Physicochemical property changes of amino acid residues that accompany missense mutations in SCN1A affect epilepsy phenotype severity.

BACKGROUND: Several different missense mutations in the voltage-gated sodium channel subunit gene SCN1A have been identified in epileptic patients with benign phenotype and patients with severe phenotype. However, the reason why similar missense mutations in SCN1A result in different phenotypes has not yet been fully clarified. OBJECTIVE: To clarify the phenotype-genotype relationship in SCN1A, a meta-analysis was performed to quantitatively determine the effect of amino acid substitutions in SCN1A on epilepsy severity phenotype using physicochemical property indices of the amino acid, and to discuss in the context of the molecular evolution of the proteins. METHODS: PubMed was searched for articles and information was extracted on localisation and types of SCN1A missense mutations in patients with benign and severe epileptic syndromes; detailed information was also extracted. RESULTS: Meta-analysis quantitatively revealed that the physicochemical properties of several amino acids significantly affected epilepsy phenotype severity. It showed that missense mutations that decreased protein hydrophobicity were significantly associated with severe epilepsy phenotypes. It also showed that the phenotype severity of SCN1A missense mutations in the transmembrane domains of SCN1A (128/155; 82.6%) could be predicted with high sensitivity and positive predictive values using the physicochemical property changes, indicating the possibility of phenotype prediction for entirely new missense mutations using analytical methods. CONCLUSIONS: The results show that changes in the physicochemical properties of amino acids affected both the phenotype and clinical symptoms of patients with SCN1A missense mutations. This meta-analysis study provides new insights into SCN1A gene functions and a new strategy for genetic diagnosis, genetic counselling and epilepsy treatment.

Effect of localization of missense mutations in SCN1A on epilepsy phenotype severity.

BACKGROUND AND METHODS: Many missense mutations in the voltage-gated sodium channel subunit gene SCN1A were identified in patients with generalized epilepsy with febrile seizures plus (GEFS+) and severe myoclonic epilepsy of infancy (SMEI), although GEFS+ is distinct from SMEI in terms of clinical symptoms, severity, prognosis, and responses to antiepileptic drugs. The authors analyzed the localization of missense mutations in SCN1A identified in patients with GEFS+ and SMEI to clarify the phenotype-genotype relationships. RESULTS: Mutations in SMEI occurred more frequently in the "pore" regions of SCN1A than did those in GEFS+. These SMEI mutations in the "pore" regions were more strongly associated than mutations in other regions with the presence of ataxia and tendency to early onset of disease. The possibility of participation of ion selectivity dysfunction of the channel in the pathogenesis of SMEI was suggested by a mutation in the pore region (R946C) identified in a SMEI patient. CONCLUSIONS: There was a significant phenotype-genotype relationship in generalized epilepsy with febrile seizures plus and severe myoclonic epilepsy of infancy with SCN1A missense mutations. More severe sodium channel dysfunctions including abnormal ion selectivity that are caused by mutations in the pore regions may be involved in the pathogenesis of SMEI.

Thermoluminescence of photostimulable materials after X irradiation below room temperature.

Thermoluminescence glow peaks in the temperature range 100 to 400 K are investigated for BaFX (X = Cl, Br) crystals after X irradiation at 100 K. A prominent glow peak of BaFCl around 210 K is found to be composed of a few recombination roots, that is, the peak corresponds to the recombination of hole trapped centres such as an O- centre and a dissociated Cl2- centre with the F (F-) centre and the O2--F(Cl-) pair defect. Another small glow peak around 270 K is likely to occur from thermal dissociation of the O2- -F(Cl-) pair defect. The main glow peak of BaFBr:O2- at 170 K may be attributed to a recombination of an O- centre with the F(Br-) centre.

Genetic variations in five genes involved in the excitement of cardiomyocytes.

We provide here 29 genetic variations, including 28 novel ones, in five genes that are potentially involved in the excitement of cardiomyocytes: we found 4 in KCNA10, 2 in KCNK1, 8 in KCNK6, 11 in SLC18A1 (VMAT1), and 4 in SLC6A2 (norepinephrine transporter). We also examined their allelic frequencies in a Japanese population of long QT syndrome-affected and nonaffected individuals. These data would be useful for genetic association studies designed to investigate acquired arrhythmias.

