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Objective: The MAST family of microtubule-associated serine–threonine kinases (STKs) have distinct expression patterns in the developing and mature human and mouse brain. To date, only MAST1 has been conclusively associated with neurological disease, with de novo variants in individuals with a neurodevelopmental disorder, including a mega corpus callosum. Methods: Using exome sequencing, we identify MAST3 missense variants in individuals with epilepsy. We also assess the effect of these variants on the ability of MAST3 to phosphorylate the target gene product ARPP-16 in HEK293T cells. Results: We identify de novo missense variants in the STK domain in 11 individuals, including 2 recurrent variants p.G510S (n = 5) and p.G515S (n = 3). All 11 individuals had developmental and epileptic encephalopathy, with 8 having normal development prior to seizure onset at

BACKGROUND Low and high grade gliomas, are the most common pediatric central nervous system (CNS) tumors and they show an extremely broad range of clinical behavior. Pediatric glioma is distinct from its adult counterpart with different genetic/epigenetic profile and biological features. Recently, several studies have shown the involvement of voltage-gated Na+ channels (VGSC) in different types of cancer, including gliomas. VGSC are multimeric transmembrane complexes, responsible for membrane depolarization in excitable cells playing an important role also in cell proliferation, migration, apoptosis and differentiation. VGSC are therapeutic targets in cardiovascular and neurological disorders and, in cancers, they could be a novel target for the development of promising anticancer therapy MATERIAL AND METHODS Firstly, we explored the genetic background of 9 pediatric glioblastomas (1–9 pGBMs), through whole-exome sequencing (WES) using HiSeq1000 platform (Illumina) with paired-end approach. On the basis of our results, we extended our study in another group of 16 epileptogenic mixed neuronal-glial tumors of pediatric age, (WHO grade I and II), through an amplicon approach (TSCA) using MiniSeq System platform (Illumina) RESULTS We identified variants in SCN1A gene in 3/9 pGBMs: case 3 had c.5782C>G in tumor and blood; case 5 showed c.2278G>T and two mosaic variants (c.5933C>T, 22% and c.4942C>T, 14%); case 6 showed c.667G>T variant only in tumor, and not in other non tumoral tissues (blood, urine and buccal swab). No variants in SCN1A were identified in a group of 16 pediatric mixed gliomas CONCLUSION In this study, we explore the genetic background of two groups of pediatric neuroepithelial brain tumors, through Next generation sequencing approach. We identified only in pGBMs variants in SCN1A gene that encoded for VGSCs and is involved in a spectrum of early-onset epileptic encephalopathies. None of our mutated patients showed history of epilepsy. Now, it is not clear the significance of these variants in pGBMs but interestingly, these variants are present in pGBM and not in mixed gliomas. Further studies on a big cohort of patients are needed to establish if they could play a role in pGBMs aggressiveness, migration and progression. Moreover, VGSCs could be a pharmacological target in pGBMs treatment

The International League against Epilepsy (ILAE) proposed a diagnostic scheme for psychogenic non-epileptic seizure (PNES). The debate on ethical aspects of the diagnostic procedures is ongoing, the treatment is not standardized and management might differ according to age group. The objective was to reach an expert and stakeholder consensus on PNES management. A board comprising adult and child neurologists, neuropsychologists, psychiatrists, pharmacologists, experts in forensic medicine and bioethics as well as patients’ representatives was formed. The board chose five main topics regarding PNES: diagnosis; ethical issues; psychiatric comorbidities; psychological treatment; and pharmacological treatment. After a systematic review of the literature, the board met in a consensus conference in Catanzaro (Italy). Further consultations using a model of Delphi panel were held. The global level of evidence for all topics was low. Even though most questions were formulated separately for children/adolescents and adults, no major age-related differences emerged. The board established that the approach to PNES diagnosis should comply with ILAE recommendations. Seizure induction was considered ethical, preferring the least invasive techniques. The board recommended looking carefully for mood disturbances, personality disorders and psychic trauma in persons with PNES and considering cognitive-behavioural therapy as a first-line psychological approach and pharmacological treatment to manage comorbid conditions, namely anxiety and depression. Psychogenic non-epileptic seizure management should be multidisciplinary. High-quality long-term studies are needed to standardize PNES management.

Summary Purpose Epilepsy is a neurologic disorder with major social impact. Surgery is a valuable option in patients who are not responding to antiepileptic drugs. The literature reports demonstrate that a proportion ranging from 40 to 100% of patients with epilepsy achieve seizure remission after surgery. A presurgical evaluation (clinical and instrumental) must be performed in all patients with drug-resistant epilepsy to assess their suitability for surgical intervention. Health Technology Assessment (HTA) represents a modern approach to the analysis of technologies used for health care. HTA could be considered a bridge between science that produces evidence and the decisions that can be taken on the basis of that evidence at different levels of the health care system. The aim of this study is the HTA of epilepsy surgery including clinical, ethical, social, and economic features. Methods The present study includes an analysis of the diagnostic and surgical workup performed at the Italian centers for the diagnosis and treatment of drug-resistant epilepsy (DRE). The study includes the following issues: (1) social, ethical impact, and costs of the disease; (2) clinical results, efficacy, and safety of surgery; (3) ethics and quality of life after surgery; and (4) economic impact and productivity regained after surgery. The cost of managing a patient with DRE included in the presurgical study was estimated by the bottom-up microcosting technique that starts from a detailed collection of data on consumption of resources and full costing. The phases analyzed were (1) noninvasive diagnostic workup; (2) neurosurgical intervention; and (3) follow-up. Key Findings The literature reports indicate epilepsy surgery as an effective treatment both on clinical results and on ethical, social, and quality of life aspects. The workup including the noninvasive presurgical study followed by surgery has a total cost of €20,827. Management of short-term follow-up increases the overhead to €22,291 at the first year, and then to €23,571 after 5 years. According to the estimates made in this survey, funding based on diagnosis-related group (DRG) tariff for the noninvasive diagnostic stage involving hospital admission is not remunerative in Italy either at regional or national levels. Effectively the difference between full cost and DRG has a delta of €3,402 and €2,537 respectively. The total cost of the presurgical, surgical, and follow-up evaluation is not remunerative for €10,554 (national data). Significance Economic surveys in Italy have shown that surgery for DRE is an advantageous treatment from the standpoint of third-party payers and is cost-effective for society. DRE presurgical evaluation and surgery are not remunerative either at regional or national levels.

