Structural and conformational similarity between synthetic peptides of curaremimetic neurotoxins and rabies virus glycoprotein.

Antibodies were raised in rabbits against synthetic peptides corresponding to loop 2, the 'toxic' loop reacting with the acetylcholine-binding site on the nicotinic acetylcholine receptor, of curaremimetic neurotoxins and the structurally similar segment of the rabies virus glycoprotein. Some of the antibodies cross-reacted with the corresponding peptides confirming the structural similarity between the neurotoxin and glycoprotein peptides. A polyclonal antibody raised against a 29 residue glycoprotein peptide (175-203) in the presence of 0.1% sodium dodecyl sulfate reacted with native alpha-bungarotoxin and rabies virus. Circular dichroism spectroscopy of the 29 residue glycoprotein peptide and a 20 residue king cobra loop 2 peptide (25-44) revealed these peptides to be conformationally similar and composed predominantly of beta sheet structure. These results show the rabies glycoprotein segment is structurally and conformationally similar to neurotoxin loop 2. This similarity may confer on the glycoprotein the capability of interacting with the neurotoxin-binding site on the acetylcholine receptor.     

Event-related potential P300 microstate topography during visual one- and two-dimensional tasks in chronic schizophrenics

Reports on left-lateralized abnormalities of component P300 of event-related brain potentials (ERP) in schizophrenics typically did not vary task difficulties. We collected 16-channel ERP in 13 chronic, medicated schizophrenics (25±4.9 years) and 13 matched controls in a visual P300 paradigm with targets defined by one or two stimulus dimensions (C1: color; C2: color and tilt); subjects key-pressed to targets. The mean target-ERP map landscapes were assessed numerically by the locations of the positive and negative map-area centroids. The centroids' time-space trajectories were searched for the P300 microstate landscape defined by the positive centroid posterior of the negative centroid. At P300 microstate centre latencies in C1, patients' maps tended to a right shift of the positive centroid (p<0.10); in C2 the anterior centroid was more posterior (p<0.07) and the posterior (positive) centroid more anterior (p<0.03), but without leftright difference. Duration of P300 microstate in C2 was shorter in patients (232 vs 347 ms;p<0.03) and the latency of maximal strength of P300 microstate increased significantly in patients (C1: 459 vs 376 ms; C2: 585 vs 525 ms). In summary only the one-dimensional task C1 supported left-sided abnormalities; the two-dimensional task C2 produced abnormal P300 microstate map landscapes in schizophrenics, but no abnormal lateralization. Thus, information processing involved clearly aberrant neural populations in schizophrenics, different when processing one and two stimulus dimensions. The lack of lateralization in the two-dimensional task supported the view that left-temporal abnormality in schizophrenics is only one of several task-dependent aberrations.     

Proton NMR spectroscopy of Canavan's disease.

Proton Nuclear Magnetic Resonance (NMR) spectroscopy was used to quantitatively determine cerebral N-Acetyl Aspartate (NAA) concentrations in four patients with Canavan's disease and in four age-matched control subjects. Macroscopic NAA concentrations (mumol/gm wet weight) were not found to be significantly different from controls. Reduced levels of choline and creatine were observed in all patients, and increased levels of lactate and inositol in the eldest three patients.     

Synthesis, conformational and pharmacological studies of glycosylated chimeric peptides of Met-enkephalin and FMRFa

Our previous study showed that a chimeric peptide of Met-enkephalin and FMRFamide, YFa (YGGFMKKKFMRFa) not only caused antinociception and potentiated morphine analgesia but also blocked the development of tolerance and physical dependence. In the continuation of that study three chimeric analogues of YFa, [Ser5]YFa, [O-Glu-Ser5]YFa and [O-Gal-Ser5]YFa, were synthesized. To increase the bioavailability and penetration of blood brain barrier (BBB), glycosylated analogues, [O-Glu-Ser5]YFa and [O-Gal-Ser5]YFa, have been synthesized by solid phase peptide synthesis by building block method using anomeric acetate activation method. Circular dichroism studies showed that all the three chimeric peptides are stable and have a propensity for adopting helical conformation in the presence of membrane mimicking solvent. In comparison of parent chimeric peptide YFa, helicity of [Ser5]YFa, [O-Glu-Ser5]YFa and [O-Gal-Ser5]YFa has decreased. Pharmacological studies using tail-flick latency in mice showed that [O-Glu-Ser5]YFa have increased analgesia and bioavailability in comparison of [O-Gal-Ser5]YFa and non-glycosylated analogue [Ser5]YFa. Exhibition of enhanced analgesia by [O-Glu-Ser5]YFa as compared to [O-Gal-Ser5]YFa seems to be due to preference of GLUT-1 transporter system for glucose.     