Multiple single-nucleotide polymorphisms (SNPs) in the Japanese population in six candidate genes for long QT syndrome.

We report here 20 single-nucleotide polymorphisms (SNPs), including 15 novel ones, in six genes that are considered to be candidates for long QT syndrome (LQTS): 2 SNPs in KCNB1, 3 in KCND3, 3 in KCNJ11, 7 in ABCC9, 3 in ADRB1, and 2 in SLC18A2. We also examined their allelic frequencies in a Japanese sample population of LQTS-affected and nonaffected individuals. These data will be useful for genetic association studies designed to investigate acquired arrhythmias.

Localization of epidermal-type fatty acid binding protein in alveolar macrophages and some alveolar type II epithelial cells in mouse lung.

Almost all alveolar macrophages in the mouse lung were strongly immunoreactive for epidermal-type fatty acid binding protein. At the electron microscope level, the immunoreactive material was localized diffusely in the cytoplasm but not within the nucleus. A certain number of alveolar type II epithelial cells were also immunoreactive for the protein with variable immunointensity, while a substantial number of the type II cells were immunonegative. No immunoreactive interstitial fibroblasts were encountered. Based on the present findings, possible roles of epidermal-type fatty acid binding protein in the host-defence mechanism played by alveolar macrophages are suggested.

Lasting changes in NMDAR1 mRNA level in various regions of cerebral cortex in epileptogenesis of amygdaloid-kindled rat.

The involvement of NMDA receptor subunit, NR1, with kindling phenomenon has been reported, but the role of NR1 in epileptogenesis is still unknown. We have examined the expression levels of NR1 mRNA in the cerebral cortices of amygdaloid-kindled rats. Northern blot analysis showed a significant increase in NR1 mRNA expression level in the ipsilateral frontal and temporal cortices at 4 weeks after the last generalized seizure. At the same time, NR1 mRNA decreased in the bilateral piriform cortices. These data suggest that NR1-mediated transmission may have an impact in the neurobiological basis of enduring epileptogenesis.

Clinical and radiological features in four adolescents with nutcracker syndrome.

We describe four adolescents with the nutcracker syndrome. In three patients, the nutcracker syndrome was detected through mass urinary screening; the other patient was diagnosed after a sudden onset of dark urine. All patients underwent magnetic resonance angiography (MRA) for diagnosis of the nutcracker syndrome, which revealed dilatation of the left renal vein ranging between 7.4 and 13 mm at the hilar portion. A renal biopsy, performed in three patients, showed no remarkable abnormalities in the glomerulus or tubulointerstitial tissue. The patients complained of physical discomfort, including headache, abdominal pain, fainting, and tachycardia mimicking clinical symptoms of an orthostatic disturbance. However, no chronic systemic diseases were detected in any of the patients after repeated laboratory examinations. An orthostatic disturbance preceded diagnosis in three patients. This report indicates that the nutcracker syndrome may cause serious physical ailments that clinically mimic an orthostatic disturbance. It may be important to identify the nutcracker syndrome among children who manifest non-specific physical complaints. MRA could be a safe and reliable method for diagnosing the nutcracker syndrome.

Functional significance of stimulatory GTP-binding protein in hippocampus is associated with kindling-elicited epileptogenesis.

In order to evaluate the involvement of the stimulatory G-protein (Gs)-related transduction system in the basic mechanisms of epilepsy, we examine the expression levels of Gsalpha mRNA and specific GTP-binding ability in the hippocampus of amygdaloid-kindled rats at various seizure stages. Northern blot analysis showed a significant increase in the Gsalpha mRNA expression level in the bilateral hippocampus at 24h after the last generalized seizure. The [3H]-GTP-binding assay with isoproterenol (IPN), a beta-receptor agonist, revealed a remarkable increase of Bmax values in the sham-operated control and partially kindled groups. However, the IPN-induced increase of Bmax values was abolished on both sides of the hippocampus at 24 h after and at 4 weeks after the last generalized seizure in fully kindled rats. These data suggest that alteration in the Gs function and beta-adrenergic receptor-Gs coupling might be implicated in the neurobiological basis of the induction mechanisms of the generalization of seizures and the mechanisms of the maintenance of enduring epileptogenesis. Conversely, the Gs-related transduction system might have a lesser impact on the acquisition process of epileptogenesis.