Barth syndrome (BTHS) is an X-linked inborn error of metabolism which affects males. The main manifestations are cardiomyopathy, myopathy, hypotonia, growth delay, intermittent neutropenia and 3-methylglutaconic aciduria. Diagnosis is confirmed by mutational analysis of the TAZ gene and biochemical dosage of the monolysocardiolipin/tetralinoleoyl cardiolipin (MLCL:L4-CL) ratio. We report a 6-year-old boy who presented with severe hypoglycemia, lactic acidosis and severe dilated cardiomyopathy soon after birth. The MLCL:L4-CL ratio confirmed BTHS (3.90 on patient's fibroblast, normal: 0-0.3). Subsequent sequencing of the TAZ gene revealed only the new synonymous variant NM_000116.3 (TAZ):c.348CT p.(Gly116Gly), which did not appear to affect the protein sequence. In silico prediction analysis suggested the new c.348CT nucleotide change could alter the TAZ mRNA splicing processing. We analyzed TAZ mRNAs in the patient's fibroblasts and found an abnormal skipping of 24 bases (NM_000116.3:c.346_371), with the consequent ablation of 8 amino acid residues in the tafazzin protein (NP_000107.1:p.Lys117_Gly124del). Molecular analysis of at risk female family members identified the patient's sister and mother as heterozygous carriers. Apparently harmless synonymous variants in the TAZ gene can damage gene expression. Such findings widen our knowledge of molecular heterogeneity in BTHS.

The Commission for Epilepsy Surgery of the Italian League Against Epilepsy (LICE) presents an overview of the techniques and methodologies of presurgical evaluation and of the surgical treatment of epilepsies. This overview is the result of the experience developed in the past years in the major Italian centers where programs of epilepsy surgery have been established, and it has the aim of offering a quick and easy reference tool for those involved in the treatment of patients with epilepsy. The sharing of different experiences has the additional aim of conforming and disseminating the employed techniques as well as the methods of selection and evaluation of patients. The synthetic coverage of the main issues concerning the presurgical workup and the available surgical options will hopefully provide a framework that may integrate and develop the contributions of every single center, in one of the more complex, challenging, and dynamic areas of neurological sciences.

Somatic and germline duplications or activating mutations of AKT3 have been reported in patients with hemimegalencephaly and megalencephaly. We performed array comparative genomic hybridization on brain tissue and blood in 16 consecutive patients with symptomatic epilepsy due to focal or multilobar malformations of cortical development who underwent surgical treatment of epilepsy. One patient with infantile spasms and a dysplastic left frontal lobe harboured a somatic trisomy of the 1q21.1-q44 chromosomal region, encompassing the AKT3 gene, in the dysplastic brain tissue but not in blood and saliva. Histopathology revealed severe cortical dyslamination, a thin cortex in the premotor area with microgyri and microsulci, immature neurons with disoriented dendrites and areas of cortical heterotopia in the sub-cortical white matter. These cytoarchitectural changes are close to those defining type Ib focal cortical dysplasia. Immunohistochemistry in brain specimens showed hyperactivation of the PI3K/AKT/mTOR pathway. These findings indicate that AKT3 upregulation may cause focal malformations of cortical development. There appears to be an etiologic continuum between hemimegalencephaly and focal cortical dysplastic lesions. The extent of brain malformations due to AKT3 upregulation may be related to the embryonic stage when the post-zygotic gene alteration occurs.

Aminoacylase 1 (ACY1) deficiency is a rare inborn error of metabolism of which less than 20 observations have been described. Patients exhibit urinary excretion of specific N-acetyl amino acids and manifest a heterogeneous clinical spectrum including intellectual disability, motor delay, seizures, moderate to severe mental retardation, absent speech, growth delay, muscular hypotonia and autistic features. Here, we report the case of ACY1 enzyme deficiency in a 6-year-old girl presenting severe intellectual disability, motor retardation, absence of spontaneous locomotor activity and severe speech delay. Urinary excretion of N-acetylated amino acids was present. Mutational analysis of ACY1 gene identified the new homozygous c.1001_1001+5del6 mutation, which alters the mRNA transcription leading to exon 13 skipping and inclusion of a premature stop codon (p.Lys308Glufs*7). A quantitative fluorescent multiplex-polymerase chain reaction (QFM-PCR) assay has been set up and confirmed homozygosity of the mutation in the patient's DNA. Biochemical analysis showed absence of ACY1 enzyme activity in the patient's fibroblasts. The structure of the mutated protein has been defined by homology modeling (HM). Our data endorse the hypothesis of a link between this inborn error of metabolism and the neurological manifestations observed in patients with ACY1 deficiency.

