Clinical and genetic spectrum of SCN2A-associated episodic ataxia.

BACKGROUND: Pathogenic variants in SCN2A are associated with various neurological disorders including epilepsy, autism spectrum disorder and intellectual disability. Few reports have recently described SCN2A-associated episodic ataxia (EA). Our study identifies its broader clinical and genetic spectrum, and describes pharmacological approaches. RESULTS: We report 21 patients with SCN2A-associated EA, of which 9 are unpublished cases. The large majority of patients present with epileptic seizures (18/21, 86%), often starting within the first three months of life (12/18, 67%). In contrast, onset of episodic ataxia ranged from 10 months to 14 years of age. The frequency of EA episodes ranged from brief, daily events up to 1-2 episodes per year each lasting several weeks. Potential triggers include minor head traumas and sleep deprivation. Cognitive outcome is favorable in most patients with normal or mildly impaired cognitive development in 17/21 patients (81%). No clear genotype-phenotype correlations were identified in this cohort. However, two mutational hotspots were identified, i.e. 7/21 patients (33%) harbor the identical pathogenic variant p.A263V, whereas 5/21 (24%) carry pathogenic variants that affect the S4 segment and its cytoplasmic loop within the domain IV. In addition, we identified six novel pathogenic variants in SCN2A. While acetazolamide was previously reported as beneficial in SCN2A-associated EA in one case, our data show a conflicting response in 8 additional patients treated with acetazolamide: three of them profited from acetazolamide treatment, while 5/8 did not. CONCLUSIONS: Our study describes the heterogeneous clinical spectrum of SCN2A-associated EA, identifies two mutational hotspots and shows positive effects of acetazolamide in about 50%.

Transcutaneous Vagus Nerve Stimulation (tVNS) for Treatment of Drug-Resistant Epilepsy: A Randomized, Double-Blind Clinical Trial (cMPsE02).

BACKGROUND: Various brain stimulation techniques are in use to treat epilepsy. These methods usually require surgical implantation procedures. Transcutaneous vagus nerve stimulation (tVNS) is a non-invasive technique to stimulate the left auricular branch of the vagus nerve at the ear conch. OBJECTIVE: We performed a randomized, double-blind controlled trial (cMPsE02) to assess efficacy and safety of tVNS vs. control stimulation in patients with drug-resistant epilepsy. METHODS: Primary objective was to demonstrate superiority of add-on therapy with tVNS (stimulation frequency 25 Hz, n = 39) versus active control (1 Hz, n = 37) in reducing seizure frequency over 20 weeks. Secondary objectives comprised reduction in seizure frequency from baseline to end of treatment, subgroup analyses and safety evaluation. RESULTS: Treatment adherence was 84% in the 1 Hz group and 88% in the 25 Hz group, respectively. Stimulation intensity significantly differed between the 1 Hz group (1.02 ± 0.83 mA) and the 25 Hz group (0.50 ± 0.47 mA; p = 0.006). Mean seizure reduction per 28 days at end of treatment was -2.9% in the 1 Hz group and 23.4% in the 25 Hz group (p = 0.146). In contrast to controls, we found a significant reduction in seizure frequency in patients of the 25 Hz group who completed the full treatment period (20 weeks; n = 26, 34.2%, p = 0.034). Responder rates (25%, 50%) were similar in both groups. Subgroup analyses for seizure type and baseline seizure frequency revealed no significant differences. Adverse events were usually mild or moderate and comprised headache, ear pain, application site erythema, vertigo, fatigue, and nausea. Four serious adverse events were reported including one sudden unexplained death in epilepsy patients (SUDEP) in the 1 Hz group which was assessed as not treatment-related. CONCLUSIONS: tVNS had a high treatment adherence and was well tolerated. Superiority of 25 Hz tVNS over 1 Hz tVNS could not be proven in this relatively small study, which might be attributed to the higher stimulation intensity in the control group. Efficacy data revealed results that justify further trials with larger patient numbers and longer observation periods.

Mutations in the sodium channel gene SCN2A cause neonatal epilepsy with late-onset episodic ataxia.