Twenty single nucleotide polymorphisms (SNPs) and their allelic frequencies in four genes that are responsible for familial long QT syndrome in the Japanese population.

We report here 20 single nucleotide polymorphisms (SNPs), including 10 novel ones, and their allelic frequencies detected in four genes that are known to be responsible for familial long QT syndrome in the Japanese population; 7 polymorphisms are in the KCNQ1 gene, 6 in the KCNH2 gene, 5 in the SCN5A gene, and 2 in the KCNE1 gene. These data will be of use for genetic association studies of acquired cardiac arrhythmias.

Inhibition of liver metastases and tumor cell invasion in spontaneous liver metastasis model (LMFS) by sodium D-glucaro-delta-lactam (ND2001).

We examined the inhibitory effect of sodium D-glucaro-delta-lactam (sodium 5-amino-5-deoxy-D-glucosaccharic acid-delta-lactam: ND2001) upon liver metastases of the LMFS tumor. A permanent cell line, LMFS, was established from a spontaneously occurring murine retroperitoneal tumor of BALB/c mouse origin, and after a subcutaneous injection, the LMFS cells proliferated at the inoculation site (100% take) with liver metastases. ND2001 had little effect on the cell growth, cell cycle and phagokinesis of the LMFS cells in vitro. However, when the invasive activity was measured by the Boydem chamber method, the number of LMFS cells was reduced, with inhibition rates of 98.0%. After the LMFS cells treated with ND2001 in vitro, the numbers of hepatic metastases of subcutaneous inoculation of treated cells were reduced dose-dependently, and those of intravenous inoculation were not found by microscopical study. When the LMFS tumor-bearing mice were treated with ND2001 (0, 30, 100 mg/kg/d) from day 1, ND2001 (30 mg/kg) inhibited the liver metastases with a rate of 56.4%, and when given from day 15, ND2001 (100 mg/kg) inhibited with a rate of 47.5%. But ND2001 showed neither cytocidal nor anti-tumor activity. Combination therapy of primary tumor resection and ND2001 administration revealed that preoperative use of ND2001 was more effective in preventing liver metastases. These results suggested that ND2001 might have a potential use as an anti-metastatic agent for operative patients without metastasis.

Up-regulation of type II adenylyl cyclase mRNA in kindling model of epilepsy in rats.

The expression level of type II adenylyl cyclase mRNA (ACII) was analyzed by northern blotting in amygdaloid kindled rats. Remarkable increases in ACII mRNA were observed in the bilateral cerebral cortex and hippocampus at 24 h after the last generalized seizure. The elevated expression level in the hippocampus persisted for 4 weeks on the stimulated side. There were no changes in expression level in single-stimulated and partially-kindled states. These results suggest that the involvement of ACII might have an effect on the mechanisms of seizure generalization and the maintenance of persistent epileptogenesis rather than on the acquisition process.

Estimation of photoparoxysmal response elicited by half-field visual stimulation.

We examined photoparoxysmal responses (PPRs) elicited by half-field visual stimulation with deep-red flicker light to determine the neurophysiological features of photosensitive epilepsy (PSE). EEG revealed two types of PPRs. One had the focal spike in the occipital region and the other in the temporo-occipital region at the contralateral hemisphere. The equivalent current dipoles of these types were located at the occipital cortex and the inferior temporal (IT) cortex, respectively. These cortices comprise one of the main pathways in the visual system, and they play important roles in color discrimination. Thus, we propose that the visual system, especially the occipital cortex and the IT cortex, might be involved in the generator mechanism of PSE.

Dipole source localization of interictal epileptiform activity in temporal lobe epilepsy with medial temporal lesion.