The aristaless-related homeobox gene (Arx) was isolated and characterized in 1997 in zebra fish and mice on the basis of its striking homology to the drosophila gene ‘aristaless.’ The discovery of the human ortholog of this gene, which is located on the short arm of the X chromosome (Xp22.13), followed in 2002, when ARX gene mutations were identified in patients with mental retardation and various form of epilepsy. Since this first report, 10 distinct disease phenotypes, with X-linked recessive inheritance, have been characterized, albeit with some clinical overlap. ARX contains five exons with four polyalanine tracts, a homeodomain, and a conserved C-terminal aristaless domain. Deletions, frameshifts, null, or missense mutations within the homeobox cause severe malformation syndromes. Trinucleotide repeat expansions in the polyalanine tracts and missense mutations outside the homeobox cause syndromes without malformations – for example, infantile spasms or West syndrome (the association of infantile spasms, hypsarrhythmia on electroencephalogram, and mental retardation). ARX sequencing is now widely available, and ARX mutation screening in boys who present with infantile spasms, mental retardation and dystonia, particularly in the presence of normal or nearly normal MRI studies, will benefit families through improved prognosis, genetic counseling, and prenatal diagnosis.

Several epilepsy syndromes associated with abnormal cortical development have been recognized for which causative gene defects have been identified. Lissencephaly (LIS) and subcortical band heterotopia (SBH) result from mutations of either the LIS1 or the DCX gene. Periventricular nodular heterotopia (PNH) features subset of neurons that have failed to migrate and therefore line the walls of the lateral ventricles. Mutations of the X-linked gene FLNA have been found in about 50% of all patients with bilateral PNH and in 100% of X-linked familial cases. A rare, recessive form of bilateral PNH is caused by mutations of the ARFGEF2 gene. There are several syndromes featuring polymicrogyria; among these syndromes, recessive bilateral frontoparietal polymicrogyria (BFPP) has been associated with mutations of the GPR56 gene and bilateral perisyilvian polymicrogyria (BPP) has been associated with SRPX2 mutations in isolated cases.

Neuropeptide S (NPS) was recently identified as the endogenous ligand of an orphan receptor, now referred to as the NPS receptor. In vivo, NPS produces a unique behavioural profile by increasing wakefulness and exerting anxiolytic-like effects. In the present study, we further evaluated the effects of in vivo supraspinal NPS in mice.Effects of NPS, injected intracerebroventricularly (i.c.v.), on locomotor activity (LA), righting reflex (RR) recovery and on anxiety states (measured with the elevated plus maze (EPM) and stress-induced hyperthermia (SIH) tests) were assessed in Swiss mice.NPS (0.01-1 nmol per mouse) caused a significant increase in LA in naive mice, in mice habituated to the test cages and in animals sedated with diazepam (5 mg kg(-1)). In the RR assay, NPS dose dependently reduced the proportion of animals losing the RR in response to diazepam (15 mg kg(-1)) and their sleeping time. In the EPM and SIH test, NPS dose dependently evoked anxiolytic-like effects by increasing the time spent by animals in the open arms and reducing the SIH response, respectively.We provide further evidence that NPS acts as a novel modulator of arousal and anxiety-related behaviours by promoting a unique pattern of effects: stimulation associated with anxiolysis. Therefore, NPS receptor ligands may represent innovative drugs for the treatment of sleep and anxiety disorders.

Current views of the role of beta-amyloid peptide fibrils range from regarding them as the cause of Alzheimer's pathology to having a protective function. In the last few years, it has also been suggested that soluble oligomers might be the most important toxic species. In all cases, the study of the conformational properties of beta-amyloid peptides in soluble form constitutes a basic approach to the design of molecules with antiamyloid activity. We have experimentally investigated the conformational path that can lead the beta-amyloid-(1-42) peptide from the native state, which is represented by an alpha-helix embedded in the membrane, to the final state in the amyloid fibrils, which is characterized by beta-sheet structures. The conformational steps were monitored by using CD and NMR spectroscopy in media of varying polarities. This was achieved by changing the composition of water and hexafluoroisopropanol (HFIP). In the presence of HFIP, conformations can be observed in solutions that have very high water content (up to 99 % water; v/v). These can be turned back to helices simply by adding the appropriate amount of HFIP. The transition of beta-amyloid peptide-(1-42) from to conformations occurs when the amount of water is higher than 80 % (v/v). The NMR structure solved in HFIP/H2O with high water content showed that, on going from very apolar to polar environments, the long N-terminal helix is essentially retained, whereas the shorter C-terminal helix is lost. The complete conformational path was investigated in detail with the aid of molecular-dynamics simulations in explicit solvent, which led to the localization of residues that might seed conformations. The structures obtained might help to find regions that are more affected by environmental conditions in vivo. This could in turn aid the design of molecules able to inhibit fibril deposition or revert oligomerization processes

BACKGROUND: Tyr-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) and Tyr-Tic-Ala were the first peptides with delta opioid antagonist activity lacking Phe, considered essential for opioid activity based on the N-terminal tripeptide sequence (Tyr-D-Xaa-Phe) of amphibian skin opioids. Analogs were then designed to restrain the rotational flexibility of Tyr by the substitution of 2,6-dimethyl-L-tyrosine (Dmt). MATERIALS AND METHODS: Tyr and Dmt peptides were synthesized by solid phase and solution methods using Fmoc technology or condensing Boc-Dmt-OH or Boc-Tyr(But)-OH with H-L-Tic-OBut or H-D-Tic-OBut, respectively. Peptides were purified (> 99%) by HPLC and characteristics determined by 1H-NMR, FAB-MS, melting point, TLC, and amino acid analyses. RESULTS: H-Dmt-Tic-OH had high affinity (Ki delta = 0.022 nM) and extraordinary selectivity (Ki mu/Ki delta = 150,000); H-Dmt-Tic-Ala-OH had a Ki delta = 0.29 nM and delta selectivity = 20,000. Affinity and selectivity increased 8700- and 1000-fold relative to H-Tyr-Tic-OH, respectively. H-Dmt-Tic-OH and H-Dmt-Tic-NH2 fitted one-site receptor binding models (eta = 0.939-0.987), while H-Dmt-Tic-ol, H-Dmt-Tic-Ala-OH and H-Dmt-Tic-Ala-NH2 best fitted two-site models (eta = 0.708-0.801, F 18.9-26.0, p < 0.0001). Amidation increased mu affinity by 10- to 100-fold and acted synergistically with D-Tic2 to reverse selectivity (delta-->mu). Dmt-Tic di- and tripeptides exhibited delta antagonist bioactivity (Ke = 4-66 nM) with mouse vas deferens and lacked agonist mu activity (> 10 microM) in guinea-pig ileum preparations. Dmt-Tic analogs weakly interacted with kappa receptors in the 1 to > 20 microM range. CONCLUSIONS: Dmt-Tic opioidmimetic peptides represent a highly potent class of opioid peptide antagonists with greater potency than the nonopioid delta antagonist naltrindole and have potential application as clinical and therapeutic compounds.