Mutations in SCN2A cause epilepsy syndromes of variable severity including neonatal-infantile seizures. In one case, we previously described additional childhood-onset episodic ataxia. Here, we corroborate and detail the latter phenotype in three further cases. We describe the clinical characteristics, identify the causative SCN2A mutations and determine their functional consequences using whole-cell patch-clamping in mammalian cells. In total, four probands presented with neonatal-onset seizures remitting after five to 13 months. In early childhood, they started to experience repeated episodes of ataxia, accompanied in part by headache or back pain lasting minutes to several hours. In two of the new cases, we detected the novel mutation p.Arg1882Gly. While this mutation occurred de novo in both patients, one of them carries an additional known variant on the same SCN2A allele, inherited from the unaffected father (p.Gly1522Ala). Whereas p.Arg1882Gly alone shifted the activation curve by -4 mV, the combination of both variants did not affect activation, but caused a depolarizing shift of voltage-dependent inactivation, and a significant increase in Na(+) current density and protein production. p.Gly1522Ala alone did not change channel gating. The third new proband carries the same de novo SCN2A gain-of-function mutation as our first published case (p.Ala263Val). Our findings broaden the clinical spectrum observed with SCN2A gain-of-function mutations, showing that fairly different biophysical mechanisms can cause a convergent clinical phenotype of neonatal seizures and later onset episodic ataxia.

[Genetics of idiopathic epilepsies]. / Genetik der idiopathischen Epilepsien.

Idiopathic epilepsies are genetically determined. They are characterized by the observed seizure types, an age-dependent onset, electroencephalographic criteria and concomitant symptoms, such as movement disorders or developmental delay. The main subtypes are the idiopathic (i) generalized, (ii) the focal epilepsies including the benign syndromes of early childhood and (iii) the epileptic encephalopathies as well as the fever-associated syndromes. In recent years, an increasing number of mutations have been identified in genes encoding ion channels, proteins associated to the vesical synaptic cycle or proteins involved in energy metabolism. These mechanisms are pathophysiologically plausible as they influence neuronal excitability. The large number of genetic defects in epilepsy complicates the genetic diagnostic analysis but novel genetic methods are available covering all known genes at a reasonable price. The proof of a genetic defect leads to a definitive diagnosis, is important for the prognostic and genetic counselling and may influence therapeutic decisions in some cases, so that genetic diagnostic testing is becoming increasingly more important and meaningful in many cases in daily clinical practice.

GLUT1 mutations are a rare cause of familial idiopathic generalized epilepsy.

OBJECTIVE: The idiopathic generalized epilepsies (IGE) are the most common genetically determined epilepsies. However, the underlying genes are largely unknown. We screened the SLC2A1 gene, encoding the glucose transporter type 1 (GLUT1), for mutations in a group of 95 European patients with familial IGE. METHODS: The affected individuals were examined clinically by EEG and brain imaging. The coding regions of SLC2A1 were sequenced in the index cases of all families. Wild-type and mutant transporters were expressed and functionally characterized in Xenopus laevis oocytes. RESULTS: We detected a novel nonsynonymous SLC2A1 mutation (c.694C>T, p.R232C) in one IGE family. Nine family members were affected mainly by absence epilepsies with a variable age at onset, from early childhood to adulthood. Childhood absence epilepsy in one individual evolved into juvenile myoclonic epilepsy. Eight affected and 4 unaffected individuals carried the mutation, revealing a reduced penetrance of 67%. The detected mutation was not found in 846 normal control subjects. Functional analysis revealed a reduced maximum uptake velocity for glucose, whereas the affinity to glucose and the protein expression were not different in wild-type and mutant transporters. CONCLUSION: Our study shows that GLUT1 defects are a rare cause of classic IGE. SLC2A1 screening should be considered in IGE featuring absence epilepsies with onset from early childhood to adult life, because this diagnosis may have important implications for treatment and genetic counseling.

Paroxysmal choreoathetosis/spasticity (DYT9) is caused by a GLUT1 defect.

