Effects of peroxisomal beta-oxidation antagonist on 2',3'-cyclic-nucleotide 3'-phosphohydrolase, membrane lipid compositions, and membrane fluidity in C-6 glial cells.

In order to understand the relationship between peroxisomal dysfunction and clinical manifestations of peroxisomal disorders, the effect of thioridazine, a peroxisomal beta-oxidation antagonist, on the differentiation, membrane lipid composition and membrane fluidity of C-6 glial cells was examined. In our study, induction of 2',3'-cyclic-nucleotide 3'-phosphohydrolase (CNP), which was considered to be a membrane-associated enzyme closely associated with myelination, was inhibited with supplementation of thioridazine, followed by an increase in the relative concentration of longer chain fatty acids in cell membrane lipids, indicating that thioridazine causes impaired differentiation in the glial stem cell system. Membrane fluidity of C-6 glial cells was examined using a fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The DPH anisotropy value was decreased in the glial cells treated with thioridazine. These results indicate that the alteration of the membrane lipid composition caused by thioridazine affects the differentiation of glial cells via the changes in membrane properties.

Enhanced expression of a-series gangliosides in fibroblasts of patients with peroxisome biogenesis disorders.

Peroxisome biogenesis disorders (PBD) are classified into Zellweger syndrome (ZS), infantile Refsum disease (IRD) and neonatal adrenoleukodystrophy. Disturbances in the differentiation of neural cells such as migration arrest are characteristic of PBD. So far the pathogenesis of these disturbances is not clearly understood. We describe an altered metabolism of glycosphingolipids in PBD which has not yet been investigated. We observed an increased amount of a-series gangliosides, GM2, GM1 and GD1a, in the fibroblasts of patients with ZS and IRD. Gangliosides GM1 and GD1a were not present in detectable amounts in normal subjects. A key step in the synthesis of a-series gangliosides is a transfer of GalNAc to ganglioside GM3, so we determined the level of ganglioside GM3 by immunohistochemical methods. We found a granular structure, which was positive toward anti-ganglioside GM3 antibody in the cytoplasm of the patients' fibroblasts. In control cells, the cell membrane was slightly positive toward anti-GM3 antibody. These results may help to clarify the pathogenesis of PBD with respect to the functional roles of glycosphingolipids in cell differentiation, proliferation and apoptosis.

Decreased membrane fluidity and unsaturated fatty acids in Niemann-Pick disease type C fibroblasts.

Niemann-Pick disease type C (NP-C) is an autosomal recessive disorder characterized by the sequestration and trapping of endocytosed cholesterol in lysosomes. The NPC1 gene on chromosome 18 was recently identified but its physiological function remains unknown. We have studied the lipid compositions of cultured human NP-C fibroblasts and mouse SPM-3T3 cell line derived from the C57BL/KsJ NP-C model mouse, which belongs to the same complementation group. Fibroblasts derived from apparently normal age-matched individuals and a subline of SPM-3T3 cells which restores cholesterol metabolism by transfer of human chromosome 18 were used as controls. Levels of free cholesterol in whole cell homogenates increased about 1.5-fold in human NP-C fibroblasts and mouse SPM-3T3 cells, while in the plasma membrane, cholesterol content did not significantly change in NP-C fibroblasts but rather decreased in SPM-3T3 cells. The total phospholipid content did not significantly change; however, among phospholipid head groups, increases in sphingomyelin and decreases in other classes were observed in human NP-C fibroblasts and mouse SPM-3T3 cells. The ratios of saturated fatty acids to unsaturated fatty acids increased in both human and mouse cells. The increase was also confirmed in the plasma membrane fraction of SPM-3T3 cells. Membrane fluidity was examined using a 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescent probe. The DPH anisotropy values were markedly increased in NP-C fibroblasts and in SPM-3T3 cells. The results suggest that a NP-C mutation causes complex alterations in cellular lipid contents and biophysical properties of the membrane.