Treatment of neurological disorders with intravenous immunoglobulin (IVIg) is an increasing feature of our practice for an expanding range of indications. For some there is evidence of benefit from randomised controlled trials, whereas for others evidence is anecdotal. The relative rarity of some of the disorders means that good randomised control trials will be difficult to deliver. Meanwhile, the treatment is costly and pressure to "do something" in often distressing disorders considerable. This review follows a 1 day meeting of the authors in November 2000 and examines current evidence for the use of IVIg in neurological conditions and comments on mechanisms of action, delivery, safety and tolerability, and health economic issues. Evidence of efficacy has been classified into levels for healthcare interventions (tables 1 and 2).

To examine the effects of nociceptin (NC) and endomorphin 1 (EM1) on electrical field stimulation (EFS)-induced contractions of the human vas deferens (hVD).Concentration-response curves to NC and EM1 were constructed in the absence and in presence of peptidase inhibitors (PI). In some experiments a NC receptor antagonist, [Phe1psi(CH2-NH)Gly2]NC(1-13)NH2 [F/G], 10 microM) or naloxone (1 microM) were included.All data are mean(95%CI). In the presence of PI, NC inhibited twitches (Emax = 67(44,90)%; pEC50 = 7.28(6.95,7.61)). NC inhibition was sensitive to [F/G]. EM1 also inhibited twitches both in the absence (Emax = 82(73,91)% pEC50 = 7.07(6.92,7.22)) and presence (Emax = 83(76,90)%; pEC50 = 7.00(6.91, 7.09)) of PI. EM1 inhibition was sensitive to naloxone.These data suggest that hVD express NC and opioid receptors that inhibit neurogenic contractions.

A series of analogs of the ORL1 receptor antagonist [Nphe1]-NC(1-13)-NH2 was prepared and tested for agonistic and antagonistic activities in the mouse vas deferens, a preparation that shows high sensitivity to nociceptin and related peptides. The purpose of this study was to determine the role of the aromatic residue at the N-terminal for antagonism and eventually identify compounds with improved potency. Results indicated that all 23 compounds are inactive as agonists, and the antagonistic potency of the initial template [Nphe1]-NC(1-13)-NH2 is high (pKB 6.43) compared with those of all other compounds except [(S)(betaMe)Nphe1]NC(1-13)-NH2 (pK(B) 6.48). The other 22 compounds can be divided into two groups: 10 show antagonistic potencies (pK(B)) ranging from 5.30 to 5.86, whereas the other 12 compounds are inactive. This study clearly shows that the aromatic ring of Nphe is very critical for the interaction with the ORL1 receptor and can not be enlarged or sterically modified without significant loss of antagonistic potency.

To address genetic predisposition to febrile convulsions (FCs) and epilepsy as an etiologic background of severe myoclonic epilepsy in infancy (SMEI).Familial antecedents of epilepsy and FCs were analyzed in four groups of patients with SMEI (65 cases), FCs (57 cases), childhood absences (67 cases), and a control group of patients with no neurologic problems (64 cases).Patients with SMEI and those with FCs had significantly increased incidence of FCs in their relatives compared with those with absence epilepsy and with the control group. The incidence of epilepsy in relatives of patients with SMEI and absence epilepsy was increased compared with that in the control group and reached statistical significance. Epilepsy in relatives with SMEI had the characteristics of idiopathic generalized epilepsy.A genetic predisposition could determine three types of epileptic syndromes: FCs, idiopathic generalized epilepsy, and SMEI.

Benign rolandic epilepsy (BRE) and childhood epilepsy with occipital paroxysms (CEOP) are overlapping in age range of presentation. Some children have been reported to manifest occipital and rolandic seizures, as distinct events. However, the presence during the same seizure of rolandic and occipital symptoms is exceptional. We present a 7-year-old boy with BRE in whom we video-EEG recorded two seizures at 4 years of age. The first episode was a classic rolandic seizure during sleep, while the second was prolonged with initial rolandic and late occipital involvement. It is possible that a rolandic seizure, in a child within the age range of both BRE and early onset CEOP, could spread to selectively involve the occipital lobe, because there is susceptibility of cortical neurons of both areas to develop seizures at this age in idiopathic partial epilepsies.

