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.

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.

Long-term ventilator-assisted children in Japan: a national survey.

Owing to improved technology and care for patients who need mechanical ventilation, the quality of life as well as the prognosis for long-term ventilator-assisted patients has improved significantly in recent years. However, the increased number of these patients has raised economic, ethical and medical problems. In order to assess the magnitude of these problems, we conducted the first nationwide survey on the status of long-term ventilator-assisted children in Japan. Questionnaires were mailed to 2524 pediatric departments at hospitals in Japan with more than 100 beds. At the time of the survey, 282 hospitals had 567 patients who had been ventilated for more than a month. Among these patients, 434 were younger than 20 years and had been ventilated for more than 3 months. The most common basic disorders were: various myopathies (n = 65), hypoxic-ischemic encephalopathy (n = 60), spinal muscular atrophy type 1 (Werdnig-Hoffmann disease, n = 55), chronic lung disorders of prematurity (n = 21), Ondine's curse (n = 22), drowning (n = 17) and congenital heart diseases (n = 16). Of these 434 patients, only 61 were ventilated at home. Although home care was considered suitable for chronic ventilator patients by many pediatricians who responded to the survey, its realization has been hampered by the lack of a system and regulations to support it. The fact that many pediatricians in Japan have actively prolonged the life of Werdnig-Hoffmann patients, from whom aggressive life saving measures have been withheld in most Western countries, has raised ethical as well as medical issues.

Acute necrotising encephalopathy of childhood: a new syndrome presenting with multifocal, symmetric brain lesions.

The clinicopathological features of a previously unrecognised type of acute encephalopathy prevalent among Japanese children is described by reviewing the records of 13 consecutive patients treated and 28 previously reported cases. The hallmark of this encephalopathy, proposed to be a novel entity termed acute necrotising encephalopathy of childhood, is multiple, necrotic brain lesions showing a symmetric distribution. The encephalopathy was noted in previously healthy children after respiratory tract infections, with presenting symptoms of coma, convulsions, vomiting, hyperpyrexia, and hepatomegaly. Laboratory examinations disclosed liver dysfunction, uraemia, and hypoproteinaemia. The histological appearance of the liver was variable and non-specific. Cerebrospinal fluid contained an increased amount of protein. Computed tomography and MRI showed the presence of symmetrically distributed brain lesions of the thalamus, cerebral white matter, brainstem, and cerebellum. Necropsy examination confirmed extensive fresh necrosis of these regions with evidence of local breakdown of the blood-brain barrier. Based on the characteristic combination of clinical and pathological findings, acute necrotising encephalopathy of childhood can be distinguished from previously known encephalopathies, including Reye's syndrome.

Application of nasal continuous positive airway pressure to early extubation in very low birthweight infants.

Using a preset protocol for early extubation, 50 babies were randomly selected to post-extubation headbox or post-extubation nasal continuous positive airway pressure (N-CPAP). All infants weighed less than 1500 g, had a gestational age of less than 34 weeks, and had been weaning from mechanical ventilation within seven days of life. The criteria for extubation included stable condition, fraction of inspired oxygen (FIO2) of < or = 35%, peak inspiratory pressure (PIP) of < or = 15 cm H2O (1.47 kPa), and ventilator rate of 6/minute. Before extubation, a loading dose of aminophylline was given followed by maintenance treatment. If reintubation was not required within 72 hours of the initial extubation the procedure was considered successful. The reintubation criteria included FIO2 > or = 70% to maintain arterial oxygen tension (PaO2) of > or = 50 mm Hg (6.67 kPa) or pulse oximetry between 90-96% and pH of < 7.25, and arterial carbon dioxide tension (PACO2) of > 60 mm Hg (8.00 kPa) and severe or recurring apnoea. The overall success rate of early extubation was 66% (33/50). The individual successful extubation rate of post-extubation in the N-CPAP group and the post-extubation headbox group were 84% (21/25) and 48% (12/25), respectively (p = 0.017; chi 2). There were no significant differences in clinical characteristics between the two groups. The most common cause of failure in early extubation was apnoea, and most occurred in the headbox group (9/12). These results suggest that application of N-CPAP to a preset protocol for extubation can achieve a better success rate of early extubation in very low birthweight (VLBW) infants.

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.

Distribution of leptomeningeal glioneuronal heterotopia in alobar holoprosencephaly.

OBJECTIVE: To clarify the pathogenetic significance of the topographic distribution of leptomeningeal glioneuronal heterotopia, a common finding in holoprosencephaly. DESIGN: Gross, histological, and immunohistochemical observations of alobar holoprosencephaly in brain specimens taken at autopsy. SETTING: Referral center. MATERIALS: Brains removed at autopsy from five consecutive patients with alobar holoprosencephaly. MAIN OUTCOME MEASURES: Immunoperoxidase staining for glial fibrillary acidic protein. RESULTS: In all brains, leptomeningeal glioneuronal heterotopia showed an identical distribution, ranging from the basal prosencephalon to the pons, with the thickest distribution occurring in the basal prosencephalon. CONCLUSION: The constant localization implicates leptomeningeal glioneuronal heterotopia in severe dysgenesis of midline prosencephalon, the basic pathogenesis of the anomaly.