Accumulation of glycolipids in mutant Chinese hamster ovary cells (Z65) with defective peroxisomal assembly and comparison of the metabolic rate of glycosphingolipids between Z65 cells and wild-type CHO-K1 cells.

The influence of peroxisomal dysfunction on glycosphingolipid metabolism was investigated using mutant Chinese hamster ovary (CHO) cells (Z65) with defective assembly of the peroxisomal membranes. In accordance with previous observations, the concentration of very long chain fatty acid (C24:0) was shown to be higher in Z65 cells than in control cells. We then compared the composition of glycolipids in Z65 cells with that in CHO-K1 cells, which are wild-type Chinese hamster ovary cells with intact peroxisomes, and found significantly increased concentrations of ceramide monohexoside (CMH) and ganglioside GM3 in Z65 cells. However, there were no differences in the concentrations of glycerophospholipids, triglycerides, free fatty acids and cholesterol between Z65 and CHO-K1 cells. Further, to investigate the metabolic rate of the major lipids, Z65 and CHO-K1 cells were pulse-labeled with [3-14C]serine. [3-14C]Serine was incorporated into phosphatidylserine, phosphatidylethanolamine and sphingomyelin more quickly in CHO-K1 than in Z65 cells. However, after 48 h, the radioactivity incorporated into those lipids, including CMH, was greater in Z65 cells than in CHO-K1 cells. Thus, the altered metabolism of glycosphingolipids, probably due to peroxisomal dysfunction, was thought to be responsible for the change in glycosphingolipid composition in Z65 cells.

Dolichol deposition in developing mammalian brain: content of free and fatty-acylated dolichol and proportion of specific isoprenologues.

Dolichol serves a critical role in N-linked glycoprotein synthesis as the carrier of saccharide moieties. In this study the deposition of dolichol was investigated in developing rat brain. The contents of both the free and fatty-acylated forms of dolichol as well as the specific isoprenologues of dolichol were determined. Two distinctive phases of rapid dolichol deposition were observed; the first during brain development, and the second, with aging. The predominant form of brain dolichol was shown to be the free derivative at all ages; however, during the two phases of rapid deposition of total dolichol, fatty-acylated dolichol became detectable and increased transiently. The rapid rate of increase of brain dolichol during development occurred later than that of brain cholesterol, which shares a common biosynthetic pathway with dolichol. Thus, the peak rate of increase of dolichol occurred at 25 days postnatal, and of cholesterol, at 18 days postnatal. A distinct ontogenetic alteration in the proportion of the individual molecular species (isoprenologues) of dolichol also was defined. These observations suggest that dolichol is particularly involved in developmental events of the third to sixth postnatal weeks in rat brain, e.g. myelination and synaptogenesis.

Dolichol kinase and the regulation of dolichyl phosphate levels in developing brain.

The developmental changes of dolichol kinase activity and dolichyl phosphate levels have been studied in rat brain. Because both dolichol kinase activity and dolichyl phosphate were enriched in microsomes, detailed study of this subcellular fraction was carried out. Dolichol kinase specific activity in brain microsomes increased postnatally 3-fold to a maximum at ca. 30 days of age. This increase was observed whether exogenous dolichol was present or not and whether Zn2+ or Ca2+ was utilized as the cation for the enzyme. Zn2+ was the most effective cation in developing brain, as we have shown previously for adult brain (Sakakihara, Y. and Volpe, J.J., J. Biol. Chem., 260 (1985) 15413-15419). Although the Vmax for the enzyme increased by three-fold with development, the Km for dolichol and for CTP did not change, indicating that the developmental increase was not related to an alteration in catalytic efficiency of the enzyme. A striking and parallel increase in dolichyl phosphate levels in brain microsomes was defined with development. Levels were lowest in one-day-old animals and then increased ca. 13-fold to a maximum at 30 days of postnatal age. The parallel increase in dolichol kinase activity and dolichyl phosphate levels in microsomes of developing brain suggests that dolichol kinase is the principal determinant of cellular levels of dolichyl phosphate, the critical intermediate in the dolichol-linked pathway to glycoproteins.