Human muscle proteins: analysis by two-dimensional electrophoresis

Proteins from single frozen sections of human muscle were separated by two-dimensional gel electrophoresis and detected by fluorography or Coomassie Blue staining. The major proteins were identical in different normal muscles obtained from either sex at different ages, and in Duchenne and myotonic dystrophy samples. Congenital myopathy, denervation atrophy, polymyositis, and Becker's muscular dystrophy samples, however, showed abnormal myosin light chain compositions, some with a decrease of fast-fiber myosin light chains and others with a decrease of slow-fiber light chains. These protein alterations did not correlate with any specific disease, and may be caused by generalized muscle-fiber damage.     

Diagnosis of muscular glycogenosis by in vivo natural abundance 13C NMR spectroscopy.

Natural abundance 13C NMR (nuclear magnetic resonance) spectroscopy was used to distinguish patients suffering from muscle glycogenosis type V (McArdle's disease) from normal subjects by measuring their muscle glycogen content at rest. Proton-decoupled 13C spectra were obtained in 10-15 min from calf muscles at rest. The ratio of the glycogen/creatine signal areas was 12.9 +/- 1.7 in four McArdle's disease patients and 2.0 +/- 0.7 in seven normal subjects. This technique thus allows the non-invasive diagnosis of muscle glycogenosis.     

Contribution of in vitro NMR spectroscopy to metabolic and neurodegenerative disorders

In vitro Nuclear Magnetic Resonance (NMR) spectroscopy is a validated biochemical tool for metabolic analyses of human body fluids and diagnosis of inborn errors of metabolism in children and adults. The technique is of special interest because it requires minimal sample preparation, it can detect simultaneously compounds of different nature and it offers structural information on the metabolites present in body fluids. In the last decade, in vitro NMR spectroscopy contributed to the identification of new inborn errors of metabolism, some of which are amenable to therapeutic intervention. Standardized analyses of body fluids, especially cerebrospinal fluid, are therefore indicated in patients affected with neurodegenerative disorders of unknown etiologies, for which extensive metabolic and genetic screening failed to identify the primary defect. In addition, the use of multivariate statistical analyses allows the comparison of the whole metabolic profile between patients with a given neurodegenerative disorder and controls. This so-called metabonomic approach yields promises for the discovery of biomarkers that can help detecting disease onset and progression, as well as evaluating therapeutic efficacy. Finally, the combination of in vitro NMR spectroscopy with genetic analytical tools may constitute a successful pathophysiological approach to investigate neurological disorders of unknown etiology.     

Exploration of exercise intolerance by 31P NMR spectroscopy of calf muscles coupled with MRI and ergometry

One hundred patients presenting with exercise intolerance or rhabdomyolysis episodes have been examined successively by 31P Nuclear Magnetic Resonance Spectroscopy (MRS) of leg plantar flexor muscles with exercise test. In all cases a muscle biopsy was performed. At the end of investigations, diagnosis of a metabolic myopathy was made in 33 patients: glycogenolysis or glycolysis deficiency in 8 cases, mitochondrial myopathy in 24 cases and CPT II deficiency in one case. Muscular dystrophy or congenital myopathy were diagnosed in 6 cases. No precise etiology could be found in 30 patients with either high CK levels or muscle biopsy abnormalities. Seven patients had rhabdomyolysis related to excessive physical activities. Twenty-four patients had functional symptoms. The principal MRS parameters used for diagnosis were the values of intracellular pH at the end of exercise and the time constant of phosphocreatine resynthesis during recovery. Lack of acidosis after exercise was observed in all patients with blockade of glycogenolysis or glycolysis. A slowing in phosphocreatine resynthesis was found in 66 p.cent of patients with definite mitochondrial myopathy. The specificity of these parameters were respectively 92.4 p.cent and 85.5 p.cent for the two groups. In conclusion (31)P MRS allows the detection of muscular glycogenoses with a sensitivity close to 100 p.cent. However, its sensitivity was lower for the detection of mitochondrial myopathies, as is also known for the other in vivo metabolic investigations, reflecting the heterogeneity of expression of mitochondrial abnormalities in a given muscle. The integration of imaging in the examination protocol may help to orientate towards the diagnostic of a dystrophy in some patients.     

