a 60-subunit core with the morphology of a pentagonal dodecahedron and consists of 20 cone-shaped trimers interconnected by 30 bridges. Frozen-hydrated and stained molecules of tE(2) in the same field vary in size approximately 20%. Analyses of the data show that the size distribution is bell-shaped, and there is an approximately 40-A difference in the diameter of the smallest and largest structures that corresponds to approximately 14 A of variation in the length of the bridge between interconnected trimers. Companion studies of mature E(2) show that the complex of the intact subunit exhibits a similar size variation. The x-ray structure of Bacillus stearothermophilus tE(2) shows that there is an approximately 10-A gap between adjacent trimers and that the trimers are interconnected by the potentially flexible C-terminal ends of two adjacent subunits. We propose that this springlike feature is involved in a thermally driven expansion and contraction of the core and, since it appears to be a common feature in the phylogeny of pyruvate dehydrogenase complexes, protein dynamics is an integral component of the function of these multienzyme complexes.

Noradrenergic dysfunction in the prefrontal cortex in depression: an [15O] H2O PET study of the neuromodulatory effects of clonidine.

BACKGROUND: Noradrenergic dysfunction has been consistently implicated in depression. Much of the evidence, though, has been indirect, such as an attenuated growth hormone response to the alpha2-adrenoceptor agonist clonidine. To more directly examine central functioning of the noradrenergic system in depression, we have used [15O] H2O positron emission tomography (PET) to measure cerebral blood flow (rCBF) in combination with clonidine as a neuromodulatory probe. METHODS: Subjects were six depressed and six healthy women, medication free and matched for age and phase of menstrual cycle. Two PET scans were acquired at baseline and two scans at 20 and 35 min following an intravenous clonidine infusion of 1.4 microg/kg while subjects performed a sustained attention task. RESULTS: The growth hormone response did not show a significant difference between groups. However, PET results revealed a difference in the right superior prefrontal cortex that was resolved as an interaction from decreased rCBF in healthy control subjects but increased rCBF in the depressed group, which was not accounted for by differences in task performance. CONCLUSIONS: This differential effect of clonidine in the right prefrontal cortex provides in vivo evidence of noradrenergic dysfunction in depression, which we postulate arises from functionally impaired presynaptic alpha2-adrenoceptors as well as regionally "supersensitive" postsynaptic cortical alpha2-adrenoceptors.

One-step purification of the recombinant catalytic subunit of pyruvate dehydrogenase phosphatase.

A facile one-step affinity chromatographic purification of the recombinant catalytic subunit (PDPc) of bovine pyruvate dehydrogenase phosphatase (PDP) to near homogeneity is described. PDPc binds in the presence of Ca(2+) to the inner lipoyl domain (L2) of the dihydrolipoamide acetyltransferase component (E2) of the mammalian pyruvate dehydrogenase complex. The affinity column consists of a glutathione S-transferase (GST)-L2 fusion protein bound to glutathione-Sepharose 4B beads. An extract of transformed Escherichia coli cells containing 50 mM Tris buffer (pH 7.5), 2 mM CaCl(2), 5 mM MgCl(2,) 150 mM NaCl, 0.5 mM dithiothreitol, 1% Triton X-100, and l M urea was passed through the affinity column, and the column was washed extensively with this buffer mixture. PDPc was eluted with 50 mM Tris buffer (pH 7.5) containing 5 mM MgCl(2), 0.5 mM dithiothreitol, and 1 mM EGTA. Approximately 22 mg of highly purified PDPc was obtained from 10 g (wet weight) of transformed cells. The preparation contained a small amount of a "nicked" form of PDPc. The cleavage is between Arg-394 and Arg-395.

Expression, purification, and structural analysis of the trimeric form of the catalytic domain of the Escherichia coli dihydrolipoamide succinyltransferase.

The dihydrolipoamide succinyltransferase (E2o) component of the alpha-ketoglutarate dehydrogenase complex catalyzes the transfer of a succinyl group from the S-succinyldihydrolipoyl moiety to coenzyme A. E2o is normally a 24-mer, but is found as a trimer when E2o is expressed with a C-terminal [His]6 tag. The crystal structure of the trimeric form of the catalytic domain (CD) of the Escherichia coli E2o has been solved to 3.0 A resolution using the Molecular Replacement method. The refined model contains an intact trimer in the asymmetric unit and has an R-factor of 0.257 (Rfree = 0.286) for 18,699 reflections between 10.0 and 3.0 A resolution. The core of tE2oCD (residues 187-396) superimposes onto that of the cubic E2oCD with an RMS difference of 0.4 A for all main-chain atoms. The C-terminal end of tE2oCD (residues 397-404) rotates by an average of 37 degrees compared to cubic E2oCD, disrupting the normal twofold interface. Despite the alteration of quaternary structure, the active site of tE2oCD shows no significant differences from that of the cubic E2oCD, although several side chains in the active site are more ordered in the trimeric form of E2oCD. Analysis of the available sequence data suggests that the majority of E2 components have active sites that resemble that of E. coli E2oCD. The remaining E2 components can be divided into three groups based on active-site sequence similarity. Analysis of the surface properties of both crystal forms of E. coli E2oCD suggests key residues that may be involved in the protein-protein contacts that occur between the catalytic and lipoyl domains of E2o.