[Phe(1)psi(CH(2)-NH)Gly(2)]noc/OFQ(1-13)-amide ([F/G]NC(1-13)NH(2)) and acetyl-RYYRIK-amide (Ac-RYYRIK-NH(2)), two peptidic ligands of the nociceptin/orphanin FQ (noc/OFQ) receptor, have been shown to exert both agonist and antagonist activity in different in vitro and in vivo systems. This is despite the observation that both peptides competitively antagonized the coupling of the activated receptor to G-proteins in brain preparations, measured in GTPgamma(35)S binding assays. In this study, [Nphe(1)]NC(1-13)-amide ([Nphe(1)]NC(1-13)NH(2)), a new noc/OFQ analog recently characterized as a pure and selective noc/OFQ receptor antagonist in several in vitro and in vivo assay systems, was shown to competitively inhibit the noc/OFQ-stimulated GTPgamma(35)S binding to rat cerebral cortex membranes with pA(2) of 7.76 (Schild analysis). This antagonism of noc/OFQ receptor G-protein coupling was selective because the peptide inhibited the noc/OFQ-evoked GTPgamma(35)S binding to rat brain membranes but not that evoked by selective agonists of the mu-, delta-, and kappa-opioid receptors. In rat cortical membranes, the effects of [F/G]NC(1-13)NH(2) and Ac-RYYRIK-NH(2) on the binding of GTPgamma(35)S were clearly differentiated from the effect of [Nphe(1)]NC(1-13)NH(2) when the concentration of GDP, competing with GTPgammaS for binding, was lowered from 100 microM (assay optimum) to 5 microM. At 5 microM GDP, the former peptides showed clear partial agonist activity, whereas [Nphe(1)]NC(1-13)NH(2) did not. These data indicate that only [Nphe(1)]NC(1-13)NH(2) was a pure antagonist of noc/OFQ receptor G-protein coupling. Furthermore, it is suggested that the variable behavior of [F/G]NC(1-13)NH(2) and Ac-RYYRIK-NH(2) (agonist, partial agonist, and antagonist) in different in vitro and in vivo systems may be explained by different partial GTP binding agonism and the existence of a GTP binding stimulus/response reserve (coupling reserve).

The modulation of [(3)H]-5-hydroxytryptamine ([(3)H]-5-HT) efflux from superfused rat cortical synaptosomes by delta, kappa, mu and ORL(1) opioid receptor agonists and antagonists was studied. Spontaneous [(3)H]-5-HT efflux was reduced (20% inhibition) by either 0.5 microM tetrodotoxin or Ca(2+)-omission. Ten mM K(+)-evoked [(3)H]-5-HT overflow was largely Ca(2+)-dependent (90%) and tetrodotoxin-sensitive (50%). The delta receptor agonist, deltorphin-I, failed to modulate the K(+)-evoked neurotransmitter efflux up to 0.3 microM. The kappa and the mu receptor agonists, U-50,488 and endomorphin-1, inhibited K(+)-evoked [(3)H]-5-HT overflow (EC(50)=112 and 7 nM, respectively; E(max)=28 and 29% inhibition, respectively) in a norBinaltorphimine- (0.3 microM) and naloxone- (1 microM) sensitive manner, respectively. None of these agonists significantly affected spontaneous [(3)H]-5-HT efflux. The ORL(1) receptor agonist nociceptin inhibited both spontaneous (EC(50)=67 nM) and K(+)-evoked (EC(50)=13 nM; E(max)=52% inhibition) [(3)H]-5-HT efflux. The effect of NC was insensitive to naloxone (up to 10 microM), but was antagonized by [Nphe(1)]nociceptin(1-13)NH(2) (a novel selective ORL(1) receptor antagonist; pA(2)=6.7) and by naloxone benzoylhydrazone (pA(2)=6.3). The ORL(1) ligand [Phe(1)psi(CH(2)-NH)Gly(2)]nociceptin(1-13)NH(2) also inhibited K(+) stimulated [(3)H]-5-HT overflow (EC(50)=64 nM; E(max)=31% inhibition), but its effect was partially antagonized by 10 microM naloxone. It is concluded that the ORL(1) receptor is the most important presynaptic modulator of neocortical 5-HT release within the opioid receptor family. This suggests that the ORL(1)/nociceptin system may have a powerful role in the control of cerebral 5-HT-mediated biological functions.

Nociceptin (NC), alias Orphanin FQ, has been recently identified as the endogenous ligand of the opioid receptor-like 1 receptor (OP(4)). This new NC/OP(4) receptor system belongs to the opioid family and has been characterized pharmacologically with functional and binding assays on native (mouse, rat, guinea-pig) and recombinant (human) receptors, by using specific and selective agonists (NC, NC(1 - 13)NH(2)) and a pure and competitive antagonist, [Nphe(1)]NC(1 - 13)NH(2). The similar order of potency of agonists and affinity values of the antagonist indicate that the same receptor is present in the four species. OP(4) is expressed in neurons, where it reduces activation of adenylyl cyclase and Ca(2+) channels while activating K(+) channels in a manner similar to opioids. In this way, OP(4) mediates inhibitory effects in the autonomic nervous system, but its activities in the central nervous system can be either similar or opposite to those of opioids. In vivo experiments have demonstrated that NC modulates a variety of biological functions ranging from nociception to food intake, from memory processes to cardiovascular and renal functions, from spontaneous locomotor activity to gastrointestinal motility, from anxiety to the control of neurotransmitter release at peripheral and central sites. These actions have been demonstrated using NC and various pharmacological tools, as antisense oligonucleotides targeting OP(4) or the peptide precursor genes, antibodies against NC, an OP(4) receptor selective antagonist and with data obtained from animals in which the receptor or the peptide precursor genes were knocked out. These new advances have contributed to better understanding of the pathophysiological role of the NC/OP(4) system, and ultimately will help to identify the therapeutic potential of new OP(4) receptor ligands.