Oxidative stress-induced metabolic alterations in rat brain astrocytes studied by multinuclear NMR spectroscopy.

Oxidative stress in cultured astrocytes exerted by 30-min treatment with 50-200 microM H(2)O(2) caused time- and concentration-dependent effects on cellular metabolism. These changes were accompanied by alterations in cellular morphology. Using (31)P nuclear magnetic resonance (NMR) spectroscopy, the data demonstrate that the energy status of the cells was greatly affected directly after the stress, as indicated by the loss of high energy phosphates, i.e., phosphocreatine (PCr) and nucleoside triphosphates (NTP). Oxidative stress also involves a dysregulation of the osmotic control in astrocytes, which is accompanied by a dramatic loss of myo-inositol, taurine, and hypotaurine, as monitored by (1)H and (13)C NMR spectroscopy. While the energy state of the cells was essentially restored during a 7-hr recovery period, the changes in osmolyte concentrations lasted longer and went on throughout the recovery period. Even after 24-hr recovery, organic osmolyte concentrations were still below the control levels. (13)C NMR spectra of astrocyte cell extracts also demonstrated an enhanced glucose metabolism via the pentose phosphate pathway (PPP) and a reduced glycolysis. Additionally, the appearance of (13)C glutamate points to a distortion of glutamine synthetase (GS), leading to the accumulation of glutamate. Glycolysis as well as GS activity were back to control levels after 7 hr recovery. Thus, in contrast to the energy metabolism, osmoregulatory processes and complex glucose metabolism was impaired not only directly after oxidative stress, but occurred with a later onset during a 2-hr recovery period, and cells only slowly recovered during the next 24 hr.     

Evaluation of muscle glycogen content by 13C NMR spectroscopy in adult-onset acid maltase deficiency

Muscle glycogen storage was measured by in vivo, natural abundance 13C nuclear magnetic resonance spectroscopy in distal and proximal lower limb segments of patients suffering from adult-onset acid maltase deficiency. Interleaved T1-weighted acquisitions of glycogen and creatine served to quantify glycogen excess. For acid maltase deficient patients (n=11), glycogen:creatine was higher than controls (n=12), (1.20+/-0.39 vs. 0.83+/-0.18, P=0.0005). Glycogen storage was above the normal 95% confidence limits in at least one site for 7/11 patients. The intra-individual coefficient of reproducibility was 12%. This totally atraumatic measurement of glycogen allows repeated measurement at different muscle sites of acid maltase deficient patients, despite selective fatty replacement of tissue. This could provide an additional parameter to follow the development of disease in individual patients, including in the perspective of forthcoming therapeutic trials. It may also offer an appropriate tool to study the role of glycogen accumulation in progression of the pathology.     

Estimation of aspartate synthesis in GABAergic neurons in mice by 13 C NMR spectroscopy.

Aspartate synthesis in GABAergic neurons was estimated following inhibition of glutamate decarboxylase (GAD) with 3-mercaptopropionic acid (3-MPA). Mice received 3-MPA, 50 mg/kg, and [1-13C]glucose or [2-13C]acetate. Brain extracts were analyzed by 13C NMR spectroscopy. GABA synthesis was inhibited by 50%, and the synthesis of [13C]aspartate subsequently decreased by 25%. This means that 50% of cerebral aspartate is labeled from metabolites formed through the GABA shunt. A large proportion of the remaining aspartate is labeled through the TCA cycle in GABAergic neurons.     