Severe delayed hemolytic transfusion reaction secondary to anti-At(a).

BACKGROUND: Anti-At(a) is a rare red cell (RBC) alloantibody found in the black population. It has been described as causing one case of mild hemolytic disease of the newborn, but its ability to cause hemolytic transfusion reactions is uncertain. CASE REPORT: The patient was a 60-year-old black female with a history of three uneventful pregnancies but no transfusions. On admission, her direct and indirect antiglobulin tests were negative, total bilirubin was 0.5 mg per dL, and lactate dehydrogenase was 224 IU per L. She received nine units of compatible RBCs in the perioperative period of a hemicolectomy. Her hemoglobin rose appropriately and stabilized at 12.6 g per dL by the 6th postoperative day. By Day 10 after surgery her hemoglobin had dropped to 6.8 g per dL, and her total bilirubin and lactate dehydrogenase had risen to 1.4 mg per dL and 783 IU per L, respectively. The direct and indirect antiglobulin tests were now newly positive with strengths of 3+. A warm hemolytic autoantibody was suspected. She was transfused two units of incompatible RBCs for a rapidly falling hemoglobin and symptomatic anemia. On Day 11, the total bilirubin rose to 3.5 mg per dL, and the lactate dehydrogenase was 1154 IU per L with a hemoglobin of 7.6 g per dL. Corticosteroids were begun. Studies of serum and an acid eluate revealed anti-At(a), but no other RBC antibodies. The patient stabilized, and further transfusion was avoided. CONCLUSION: Although anti-At(a) was previously described as being of uncertain clinical significance, this patient demonstrated the ability of the antibody to cause a severe delayed hemolytic transfusion reaction.

FDG-PET analysis and findings in amnesia resulting from hypoxia.

The assumptions underlying neuroimaging, and problems in its analysis and interpretation, are commonly underestimated in neuropsychology. The ways in which fluoro-deoxy-glucose (FDG) positron emission tomography (PET) data can be analysed are discussed. PET findings from four patients who had suffered severe amnesia, following episodes of acute hypoxia, are presented. These patients had shown evidence of medial temporal (hippocampal and parahippocampal) atrophy on MRI brain scans. The PET data were analysed in several different ways. The converging findings were that the patients showed bilateral thalamic hypometabolism, and there was also evidence of retrosplenial hypometabolism bilaterally. Cognitively, these patients performed most like other patients with medial temporal lesions, but the results indicate that structural lesions can have distal metabolic effects on structures elsewhere. These findings are interpreted in the light of neuroanatomical observations concerning parallel projections between medial temporal lobe structures and the thalamus, some of which pass via the retrosplenium.

Crystal structure of the truncated cubic core component of the Escherichia coli 2-oxoglutarate dehydrogenase multienzyme complex.

The dihydrolipoamide succinyltransferase (E2o) component of the 2-oxoglutarate dehydrogenase multienzyme complex is composed of 24 subunits arranged with 432 point group symmetry. The catalytic domain (CD) of the E2o component catalyzes the transfer of a succinyl group from the S-succinyldihydrolipoyl moiety to coenzyme A. The crystal structure of the Escherichia coli E2oCD has been solved to 3.0 A resolution using molecular replacement phases derived from the structure of the catalytic domain from the Azotobacter vinelandii dihydrolipoamide acetyltransferase (E2pCD). The refined model of the E. coli E2oCD consists of residues 172 to 404 and has an R-factor of 0.205 (Rfree=0.249) for 9696 reflections between 20.0 and 3.0 A resolution. Although both E2oCD and E2pCD form 24mers, subtle changes in the orientations of two helices in E2oCD increase the stability of the E2oCD 24mer in comparison to the less stable A. vinelandii E2pCD 24mer. Like E2pCD and chloramphenicol acetyltransferase (CAT), the active site of E2oCD is located in the middle of a channel formed at the interface between two 3-fold related subunits. Two of the active-site residues (His375 and Thr323) have a similar orientation to their counterparts in E2pCD and CAT. A third catalytic residue (Asp379) assumes a conformation similar to the corresponding residue in E2pCD (Asn614), but different from its counterpart in CAT (Asp199). Binding of the substrates to E2oCD is proposed to induce a change in the conformation of Asp379, allowing this residue to form a salt bridge with Arg184 that is analogous to that formed between Asp199 and Arg18 in CAT. Computer models of the active site of E2o complexed with dihydrolipoamide and with coenzyme A led to the identification of the probable succinyl-binding pocket. The residues which form this pocket (Ser330, Ser333, and His348) are probably responsible for E2o's substrate specificity.

Crystal structure of eucaryotic E3, lipoamide dehydrogenase from yeast.

The crystal structure of eucaryotic lipoamide dehydrogenase from yeast has been determined by an X-ray analysis at 2.7 (partially at 2.4) A resolution. The enzyme has two identical subunits related by a pseudo twofold symmetry. The tertiary structure is similar to those of other procaryotic enzymes. The active site, consisting of FAD, Cys44, and Cys49 from one subunit and His457' from the other subunit, is highly conserved. This enzyme is directly bound to the core protein E2 of the 2-oxoglutarate dehydrogenase complex, whereas it is bound to the pyruvate dehydrogenase complex through a protein X. The calculated electrostatic potential suggests two characteristic regions for binding with these two proteins.