OBJECTIVE: Mutations in SLC2A1, encoding the glucose transporter type 1 (GLUT1), cause a broad spectrum of neurologic disorders including classic GLUT1 deficiency syndrome, paroxysmal exercise-induced dyskinesia (PED, DYT18), and absence epilepsy. A large German/Dutch pedigree has formerly been described as paroxysmal choreoathetosis/spasticity (DYT9) and linked close to but not including the SLC2A1 locus on chromosome 1p. We tested whether 1) progressive spastic paraparesis, in addition to PED, as described in DYT9, and 2) autosomal dominant forms of hereditary spastic paraparesis (HSP) without PED are caused by SLC2A1 defects. METHODS: The German/Dutch family and an Australian monozygotic twin pair were clinically (re-)investigated, and 139 index cases with dominant or sporadic HSP in which relevant dominant genes were partially excluded were identified from databanks. SLC2A1 was sequenced in all cases in this observational study and the functional effects of identified sequence variations were tested in glucose uptake and protein expression assays. RESULTS: We identified causative mutations in SLC2A1 in both families, which were absent in 400 control chromosomes, cosegregated with the affection status, and decreased glucose uptake in functional assays. In the 139 index patients with HSP without paroxysmal dyskinesias, we only identified one sequence variation, which, however, neither decreased glucose uptake nor altered protein expression. CONCLUSIONS: This study shows that DYT9 and DYT18 are allelic disorders and enlarges the spectrum of GLUT1 phenotypes, now also including slowly progressive spastic paraparesis combined with PED. SLC2A1 mutations were excluded as a cause of HSP without PED in our cohort.

[New developments in epileptogenesis and therapeutic perspectives]. / Neue Entwicklungen der Epileptogenese und therapeutische Perspektiven.

Epileptogenesis describes the mechanisms of how epilepsies are generated. We have chosen four areas in which significant progress has been achieved in understanding epileptogenesis. Those are (1) inflammatory processes which play an increasingly important role for the generation of temporal lobe epilepsy with hippocampal sclerosis (TLE with HS), (2) disturbances of intrinsic properties of neuronal compartments, in particular acquired defects of ion channels of which those in dendrites are described here for TLE with HS, (3) epigenetic effects, which affect for example the methylation of promoters and secondarily can change the expression of specific genes in TLE with HS, and finally (4) the epileptogenesis of idiopathic epilepsies which are caused by inborn genetic alterations affecting mainly ion channels. Apart from aspects of basic research, we will describe clinical consequences and therapeutic perspectives.

[Muscle channelopathies. Myotonias and periodic paralyses]. / Muskuläre Kanalopathien. Myotonien und periodische Paralysen.

The myotonias and familial periodic paralyses are muscle channelopathies. They have in common an impaired muscle excitation that is caused by mutations in voltage-gated Na(+), K(+), Ca(2+), and Cl(-) channels. Membrane hyperexcitability usually results in myotonic stiffness; with increasing membrane depolarization hyperexcitability can be transiently turned into hypoexcitability causing transient weakness as in severe myotonia. Hypoexcitability due to long-lasting depolarization that inhibits action potential generation is the common mechanism for the periodic paralyses. Interictally, the ion channel malfunction may be compensated, so that specific exogenous or endogenous provocative factors are required to produce symptoms in the patients. An especially obvious triggering agent is the level of serum potassium, the ion decisive for resting membrane potential and degree of excitability. Periodic paralysis mutations for which the ion channel malfunction is not fully compensated interictally cause progressive myopathy.

SCN2A mutation associated with neonatal epilepsy, late-onset episodic ataxia, myoclonus, and pain.