CSF proteome analysis in multiple sclerosis patients by two-dimensional electrophoresis

Background and purpose:  In recent years, different approaches have been used to investigate changes of cerebrospinal fluid (CSF) proteome in patients affected by multiple sclerosis (MS) with the aim to identify protein markers with potential diagnostic or prognostic value. Because of the lack of standardization of current proteomic techniques, contrasting results were achieved until now in different laboratories. In this study, we compare CSF proteome of 10 relapsing–remitting MS (RR-MS) patients, 11 patients with clinically isolated syndrome (CIS), and 10 control subjects without neurological or systemic diseases.
Methods:  The differential expression of CSF proteins amongst these cohorts of patients was investigated by using two-dimensional electrophoresis and mass spectrometry.
Results and conclusions:  We found an overexpression of IgG free kappa light chain protein in both CIS and RR-MS patients, compared with control subjects and an increased expression of an apolipoprotein E isoform in RR-MS patients, compared with CIS and control groups. Our results confirm the presence of CSF proteome changes in MS patients. Future research should be aimed to investigate the role of these candidate CSF markers in larger cohorts of CIS and MS patients.     

Phosphorus NMR spectroscopy study of muscular enzyme deficiencies involving glycogenolysis and glycolysis

We used phosphorus NMR spectroscopy to study 16 patients with muscular enzyme deficiencies affecting glycogenolysis and glycolysis. Study of phosphomonoester (Pm) kinetics and intracellular pH during exercise and recovery provided criteria for the distinction of these metabolic myopathies by NMR spectroscopy. The Pm peak was undetectable in patients lacking debrancher enzyme or phosphorylase. By contrast, in phosphofructokinase (PFK) or phosphoglycerate kinase (PGK) deficiency, the Pm peak was larger than that of inorganic phosphate in exercise, whereas it was always smaller in normal subjects. During recovery, the disappearance of Pm was slower in PGK than in PFK deficiency.     

Apert syndrome: analysis of associated brain malformations and conformational changes determined by surgical treatment

Apert Syndrome, also called acrocephalosyndactylia type 1, is characterized by craniostenosis with early fusion of sutures of the vault and/or cranial base, associated to mid-face hypoplasia, symmetric syndactylia of the hands and feet and other systemic malformations. CNS malformations and intracranial hypertension are frequently observed in these patients. Early surgical treatment aims to minimize the deleterious effects of intracranial hypertension. Fronto-orbital advancement, the usual surgical technique, increases the intracranial Volume and improves the disposition of encephalic structures previously deformed by a short skull. This study analyzes CNS alterations revealed by magnetic resonance in 18 patients presenting Apert Syndrome, and the conformational alterations in the encephalic structures after surgical treatment. The patients' age in February 2001 ranged from 14 to 322 Months (m=107). Image study included brain magnetic resonance showing ventricular enlargement in five cases (27.8%), corpus callosum hypoplasia in five cases (27.8%), septum pellucidum hypoplasia in five cases (27.8%), cavum vergae in two cases (11.1%) and, arachnoid cyst in the posterior fossa in two cases (11.1%). Absence of CNS alterations was noted in 44.4% of cases. A corpus callosum morphologic index was established by dividing its height by its length, which revealed values that ranged from 0.4409 to 1.0237. The values of this index were correlated to the occurrence or absence of surgical treatment (p=0.012; t=2.83). Data analysis allowed the conclusion that the corpus callosum morphologic measure quantified the conformational alterations of the cerebral structures determined by the surgical treatment.     

Investigating glycogenosis type III patients with multi-parametric functional NMR imaging and spectroscopy

Debranching enzyme deficiency (Glycogen storage disease (GSD) type III) causes progressive muscle wasting myopathy. A comprehensive nuclear magnetic resonance study involving spectroscopy (NMRS) and imaging (NMRI) evaluated status and function of calf muscles in 18 GSDIII patients. At rest, ³¹P NMRS showed elevated pH and accumulation of anomalous phosphomonoesters, ¹³C NMRS quantified excess glycogen accumulation and NMRI demonstrated progressive fat replacement that paralleled muscle weakness. Multi-parametric functional NMR, performed at recovery from a single bout of aerobic exercise, simultaneously assessed oxidative phosphorylation from ³¹P NMRS, muscle perfusion and BOLD, a marker of blood oxygenation, from arterial spin labeled NMRI, and oxygen uptake from deoxymyoglobin proton NMRS. While blocked glycogenolysis caused inadequate substrate supply to the mitochondria, combined measurements suggested that altered perfusion was also responsible for impaired post-exercise phosphocreatine recovery and could contribute to exercise intolerance in GSDIII. These non-invasive investigations provide new indices to quantify the progression of GSDIII.     