Dipole sources of interictal epileptiform activities recorded by conventional electroencephalogram (EEG) were estimated using the dipole tracing method. Four cases of temporal lobe epilepsy with medial temporal lesions were studied. Two patients with hippocampal sclerosis, one patient with granulation in the hippocampus and one patient with cavernous angioma were involved in the study. Interictal epileptiform activities were classified into two patterns according to the topography of spikes. They were widespread spikes over the parasagittal electrodes (parasagittal spikes) and restricted spikes at the temporal electrodes (temporal spikes). Dipole sources of parasagittal spikes were localized in the medio-basal temporal lobe with vertically orientated vector moment. Dipole sources of temporal spikes were localized in the medio-basal temporal lobe with horizontally orientated vector moment. Locations of dipoles and directions of vector moments were consistent with topography and polarity of spikes. The difference in the two patterns of interictal epileptiform activities was derived from the difference in the direction of the vector moment of dipole sources. There was no difference in the location of dipole sources. Both the dipole sources and the lesions were localized in the same medio-basal temporal lobe. Dipole tracing was very useful in localizing the dipole sources of interictal epileptiform activities and in understanding the neurophysiological background.

[Effects of HCFU and TNP 470 on liver metastasis of BALB/c retroperitoneal sarcoma (LMFS)].

Combination therapy using HCFU and TNP 470, which inhibits angiogenesis, was examined for effectiveness against the footpad injection model of LMFS tumor. This LMFS, a retroperitoneal sarcoma of BALB/c mice, proliferated at the inoculation site (100% take) and all mice operated on after day 15 had spontaneous metastatic nodules in the liver. Mice with the LMFS tumor were given HCFU and 5-FU (5 days/week), and/or TNP 470 (3 days/week) from day 3 for 3 weeks and sacrified at day 28 under anesthesia. Seven of 10 mice receiving 5-FU and TNP 470 died from the side effects of the drugs. Mean tumor weight and liver metastatic nodules were determined and compared with a control group. The results were as follows: HCFU group: 94.6%, 11.8%, 5 FU group: 73.9%, 28.8%, TNP 470 group: 67.6%, 44%, HCFU and TNP 470 group: 33.3%, 6.4%. Mice with LMFS were given HCFU and/or TNP 470 from day 3 for 4 weeks. The foot on the injected side was amputated on day 15, and the animals were sacrified on day 35. Liver metastatic nodules compared with those of the operation (OP) group as follows: OP + HCFU group: 14.4%, OP + TNP group: 39.1%, and OP + HCFU + TNP 470 group: 5.4%. Histologically, 5 of 5 mice of the OP group, 3 of 5 of the OP + HCFU group, 4 of 5 of the OP + TNP 470 group and 1 of 5 of the OP + HCFU + TNP 470 group had liver metastases. These results show that while either HCFU or TNP 470 is effective by itself, a combination of these drugs is more effective against liver metastasis.

Attention changes the peak latency of the visual gamma-band oscillation of the EEG.

To investigate the physiological role of visual gamma-band oscillation (GBO), we calculated the event-related dynamics of the EEG power-spectrum for paired visual stimuli (S1 and S2) with or without attention in 12 subjects. The visual stimuli elicited transient increases in the GBO power (around 40 Hz), which were maximal over the parietal area. The peak GBO increase appeared around 300 ms after stimulus onset, but its latency was shorter after S1 and longer after S2 under the 'with attention' than under the 'without attention' condition. This transient increase in the visual GBO is thought to reflect attention and to reset the activity of the visual system in preparation for a new stimulus.

Differences in the width of the intercellular spaces in the epithelial basal infolding and the renal glomerular filtration site between freeze-substitution and conventional fixation.

After aldehyde prefixation, pretreatment with cryoprotectant and subsequent freeze-substitution with OsO4 in acetone (AC-FS), extensive gap junction-like close membrane appositions are frequently found in the basal infolding of the salivary gland epithelium, although the desmosomal intercellular space had the same width as with conventional electron microscopy. The intercellular space between podocyte pedicles and endothelial cells at the renal glomerular filtration site was narrower by the total width of 2 laminae lucidae following AC-FS than with conventional electron microscopy and was occupied by a homogeneous lamina densa without a lamina lucida, although no marked difference was discernable in the thickness of the lamina densa itself between the 2 preparative procedures. In addition, a decrease in the thickness of the glycocalyx was evident in the intestinal epithelial microvilli following AC-FS. It is thus likely that osmication in acetone at freezing temperatures remove the glycocalyx and related structures to a variable extent, and that this loss is responsible for reducing the intercellular spaces at some of the simple appositions narrower to the dimensions of the gap junction. It is also responsible for disappearance of the lamina lucida of the basement membrane.