BACKGROUND: Inherited and de novo mutations in sodium channel genes underlie a variety of channelopathies. Mutations in SCN2A, encoding the brain sodium channel Na(V)1.2, have previously been reported to be associated with benign familial neonatal infantile seizures, febrile seizures plus, and intractable epilepsy of infancy. METHODS: We evaluated the clinical characteristics in a patient with a neonatal-onset complex episodic neurologic phenotype. We screened SCN2A for mutations and carried out in vitro electrophysiologic analyses to study the consequences of the identified mutation. We studied the developmental expression of Na(V)1.2 in cerebellum by immunohistochemical analysis. RESULTS: The patient presented with neonatal-onset seizures and variable episodes of ataxia, myoclonia, headache, and back pain after 18 months of age. The patient carries a de novo missense mutation (p.Ala263Val) in SCN2A, which leads to a pronounced gain-of-function, in particular an increased persistent Na(+) current. Immunohistochemical studies suggest a developmentally increasing expression of Na(V)1.2 in granule cell axons projecting to Purkinje neurons. CONCLUSIONS: These results can explain a neuronal hyperexcitability resulting in seizures and other episodic symptoms extending the spectrum of SCN2A-associated phenotypes. The developmentally increasing expression of Na(V)1.2 in cerebellum may be responsible for the later onset of episodic ataxia.

Efficacy and safety of adjunctive ezogabine (retigabine) in refractory partial epilepsy.

OBJECTIVE: This study assessed the efficacy and safety of the neuronal potassium channel opener ezogabine (US adopted name; EZG)/retigabine (international nonproprietary name; RTG) as adjunctive therapy for refractory partial-onset seizures. METHODS: This was a multicenter, randomized, double-blind, placebo-controlled trial in adults with ≥4 partial-onset seizures per month receiving 1 to 3 antiepileptic drugs. EZG (RTG) or placebo, 3 times daily, was titrated to 600 or 900 mg/d over 4 weeks, and continued during a 12-week maintenance phase. Median percentage seizure reductions from baseline and responder rates (≥50% reduction in baseline seizure frequency) were assessed. RESULTS: The intention-to-treat population comprised 538 patients (placebo, n = 179; 600 mg, n = 181; 900 mg, n = 178), 471 of whom (placebo, n = 164; 600 mg, n = 158; 900 mg, n = 149) entered the maintenance phase. Median percentage seizure reductions were greater in EZG (RTG)-treated patients (600 mg, 27.9%, p = 0.007; 900 mg, 39.9%, p < 0.001) compared with placebo (15.9%). Responder rates were higher in EZG (RTG)-treated patients (600 mg, 38.6%, p < 0.001; 900 mg, 47.0%, p < 0.001) than with placebo (18.9%). Treatment discontinuations due to adverse events (AEs) were more likely with EZG (RTG) than with placebo (placebo, 8%; 600 mg, 17%, 900 mg, 26%). The most commonly reported (>10%) AEs in the placebo, EZG (RTG) 600 mg/d, and EZG (RTG) 900 mg/d groups were dizziness (7%, 17%, 26%), somnolence (10%, 14%, 26%), headache (15%, 11%, 17%), and fatigue (3%, 15%, 17%). CONCLUSIONS: In this dose-ranging, placebo-controlled trial, adjunctive EZG (RTG) was effective and generally well tolerated in adults with refractory partial-onset seizures. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that adjunctive EZG/RTG reduces the occurrence of partial-onset seizures.

Comparative analysis of brain structure, metabolism, and cognition in myotonic dystrophy 1 and 2.

OBJECTIVE: Myotonic dystrophy type 1 and 2 (DM1/DM2) are multisystemic diseases with common cognitive deficits beside the cardinal muscular symptoms. We performed a comprehensive analysis of cerebral abnormalities to compare the neuropsychological defects with findings in different imaging methods in the same cohort of patients. METHODS: Neuropsychological investigations, structural cerebral MRI including brain parenchymal fraction (BPF) and voxel-based morphometry (VBM), and (18)F-deoxy-glucose PET (FDG-PET) were performed in patients (20 DM1 and 9 DM2) and matched healthy controls, and analyzed using statistical parametric mapping (SPM2). RESULTS: DM1 and DM2 patients showed typical neuropsychological deficits with a pronounced impairment of nonverbal episodic memory. Both patient groups showed a reduction of the global gray matter (measured by BPF), which could be localized to the frontal and parietal lobes by VBM. Interestingly, VBM revealed a bilateral hippocampal volume reduction that was correlated specifically to both a clinical score and episodic memory deficits. VBM also revealed a pronounced change of thalamic gray matter. White matter lesions were found in >50% of patients and their extent was correlated to psychomotor speed. FDG-PET revealed a frontotemporal hypometabolism, independent of the decrease in cortical gray matter. All abnormalities were similar in both patient groups but more pronounced for DM1. CONCLUSIONS: Our results suggest that 1) some of the characteristic cognitive deficits of these patients are linked to specific structural cerebral changes, 2) decreases in gray matter and metabolism are independent processes, and 3) the widespread brain abnormalities are more pronounced in DM1.