Deducing the bioactive face of hydantoin anticonvulsant drugs using NMR spectroscopy

BACKGROUND: The general purpose of this study was to deduce the geometry of the bioactive face (pharmacophore) for the hydantoin class of anticonvulsants. METHODS: Six hydantoin analogs, selected as probes of hydantoin structure, were synthesized. Nuclear magnetic resonance spectroscopy and molecular modelling calculations were used to determine the geometric relationship between the aromatic group and the amide group in the hydantoin pharmacophore. RESULTS: In accord with both theoretical and experimental results, the biologically inactive hydantoin analogs containing a benzyl substituent existed in a folded conformation with the benzene flopped over the hydantoin ring. Conversely the biologically active hydantoins had a phenyl ring extended away from the hydantoin ring. CONCLUSIONS: The bioactive face for hydantoins consists of a N(H)-C(=O)-X-phenyl molecular fragment, where X is a carbon or nitrogen atom and where the distance between the centre of the amide bond and the centroid of the phenyl ring is 4.3 A.     

Preliminary observations on brain energy metabolism in migraine studied by in vivo phosphorus 31 NMR spectroscopy

We measured brain energy phosphate metabolism and intracellular pH (pHi) in a cross-sectional study of migraine patients by in vivo phosphorus 31 NMR spectroscopy. During a migraine attack the ratio ATP/total phosphate signal (mole % ATP) was preserved, but there was a decrease in mole % phosphocreatine (PCr) and an increase in mole % inorganic phosphate (Pi) resulting in a decrease of the PCr/Pi ratio, an index of brain phosphorylation potential. This was found in classic but not common migraine. Mole % Pi was also increased in combined brain regions between attacks. There was no alteration in brain pHi during or between attacks. Energy phosphate metabolism but not pHi appears disordered during a migraine attack.     

Protein analysis in the rat auditory brainstem by two-dimensional gel electrophoresis and mass spectrometry

A catalogue of the protein repertoire of processing centres in the central auditory system would greatly foster our knowledge on the anatomical and functional properties of this sensory system. Towards this goal, we report on the first mapping study of the protein content in the superior olivary complex (SOC) and the inferior colliculus (IC) of the rat auditory brainstem. The protein content of these two structures was assessed by means of two-dimensional gel electrophoresis (2-DGE) and mass spectrometry. To do so, proteins were first separated into four fractions by differential centrifugation. For comparison, corresponding cerebellar fractions were also analysed. Immunoblot analysis revealed highly enriched microsomal and cytosolic fractions; the other two fractions were mixtures of various subcellular compartments. Separation of the 800 g pellets (enriched for nuclear and plasma membrane proteins) and the 100,000 g supernatants (enriched for cytosolic proteins) by 2-DGE yielded between 456 and 674 distinct protein spots after silver staining. The overall protein pattern of all three tissues was similar for a given fraction. Fifty prominent protein spots of the SOC cytosolic fraction were identified by mass spectrometry and yielded information on thirty different genes with various cellular functions, e.g. primary metabolism, cytoarchitecture, and signal transduction. Sequencing of eleven corresponding spots from the SOC and IC cytosolic fractions confirmed the great similarity between the two samples. The results of this analysis are part of a novel integrated database of the gene repertoire of the auditory brainstem (ID-GRAB), that is publicly available (http://www.id-grab.de).     

Assessment of GABA concentration in human brain using two-dimensional proton magnetic resonance spectroscopy

A quantitative method to assess in vivo brain gamma-aminobutyric acid (GABA) levels is proposed using a J-resolved, two-dimensional (2D) magnetic resonance spectroscopy (MRS) technique. Localized, J-resolved 2D MR spectra were obtained from a 12-cm(3) voxel in the occipital lobe of 36 healthy volunteers (18 male and 18 female, age: 25.1+/-4.8 years). Based on phantom measurements, a GABA resonance peak located at 2.94 ppm, 7.45 Hz, in J-resolved 2D MRS overlaps the least with other resonance peaks which arise from N-acetylaspartate, choline, creatine, glutamate and glutamine. Measurements of this resonance peak yield in vivo GABA concentrations of 1.01+/-0.36 micromol/cm(3) for male and 1.16+/-0.43 micromol/cm(3) for female volunteers, without correction for T1 and T2 relaxation effects. These results are in good agreement with previously reported data and suggest that, with further development, 2D MRS may provide a practical means to estimate the concentration of this important neurotransmitter.