1.. Nociceptin (orphanin FQ) is a novel neuropeptide capable of inducing a variety of biological actions via activation of a specific G-protein coupled receptor. However, the lack of a selective nociceptin receptor antagonist has hampered our understanding of nociceptin actions and the role of this peptide in pathophysiological states. As part of a broader programme of research, geared to the identification and characterization of nociceptin receptor ligands, we report that the novel peptide [Nphe(1)]nociceptin(1-13)NH(2) acts as the first truly selective and competitive nociceptin receptor antagonist and is devoid of any residual agonist activity. 2. [Nphe(1)]nociceptin(1-13)NH(2) binds selectively to recombinant nociceptin receptors expressed in Chinese hamster ovary (CHO) cells (pK(i) 8.4) and competitively antagonizes the inhibitory effects of nociceptin (i) on cyclic AMP accumulation in CHO cells (pA(2) 6.0) and (ii) on electrically evoked contractions in isolated tissues of the mouse, rat and guinea-pig with pA(2) values ranging from 6.0 to 6.4. 3. [Nphe(1)]nociceptin(1-13)NH(2) is also active in vivo, where it prevents the pronociceptive and antimorphine actions of intracerebroventricularly applied nociceptin, measured in the mouse tail withdrawal assay. Moreover, [Nphe(1)]nociceptin(1-13)NH(2) produces per se a dose dependent, naloxone resistant antinociceptive action and, at relatively low doses, potentiates morphine-induced analgesia. 4. Collectively our data indicate that [Nphe(1)]nociceptin(1-13)NH(2), acting as a nociceptin receptor antagonist, may be the prototype of a new class of analgesics.

Mutations in the X-linked doublecortin gene appear in many sporadic cases of double cortex (DC; also known as subcortical band heterotopia), a neuronal migration disorder causing epilepsy and mental retardation. The purpose of this study was to examine why a significant percentage of sporadic DC patients had been found not to harbor doublecortin mutations and to determine whether clinical features or magnetic resonance imaging scan appearance could distinguish between patients with and without doublecortin mutations. Magnetic resonance imaging scan analysis differentiated patients into the following four groups: anterior biased/global DC with doublecortin mutation (16 of 30; 53%), anterior biased/global DC without mutation (8 of 30; 27%), posterior biased DC without mutation (3 of 30; 10%), and limited/unilateral DC without mutation (3 of 30; 10%). The presence of these atypical phenotypes suggests that other genetic loci or mosaicism at the doublecortin locus may be responsible for this diversity of DC cases.

Mutations in the X-encoded gene ATRX are known to give rise to profound syndromal mental retardation (MR). Here, we describe a pedigree, including 4 affected family members with a 324C--T nonsense mutation in the ATRX gene. Although 2 patients have moderate to profound MR and the typical facial features of ATR-X syndrome, the other 2 patients presented with mild MR and epilepsy but without the characteristic facial dysmorphism. Mutations in the ATRX gene should be considered as a cause of mild MR in male patients lacking specific diagnostic features.

The modulation by delta-, kappa-, mu-, and opioid receptor like-1 (ORL(1)) agonists and antagonists of L-glutamate (L-Glu) and gamma-aminobutyric acid (GABA) efflux from superfused rat cerebrocortical synaptosomes was studied. Tetrodotoxin (0.5 microM) inhibited the spontaneous efflux of both transmitters by 20%. Ca(2+) omission decreased GABA and facilitated L-Glu efflux. The neurotransmitter overflow evoked by K(+) concentrations in the 7.5- to 10-mM range was largely Ca(2+) dependent and tetrodotoxin sensitive. Neither the delta-receptor agonist deltorphin (up to 0.3 microM) nor the ORL(1) receptor agonist nociceptin (up to 1 microM) significantly affected either spontaneous or K(+)-evoked neurotransmitter efflux. Conversely, the ORL(1) ligand [Phe(1)(CH(2)-NH)Gly(2)]nociceptin(1-13)NH(2) (0.3 microM) caused a naloxone-sensitive inhibition of both L-Glu- and GABA-stimulated overflow. The kappa-receptor agonist (-)-U50,488 failed to modulate spontaneous L-Glu and GABA efflux. However, it similarly inhibited the K(+)-evoked overflow of both neurotransmitters (EC(50) approximately 100 nM; E(max) approximately 25-30% inhibition) in a norbinaltorphimine-sensitive manner. The selective mu-receptor agonist endomorphin 1 inhibited both spontaneous (EC(50) approximately 50 nM) and K(+)-evoked (EC(50) approximately 10 nM; E(max) approximately 50% inhibition) L-Glu efflux in a naloxone-sensitive manner. Conversely, it significantly inhibited only K(+)-evoked GABA efflux (EC(50) approximately 10 nM), although with a lower maximal effect (E(max) approximately 25-30% inhibition). It is concluded that, in the rat cerebral cortex, L-Glu and GABA efflux from nerve terminals is under the direct inhibitory control of kappa- and mu- (but not delta- or ORL(1)) receptors. Because glutamatergic terminals emerged as a preferential target of mu-receptor agonists, the activation of this receptor may advocate both relevant analgesic and neuroprotective effects.

Nociceptin(NC) is the endogenous ligand for the opioid receptor like-1 receptor (NC-receptor). [Phe1(psi)(CH2-NH)Gly2]Nociceptin(1-13)NH2 ([F/G]NC(1-13)NH2) has been reported to antagonize NC actions in peripheral guinea-pig and mouse tissues. In this study, we investigated the effects of a range of NC C-terminal truncated fragments and [F/G]NC(1-13)NH2 on NC receptor binding, glutamate release from rat cerebrocortical slices (rCX), inhibition of cyclic AMP accumulation in CHO cells expressing the NC receptor (CHO(NCR)) and electrically evoked contractions of the rat vas deferens (rVD). In radioligand binding assays, a range of ligands inhibited [125I]-Tyr14-NC binding in membranes from rCX and CHO(NCR) cells. As the peptide was truncated there was a general decline in pKi. [F/G]NC(1-13)NH2 was as potent as NC(1-13)NH2. The order of potency for NC fragments to inhibit cyclic AMP accumulation in whole CHO(NCR) cells was NCNH2or =NC=NC(1-13)NH2NC(1-12)NH2NC(1-11)NH2. [F/G]NC(1-13)NH2 was a full agonist with a pEC50 value of 8.65. NCNH2 and [F/G]NC(1-13)NH2 both inhibited K+ evoked glutamate release from rCX with pEC50 and maximum inhibition of 8.16, 48.5+/-4.9% and 7.39, 58.9+/-6.8% respectively. In rVD NC inhibited electrically evoked contractions with a pEC50 of 6.63. Although [F/G]NC(1-13)NH2, displayed a small (instrinsic activity alpha = 0.19) but consistent residual agonist activity, it acted as a competitive antagonist (pA2 6.76) in the rVD. The differences between [F/G]NC(1-13)NH2 action on central and peripheral NC signalling could be explained if [F/G]NC(1-13)NH2 was a partial agonist with high strength of coupling in the CNS and low in the periphery. An alternative explanation could be the existence of central and peripheral receptor isoforms.