Pernambucone, a new tropone derivative from Croton argyroglossum.

Two tropone derivatives, orobanone (1), previously isolated from Orobanche rapum-genistae, and the new natural product pernambucone (3,8-dimethyl-5-isopropyl-2,3-dihydro-1H-azulene-1,6-dione, 2), were isolated from the sterm bark of Croton argyroglossum. The structures were elucidated from spectroscopic data.

The Wada test in Austrian, Dutch, German, and Swiss epilepsy centers from 2000 to 2005: a review of 1421 procedures.

Twenty-six Austrian, Dutch, German, and Swiss epilepsy centers were asked to report on use of the Wada test (intracarotid amobarbital procedure, IAP) from 2000 to 2005 and to give their opinion regarding its role in the presurgical diagnosis of epilepsy. Sixteen of the 23 centers providing information had performed 1421 Wada tests, predominantly the classic bilateral procedure (73%). A slight nonsignificant decrease over time in Wada test frequency, despite slightly increasing numbers of resective procedures, could be observed. Complication rates were relatively low (1.09%; 0.36% with permanent deficit). Test protocols were similar even though no universal standard protocol exists. Clinicians rated the Wada test as having good reliability and validity for language determination, whereas they questioned its reliability and validity for memory lateralization. Several noninvasive functional imaging techniques are already in use. However, clinicians currently do not want to rely solely on noninvasive functional imaging in all patients.

Peripheral nerve hyperexcitability due to dominant-negative KCNQ2 mutations.

BACKGROUND: Peripheral nerve hyperexcitability (PNH) is characterized by muscle overactivity due to spontaneous discharges of lower motor neurons usually associated with antibodies against voltage-gated potassium channels. PNH may also occur in combination with episodic ataxia or epilepsy caused by mutations in K(V)1.1 or K(V)7.2 channels. Only one PNH-associated mutation has been described so far in K(V)7.2 (R207W), in a family with both PNH and neonatal seizures. METHODS: PNH was characterized by video and electromyography. The KCNQ2 gene was sequenced and K(V)7.2 channels were functionally characterized using two-microelectrode voltage-clamping in Xenopus oocytes. RESULTS: In a patient with PNH without other neurologic symptoms, we identified a novel KCNQ2 mutation predicting loss of a charged residue within the voltage sensor of K(V)7.2 (R207Q). Functional analysis of both PNH-associated mutants revealed large depolarizing shifts of the conductance-voltage relationships and marked slowing of the activation time course compared to wild type (WT) channels, less pronounced for R207Q than R207W. Co-expression of both mutant with WT channels revealed a dominant negative effect reducing the relative current amplitudes after short depolarizations by >70%. The anticonvulsant retigabine, an activator of neuronal K(V)7 channels, reversed the depolarizing shift. CONCLUSIONS: Mutations in KCNQ2 can cause idiopathic PNH alone and should be considered in sporadic cases. Both K(V)7.2 mutants produce PNH by changing voltage-dependent activation with a dominant negative effect on the WT channel. This distinguishes them from all hitherto examined Kv7.2 or K(V)7.3 mutations which cause neonatal seizures by haploinsufficiency. Retigabine may be beneficial in treating PNH.

Ion channels and epilepsy.