Dolichol-linked glycoprotein synthesis in developing mammalian brain: maturational changes of the N-acetylglucosaminylphosphotransferase.

The enzyme UDP-N-acetylglucosamine:dolichyl phosphate, N-acetylglucosamine-1-phosphate transferase (GlcNAc-1-P transferase), the first committed step in the dolichol-linked oligosaccharide pathway for glycoprotein biosynthesis, has been studied in developing rat brain. The enzyme was shown to be localized in microsomes, particularly heavy microsomes, and to be activated by Mg2+ and inhibited by tunicamycin. Study of the enzyme with brain development demonstrated two prominent findings. First, the accentuation of enzymatic activity caused by addition of a saturating concentration of dolichyl phosphate was greater in brain of older (3-4 weeks of age and subsequently) animals (25-fold) than in brain of younger (less than two weeks of age) animals (10-fold). This difference suggests that dolichyl phosphate may be limiting for GlcNAc-1-P transferase activity in endoplasmic reticulum of the older animals. Second, a marked (3.5-fold) increase in activity occurred over a discrete time period (3-4 weeks of postnatal life) during brain development. That this increase reflected an increase in enzyme amount rather than in catalytic efficiency was suggested by kinetic studies. Coupled with our previous demonstrations of increases in brain dolichol, dolichol kinase activity, and dolichyl phosphate levels during approximately the same developmental period (Sakakihara, Y. and Volpe, J.J., Dev. Brain Res., 14 (1984) 225-262; Volpe, J.J. et al., Dev. Brain Res., in press), the data suggest a temporally discrete period of activation of the dolichol-linked pathway to glycoproteins. Whether the pathway is regulated coordinately or sequentially is a fertile topic for future study.

Magnetoencephalographic study of giant somatosensory evoked responses in patients with rolandic epilepsy.

We report five patients with rolandic epilepsy associated with giant somatosensory responses to median nerve stimulation, in whom we analyzed the pathophysiologic relationship between rolandic discharges and the somatosensory responses using magnetoencephalography. Four of the five patients showed giant P30m, the current source of which was localized in the primary somatosensory cortex, while the first cortical response, N20m, was not enhanced, except in one patient. The current source of the giant middle-latency component, N70m, was localized posterior to that of N20m, possibly in the posterior parietal cortex, in all patients. The initial positive peak and large negative peak of rolandic discharges were identical to P30m and N70m with respect to the current source localization, wave form, topographic pattern, and time relationship in the electroencephalogram and magnetoencephalogram, and somatosensory evoked magnetic field and somatosensory evoked potential records, respectively. In addition, the secondary sensory cortex was considered to be the generator of the middle-latency component in one patient. In one patient, the current intensity of the N70m was normalized along with clinical improvement and the disappearance of rolandic discharges, whereas those of other somatosensory evoked magnetic field components remained unchanged. Our data suggest that the rolandic discharge generator mechanism in these patients could be closely related to the developmental alteration of excitability in the primary somatosensory cortex, posterior parietal cortex, and secondary somatosensory cortex, which decreased with age, and it could share a common neuronal pathway, at least in part, with the giant P30m-N70m (N90m) in the somatosensory evoked magnetic field through the sequential and parallel processing of somatosensory information.

Ethical attitudes of Japanese physicians regarding life-sustaining treatment for children with severe neurological disabilities.