This paper reviews existing publications on the use of piracetam for the treatment of cortical myoclonus of various etiologies and includes the personal experience of the authors in progressive myoclonus epilepsy. Two double-blind comparisons with placebo provided results which allow recommendations for the dosage and usage of piracetam in cortical myoclonus. Wide individual variation (7-24g daily) exists in dosage requirements but responses are dose-related so that dosage should be increased until an optimum effect is obtained. Tolerability after long-term use of piracetam in high dosage has been very good and without toxicity or serious adverse effects. Side effects have been occasional, mild and transient. The authors present their experience of 12 patients with progressive myoclonus epilepsy in whom the administration of up to 45 g piracetam daily, when added to existing anti-epileptic treatment, caused marked and sometimes spectacular improvement and was without significant adverse effects. Improvement was maintained for up to 7 years. The use of piracetam for disabling cortical myoclonus of any etiology, either as an addition to existing antimyoclonic drugs or as monotherapy, may bring about profound improvement in disability and quality of life. Piracetam should be considered a first-line drug for the treatment of cortical myoclonus.

Mutations in the X-linked gene doublecortin, which encodes a protein with no dear structural homologues, are found in pedigrees in which affected females show "double cortex" syndrome (DC; also known as subcortical band heterotopia or laminar heterotopia) and affected males show X-linked lissencephaly. Mutations in doublecortin also cause sporadic DC in females. To determine the incidence of doublecortin mutations in DC, we investigated a cohort of eight pedigrees and 47 sporadic patients with DC for mutations in the doublecortin open reading frame as assessed by single-stranded conformational polymorphism analysis. Mutations were identified in each of the eight DC pedigrees (100%), and in 18 of the 47 sporadic DC patients (38%). Identified mutations were of two types, protein truncation mutations and single amino acid substitution mutations. However, pedigrees with DC displayed almost exclusively single amino acid substitution mutations, suggesting that patients with these mutations may have less of a reproductive disadvantage versus those patients with protein truncation mutations. Single amino acid substitution mutations were tightly clustered in two regions of the open reading frame, suggesting that these two regions are critical for the function of the Doublecortin protein.

The present study describes the labelling of the nociceptin (NC) receptor, ORL1, in mouse forebrain membranes with a new ligand partially protected from metabolic degradation at the C-terminal; the ligand, [3H]-NC-NH2, has a specific activity of 24.5 Ci mmol(-1). Saturation experiments revealed a single class of binding sites with a KD value of 0.55 nM and Bmax of 94 fmol mg(-1) of protein. Non specific binding was 30% of total binding. Kinetic binding studies yielded the following rate constants: Kobs = 0.104 min(-1); K1 =0.034 min(-1): T1/2=20 min; K(+1)=0.07 min nM(-1). Thermodynamic analyses indicated that [3H]-NC-NH2 binding to the mouse ORL1 is totally entropy driven, similar to what has been observed for the labelled agonists to the opioid receptors OP1(delta), OP2(kappa) and OP3(mu). Receptor affinities of several NC fragments and analogues, including the newly discovered ORL-1 receptor antagonist [Phe1psi(CH2-NH)Gly2]NC(1-13)-NH2([F/G]NC(1-13)-NH2), were also evaluated in displacement experiments. The competition curves for these compounds were found to be parallel to that of NC and the following order of potency was determined for NC fragments: NC-OH = NC-NH2-NC(1-13)-NH2NC(1-12)-NH2NC(1-13)-OHNC(1-11)-NH2, and for NC and NC(1-13)-NH2 analogues: [Tyr1]NC-NH2or = [Leu1]NC(1-13)-NH2or = [Tyr1]NC(1-13)-NH2or = [F/G]NC(1-13)-NH2[Phe3]NC(1-13)-NH2[DF/G]NC(1-13)-NH2. Standard opioid receptor ligands (either agonists or antagonists) were unable to displace [3H]-NC-NH2 binding when applied at concentrations up to 10 microM indicating that this new radioligand interacts with a non opioid site, probably the ORL1 receptor.

Par j 1.0101 is one of the two major allergens of the Parietaria judaica (Pj) pollen, and its three-dimensional structure was built by three-dimensional structural homology modeling. The resultant model was used to identify putative IgE binding regions. Western blot analysis of gene fragmentation products showed that the 1 to 30 region was capable of binding specific IgE from a pool of sera (n = 30) of patients allergic to Pj pollen. Using the structural model as a guide, deletion and site-directed mutagenesis of the 1 to 30 region was performed, and the amino acids involved in IgE binding were identified. In addition, a synthetic peptide covering the 1 to 30 region was capable of binding human IgE without triggering histamine release from basophils of Pj allergic patients (n = 6) and thus represents a haptenic molecule with potential use as an immunotolerant agent. This epitope is also present on the Par j 2.0101 major allergen representing a common IgE epitope. It is an immunodominant epitope, since it was capable of inhibiting 30% of all specific IgE against the Pj major allergens, and therefore, it might be a candidate for the future development of immunotherapeutics.