Ion channels provide the basis for the regulation of excitability in the central nervous system and in other excitable tissues such as skeletal and heart muscle. Consequently, mutations in ion channel encoding genes are found in a variety of inherited diseases associated with hyper- or hypoexcitability of the affected tissue, the so-called 'channelopathies.' An increasing number of epileptic syndromes belongs to this group of rare disorders: Autosomal dominant nocturnal frontal lobe epilepsy is caused by mutations in a neuronal nicotinic acetylcholine receptor (affected genes: CHRNA4, CHRNB2), benign familial neonatal convulsions by mutations in potassium channels constituting the M-current (KCNQ2, KCNQ3), generalized epilepsy with febrile seizures plus by mutations in subunits of the voltage-gated sodium channel or the GABA(A) receptor (SCN1B, SCN1A, GABRG2), and episodic ataxia type 1-which is associated with epilepsy in a few patients--by mutations within another voltage-gated potassium channel (KCNA1). These rare disorders provide interesting models to study the etiology and pathophysiology of disturbed excitability in molecular detail. On the basis of genetic and electrophysiologic studies of the channelopathies, novel therapeutic strategies can be developed, as has been shown recently for the antiepileptic drug retigabine activating neuronal KCNQ potassium channels.

Generalized epilepsy with febrile seizures plus: further heterogeneity in a large family.

BACKGROUND: Generalized epilepsy with febrile seizures plus (GEFS(+)) is a recently described benign childhood-onset epileptic syndrome with autosomal dominant inheritance. The most common phenotypes are febrile seizures (FS) often with accessory afebrile generalized tonic-clonic seizures (GTCS, FS(+)). In about one third, additional seizure types occur, such as absences, myoclonic, or atonic seizures. So far, three mutations within genes encoding subunits of neuronal voltage-gated Na(+) channels have been found in GEFS(+) families, one in SCN1B (beta(1)-subunit) and two in SCN1A (alpha-subunit). METHODS: The authors examined the phenotypic variability of GEFS(+) in a five-generation German family with 18 affected individuals. Genetic linkage analysis was performed to exclude candidate loci. RESULTS: Inheritance was autosomal dominant with a penetrance of about 80%. A variety of epilepsy phenotypes occurred predominantly during childhood. Only four individuals showed the FS or FS(+) phenotype. The others presented with different combinations of GTCS, tonic seizures, atonic seizures, and absences, only in part associated with fever. The age at onset was 2.8 +/- 1.3 years. Interictal EEG recordings showed rare, 1- to 2-second-long generalized, irregular spike-and-wave discharges of 2.5 to 5 Hz in eight cases and additional focal parietal discharges in one case. Linkage analysis excluded the previously described loci on chromosomes 2q21-33 and 19q13. All other chromosomal regions containing known genes encoding neuronal Na(+) channel subunits on chromosomes 3p21-24, 11q23, and 12q13 and described loci for febrile convulsions on chromosomes 5q14-15, 8q13-21, and 19p13.3 were also excluded. CONCLUSION: These results indicate further clinical and genetic heterogeneity in GEFS(+).

Enhanced inactivation and acceleration of activation of the sodium channel associated with epilepsy in man.

Generalized epilepsy with febrile seizures-plus (GEFS+) is a benign Mendelian syndrome characterized by childhood-onset febrile and afebrile seizures. Three point mutations within two voltage-gated sodium channel genes have been identified so far: in GEFS+ type 1 a mutation in the beta1-subunit gene SCN1B, and in GEFS+ type 2 two mutations within the neuronal alpha-subunit gene SCN1A. Functional expression of the SCN1B and one of the SCN1A mutations revealed defects in fast channel inactivation which are in line with previous findings on myotonia causing mutations in SCN4A, the skeletal muscle sodium channel alpha-subunit gene, all showing an impaired fast inactivation. We now studied the second GEFS+ mutation (T875M in SCN1A), using the highly homologous SCN4A gene (mutation T685M). Unexpectedly, the experiments revealed a pronounced enhancement of both fast and slow inactivation and a defect of channel activation for T685M compared to wild-type channels. Steady-state fast and slow inactivation curves were shifted in the hyperpolarizing direction, entry into slow inactivation was threefold accelerated, recovery from slow inactivation was slowed by threefold and the time course of activation was slightly but significantly accelerated. In contrast to other disease-causing mutations in SCN1A, SCN1B and SCN4A, the only mechanism that could explain hyperexcitability of the cell membrane would be the acceleration of activation. Because the enhancement of slow inactivation was the most obvious alteration in gating found for T685M, this might be the disease-causing mechanism for that mutation. In this case, the occurrence of epileptic seizures could be explained by a decrease of excitability of inhibitory neurons.