Ethical attitudes of Japanese physicians regarding life-sustaining treatment for children with severe neurological disabilities (SND) were investigated by mailing a translated questionnaire which the Child Neurology Society (CNS) of the United States used for their survey. The questionnaire was sent to 202 council members of the Japanese Society of Child Neurology (JSCN), and the answers of 147 respondents (72.8%) were analyzed. It was found that the majority (85. 0%) of respondents believed that the same level of care should be provided to children with SND as those without it. However, fewer respondents (15.6%) believed that cardiopulmonary resuscitation was indicated for children with progressive or degenerative brain disorders. With respect to the authoritative role of medical indications and family/guardian's wishes in clinical decision-making for children with SND, about 30% of respondents believed that medical indications should override family/guardian's wishes. However, almost as many respondents (29.9%) chose an ambivalent answer. If compared with the results of the preceding CNS survey, considerably more respondents gave ambivalent answers (average 26. 6%) than in the CNS survey (5.8%). About half of the respondents (49. 0%) acknowledged the need for ethical guidelines to help physicians make ethically difficult decisions. Although statistical comparison was not possible, there were considerable differences between the results of the current study and those of the CNS survey.

Changing spectrum of pediatric neurologic disorders during 18 selected years, 1900-1980, at the Hospital of University of Tokyo.

In order to assess the changing pattern of pediatric neurologic disorders during this century, we retrieved and analyzed the medical records of hospitalized patients with neurologic disorders in our hospital from 1900 to 1980. It was demonstrated that bacterial meningitis had been by far the most common cause of death until 1950. After 1950, both the incidence and the mortality rate of bacterial meningitis declined rapidly probably because of the improved medical care and introduction of various antibiotics. We noticed several interesting features in the changing spectrum of bacterial meningitis as described below. First it was demonstrated that the incidence of tuberculous meningitis declined almost a decade later than those of other kinds of purulent meningitis. Second, the incidence of bacterial meningitis declined even before the introduction of antibiotics. Although the development of antibiotics was the main contributing factor in improving the prognosis for bacterial meningitis, it is suggested from our data that other factors such as improved general supportive care and carrying out of public health programs also played an important role in improving the overall prognosis for bacterial meningitis.

Reduction of seizure frequency with clomipramine in patients with complex partial seizures.

Two patients with complex partial seizures who had been refractory to various antiepileptics were treated with clomipramine. The frequency of the seizures was reduced to 0-30% of the original levels. It has been reported that imipramine is effective in absence and minor motor seizures, and its antiepileptic effect is thought to be related to the inhibition of the presynaptic re-uptake of serotonin and norepinephrine. The basic effect of clomipramine is the same as that of imipramine except that the inhibitory action of clomipramine on serotonin re-uptake is 5- to 10-times more potent than that of imipramine. It is implied that clomipramine may be of use in the treatment of partial epilepsies.

A case of parietal lobe epilepsy with distinctive clinical and neuroradiological features.

We present a case of parietal lobe epilepsy, the epileptogenic focus of which was conspicuously demonstrated on magnetoencephalography (MEG). The remarkable fluctuation in the seizure frequency and the presence of atonic seizures were suggestive of atypical partial benign epilepsy of childhood (APBEC). An interictal positron emission tomography (PET) scan performed during the cluster of fits revealed hypermetabolism around the epileptogenic focus, which might be related to the marked tendency of clustering of seizures in this patient.

Varicella-associated acute necrotizing encephalopathy with a good prognosis.

A patient with acute necrotizing encephalophathy (ANE) following varicella infection with a good prognosis is reported. A somatosensory evoked magnetic field (SEF) study using a 37-channel-magnetoencephalography system demonstrated normal latency and strength of the first component (N20m) elicited by median nerve stimulation, despite bilateral symmetrical thalamic lesions on MRI. The normal SEF findings and the good prognosis suggested a reversible breakdown of the blood-brain barrier, and an edematous process as the brain pathology. Furthermore, our results support the idea of distinct generators for the three earliest cortical SEF components (N20m, P30m, N45m).

Temporary improvement of neurological symptoms with gammaglobulin therapy in a boy with adrenoleukodystrophy.