Substitution of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) in place of Gly2 in dynorphin A-(1-13)-NH2 and -(1-11)-NH2 (DYN) analogues (1 and 2) decreased the affinity to the kappa, delta, and mu receptors, and kappa selectivity. The analogue [D-Ala2, des-Gly3]DYN (4), a chimera between deltorphin/dermorphin N-terminal tripeptide and DYN, was virtually inactive for kappa-sites while the affinities for delta- and mu-receptors remained essentially unchanged. The doubly substituted analogue [2',6'-dimethyl-L-tyrosine (Dmt1)-Tic2]DYN (3) exhibited high delta-affinity (Ki=0.39 nM) while mu- and kappa-affinities were only an order of magnitude less (4-5 nM). Bioactivity of [Tic2]DYN peptides (1-3) on guinea-pig ileum and rabbit jejunum revealed potent delta- and kappa-antagonism, while the delta agonist potency of 4 was comparable to DYN. Thus, conversion from a kappa-agonist to antagonist occurred with the inclusion of Tic into DYN analogues, similar to the appearance of antagonist properties with delta- and mu-opioid agonists containing a Tic2 residue.

The newly discovered neuropeptide nociceptin (NC) (alias orphanin FQ) was tested for its potential direct effects as well as for its ability to modify the electrically evoked contractions in several isolated organs suspended in vitro. NC was inactive both as stimulant and as inhibitor of smooth muscle in several preparations, whereas it inhibited the contractions induced by electrical field stimulation in the mouse vas deferens and guinea pig ileum. NC showed the same potency (IC50 = 10 nM) in the two preparations. However, it was significantly more effective in the mouse vas deferens (maximal effect -80%) than in the guinea pig ileum (maximal effect -50%). The inhibitory effect exerted by NC in the two preparations was not affected by naloxone or more selective opioid receptor antagonists. Moreover, by truncation of C-terminal sequences, NC fragments were designed. These fragments were subsequently tested in the mouse vas deferens and in the guinea pig ileum: NC(1-13)-NH2 was the smallest peptide maintaining the same efficacy and potency as the natural peptide. Finally, NC-NH2 and its fragments NC(1-13)-NH2 and NC(1-9)-NH2 were modified by substituting the phenylalanine 1 residue with a tyrosine. These peptides were tested in the guinea pig ileum, where they behaved as mixed NC-opioid receptor agonists ([Tyr1]NC-NH2 and [Tyr1]NC(1-13)-NH2) or as pure opioid receptor agonists ([Tyr1]NC(1-9)-NH2. In conclusion, the present paper demonstrated that the electrically stimulated mouse vas deferens and guinea pig ileum can be used as a sensitive bioassay for studying the pharmacology of NC and related compounds.

We describe 9 patients with a bilateral malformation of cortical development, centered around the parasagittal and mesial aspects of the parietooccipital cortex, with magnetic resonance imaging findings suggestive of polymicrogyria. No familial distribution or etiologic factors were identified. Location in a watershed area between anterior and posterior cerebral arteries suggests postmigratory perfusion failure as the underlying cause. In most patients the malformation was detected by magnetic resonance imaging after computed tomography scans with 10-mm-thick sections were considered normal. Seizures, present in all, had started between the ages of 20 months and 15 years (mean, 9 years) and were intractable in 7. Complex partial seizures with or without minor automatisms were the most frequent ictal pattern. In only 4 patients these were preceded by symptoms indicating posterior onset. Interictal electroencephalograms showed both diffuse and bilateral parietooccipital or temporal abnormalities. The range of IQ scores indicated average intelligence to mild retardation. Several patients presented deficits on neuropsychological tasks requiring performance under time constraints, suggesting that the malformation may result in cognitive slowing. Early diagnosis of this malformation may be difficult because of the lack of neurological signs, relatively late seizure onset, difficulty in localizing seizure onset, and inability to recognize the cortical abnormality on computed tomography scans.

In this study we compare the peculiarities of psychopathological risk in children with partial epilepsy versus children with chronic illness. Sixteen children affected by epilepsy with partial seizures, symptomatic or cryptogenetic (group A), are compared with 16 children affected by diabetes mellitus type 1 (group B) and with 16 early treated congenital hypothyroid children (group C). For the valuation we have used: 1) Rutter scales for parents; 2) questionnaire on maternal attitude towards epilepsy, modified for diabetes and hypothyroid children; 3) anamnestic interviews for epileptic patients; 4) individual observations. We have emphasized that: 1) the quality of life in children with epilepsy and their families is poorer compared to the ones of the other chronically ill children; 2) the attack is the most characterising factor of life quality of both group A and group B; 3) both group A and group B show an important anxiety for cognitive and behavioural disturbances; 4) in epilepsy families several factors contribute to the increased anxiety towards social life: occurrence of seizures, side effects of drugs, real or supposed underlying encephalopathy.

We studied the frequency, clinical and EEG characteristics, and outcome of the epileptic syndrome in 31 patients with a congenital neurologic syndrome characterized by pseudobulbar palsy, cognitive deficits, and bilateral perisylvian polymicrogyria. Seizures were present in 27 of 31 patients (87%) and usually began between the ages of 4 and 12 years; they commonly consisted of atypical absence, atonic/tonic, and generalized tonic-clonic seizures. Partial attacks were present in 26%. EEG demonstrated generalized spike and wave abnormalities and, less frequently, multifocal discharges, predominantly in centro-parietal regions. Seizures were poorly controlled in 65%, with the remaining patients well controlled. Seven patients underwent callosotomy, which resulted in seizure improvement. This study indicates that the epileptic spectrum in this syndrome is broad but follows predictable patterns. Callosotomy is a valuable treatment strategy in those with intractable drop attacks.