Two mutations in the IV/S4-S5 segment of the human skeletal muscle Na+ channel disrupt fast and enhance slow inactivation.

Fast and slow inactivation (FI, SI) of the voltage-gated Na+ channel are two kinetically distinct and structurally dissociated processes. The voltage sensor IV/S4 and the intracellular IV/S4-S5 loop have been shown to play an important role in FI mediating the coupling between activation and inactivation. Two mutations in IV/S4-S5 of the human muscle Na+ channel, L1482C/A, disrupt FI by inducing a persistent Na+ current, shifting steady-state inactivation in the depolarizing direction and accelerating its recovery. These effects were more pronounced for L1482A. In contrast, SI of L1482C/A channels was enhanced showing a more complete SI and a 3-fold slowing of its recovery. Effects on SI were more pronounced for L1482C. The results indicate an important role of the IV/S4-S5 loop not only in FI but also in SI of the Na+ channel.

Formation of a novel colored product during the Maillard reaction of D-glucose.

Reactions between reducing sugars and proteins or amino acids (Maillard reaction) lead to the formation of yellow to brown products (melanoidins) that are important for food preparation and processing, such as baking, roasting, or malt production. Thus far, the structures of the melanoidins have not been elucidated, although some structural insights have been gained from model reactions. In this study, D-glucose was heated with an amine and two colored compounds were detected by HPLC/UV--vis. After purification, the main product was identified as [(4aS,6R,7S,8R,8aR)-4,4a,6,7,8,8a-hexahydro-7,8-dihydroxy-6-hydroxymethyl-1,4-dipropyl-1H-pyrano[2,3-b]pyrazine-2-yl]-1-hydroxy-3-buten-2-one (1a). For the minor compound (2a), some spectral data were obtained, but the structure was not fully characterized. 1a and 2a are the main colored compounds when the reaction is performed in alcoholic solution or on a cellulose surface. Thus, it was concluded that products with an analogous structure are important for the color formation of foodstuffs with low water activity.

A sodium channel mutation causing epilepsy in man exhibits subtle defects in fast inactivation and activation in vitro.

Generalized epilepsy with febrile seizures plus (GEFS+) is a benign epileptic syndrome of humans. It is characterized by febrile and afebrile generalized seizures that occur predominantly in childhood and respond well to standard antiepileptic therapy. A mutation in the b1-subunit of the voltage-gated sodium channel, linked to chromosome 19q13 (GEFS+ type 1) has been found in one family. For four other families, linkage was found to chromosome 2q21-33 (GEFS+ type 2) where three genes encoding neuronal sodium channel a-subunits are located (SCN1-3A). Recently, the first two mutations were identified in SCN1A. We introduced one of these mutations, which is highly conserved to SCN1A, into the cDNA of the gene SCN4A encoding the a-subunit of the human skeletal muscle sodium channel (hSkm1). The mutation is located in the S4 voltage sensor of domain IV, predicting substitution of histidine for the fifth of eight arginines (R1460H in hSkm1). Functional studies were performed by expressing the a-subunit alone in the mammalian tsA201 cell line using the whole-cell patch clamp technique. Compared to wild-type (WT), mutant R1460H channels showed small defects in fast inactivation. The time course of inactivation was slightly (1.5-fold) slowed and its voltage dependence reduced, and recovery from inactivation was accelerated 3-fold. However, there was no increase in persistent sodium current as observed for SCN4A mutations causing myotonia or periodic paralysis. The activation time course of R1460H channels was slightly accelerated. Slow inactivation was slightly but significantly stabilized, confirming the importance of this region for slow inactivation. The combination of activation and fast inactivation defects can explain the occurrence of epileptic seizures, but the effects were much more subtle than the inactivation defects described previously for mutations in SCN4A causing disease in skeletal muscle. Hence, with regard to pathological excitability, our results suggest a greater vulnerability of the central nervous system compared to muscle tissue.