A 10-year-old boy with adrenoleukodystrophy was treated with gammaglobulin in conjunction with a mixture of glyceryl trioleate and glyceryl trierucate. With a high dose of gammaglobulin, clinical improvement, including the reduction of visual field defects, was noted. On magnetic resonance imaging, attenuation of the enhancement of the rim with gadolinium was observed, suggesting repair of the blood-brain barrier. When auditory agnosia developed later, a temporary improvement was again obtained with gammaglobulin. Although the progress of the disease could not be arrested permanently, gammaglobulin therapy seemed to have been associated with temporary improvement of the clinical symptoms in this patient with adrenoleukodystrophy.

Zonisamide - induced urinary lithiasis in patients with intractable epilepsy.

We report here three patients with intractable epilepsy who developed urinary lithiasis during zonisamide (ZNS) treatment. Abdominal pain due to left-sided hydronephrosis was the initial symptom in the first patient, and it was resolved after the excretion of a stone. The second patient, who had no specific symptoms, was found to have a thick sludge of calcium phosphate in the bladder when he suffered from aspiration pneumonia and dehydration. The third patient, who had a history of recurrent urinary obstruction, was also found to have a thick sludge of calcium oxalate in the bladder. The urinalysis of the three patients revealed alkaline urine and hypercalciuria. Although their urinary lithiasis was resolved by discontinuation of ZNS and supportive therapy, routine examination of urine parameters such as pH and sediments, and daily urine-output checks are thought to be necessary during treatment with ZNS, especially for patients who are bedridden for a long time and receive multiple antiepileptic drugs.

Transcranial magnetic stimulation in an adrenoleukodystrophy patient.

We experienced an 11-year-old boy diagnosed as having adrenoleukodystrophy (ALD), and studied his motor evoked potentials (MEPs) elicited by transcranial magnetic stimulation (TMS). He had intellectual and visual impairment, and MRI revealed high intensity of the parieto-occipital white matter. On evaluation of the long tracts, slight spasticity with equivocal Babinski signs was noted: however, the long tracts appeared intact on MRI, and short latency somatosensory evoked potentials (SSEPs) were completely normal. On TMS delivered through a circular coil, MEPs recorded from the relaxed first dorsal interosseous muscle showed that only the duration was significantly prolonged, which may be due to temporal dispersion of descending volleys in the pyramidal tracts, while the latency was not prolonged. TMS in ALD was considered sensitive and useful for detecting subtle involvement of the long tracts.

The gene copy ratios of SMN1/SMN2 in Japanese carriers with type I spinal muscular atrophy.

Spinal muscular atrophy is an autosomal recessive neurodegenerative disorder with progressive weakness and atrophy of voluntary muscles. The survival motor neuron gene (SMN) is present in two highly homologous copies (SMN1 and SMN2) on chromosome 5q13. Homozygous deletion of exons 7 and 8 of SMN1 is responsible for spinal muscular atrophy. In spinal muscular atrophy patients, SMN2 partially compensates for the lack of SMN1. Previously, we reported the relatively high incidence of a large deletion including the SMN1 region in Japanese spinal muscular atrophy type I patients. In order to further establish the genetic background of Japanese spinal muscular atrophy type I patients, we investigated the SMN1/SMN2 ratio in the carriers. In normal individuals, there is one copy of each gene on the chromosome (the SMN1/SMN2 ratio was 1). Among 15 carriers (14 parents and one carrier sibling of Japanese type I spinal muscular atrophy patients with homozygous deletion of exons 7 and 8 of SMN1), we found that the SMN1/SMN2 ratio was 0.5 or 1 in 11 (73.3%) carriers. The remaining four carriers had an SMN1/SMN2 ratio of 1/3. This finding supports the idea that deletion rather than conversion is the main genetic event in type I spinal muscular atrophy. In addition, the ratio of SMN1/SMN2 among Japanese carriers, which was thought to be higher than that of the Western population, was compatible with the results obtained in Western populations. For further insight into the characteristic genetic background of spinal muscular atrophy in Japanese, determination of the gene copy number is essential.