Liveborn twin with intrauterine death of one twin: report of two cases.

Twin pregnancies carry a greater mortality and morbidity rate than singleton pregnancies. In case of an intrauterine fetal death (IUFD), the risk of mortality and morbidity of the surviving twin is increased. The pathogenesis is usually due to twin to twin transfusion. The donor twin is hypovolemic, anemic and often shows a growth retardation or even severe enough to cause an IUFD. The recipient twin is hypervolemic, polycythemic and weighs more than its co-twin. In this paper we reported two cases of liveborn twin complicated by an IUFD of its co-twin. Both cases were monochorionic twins. The first case was born at 29 weeks and 6 days of gestation, a male infant weighed 1054 g. His co-twin was stillborn and weighed 722 g. At birth the surviving infant showed respiratory distress syndrome (RDS), anemia and bilateral periventricular echogenicity (PVE). The second case was a female infant with a gestational age of 26 weeks and 2 days and weighed 850 g. Her stillborn co-twin weighed 804 g. Both twins showed signs of hydrops, and the liveborn infant had RDS, marked anemia (Hb 6 g/dl) and hypoalbuminemia. We will discuss the possible pathogenesis in our cases and also review some literature.

Nasal continuous positive airway pressure following surfactant replacement for the treatment of neonatal respiratory distress syndrome.

To evaluate whether nasal continuous positive airway pressure (N-CPAP) could be an alternative to ventilator treatment in infants with severe respiratory distress syndrome (RDS) after surfactant treatment, we performed the trial on 15 newborn babies with birth weight > or = 1500 g. All babies were put on N-CPAP as soon as the diagnosis of RDS was established. The N-CPAP system that we used in this study consisted of no ventilator. When FiO2 > or = 0.7 was required for maintaining PaO2 > 50 mmHg, surfactant treatment was decided. After the tracheal instillation of surfactant (120 mg/kg body weight), the babies were randomly assigned into 2 groups. In the ventilator group (n = 7), the babies were connected to mechanical ventilation following surfactant instillation. In the N-CPAP group (n = 8), the babies were extubated immediately after instillation of surfactant and were connected to N-CPAP. There was a marked improvement in the ratio of arterial PO2 to alveolar PO2 (a/A PO2) immediately following surfactant treatment and the effect was sustained during the study period of 72 hours in both groups. No significant differences in a/A PO2 were noted in 2 groups. There was a significantly higher PaCO2 in the N-CPAP group than in the ventilator group. PaCO2 declined significantly in the ventilator group 8 hours after surfactant treatment. A delayed decline in PaCO2 until 48 hours after surfactant instillation in the N-CPAP group was noted.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of Bcl-2 protein in murine neural cells in culture.

To explore the role of the protooncogene bcl-2 in the prevention of programmed cell death in the nervous system, we investigated its expression in mouse neural cells in primary culture. The 26 kDa protein product, Bcl-2, was detected by immunocytochemistry and immunoblotting in cultured neurons, astrocytes and oligodendrocytes, but the immunoreactivity of microglial cells was not detectable by immunoblotting. The subcellular distribution of Bcl-2 was similar between in vivo (brain) and in vitro (culture) and between cultured neurons and astrocytes, while the content was higher in astrocytes than in neurons. The substantial expression of bcl-2 in primary cultured brain cells suggests that it has some physiological control in the brain over programmed cell death, which may be exerted not only in neurons but also in some glial cells such as astrocytes.

Elevated levels of dolichol in the brains of mucopolysaccharidosis and related disorders.

The contents of total dolichol were measured in the cerebral cortex of various patients with lysosomal storage disorders, including mucopolysaccharidosis. Strikingly high levels of dolichol were demonstrated in GM1-gangliosides, Sanfilippo B syndrome, and a severe type of Hunter syndrome as well as neuronal ceroid-lipofuscinosis. An increased level of dolichol in cerebral cortex in neuronal ceroid-lipofuscinosis (NCL) was once regarded as pathognomonic for NCL. Our data, however, suggest that an increased level of dolichol in cerebral cortex is a nonspecific phenomenon related to some lysosomal dysfunction secondary to various neurodegenerative disorders.

Ventricle works as a converting organ of atrial blood flow: physiological significance of mean ventricular pressure.

The roles and characteristics of the ventricle were examined using mean ventricular pressure (MVP) in ventricular-vascular association. One hundred and two patients with congenital heart diseases who had undergone cardiac catheterization were studied. They were divided into five groups: Group 1, atrial septal defect without pulmonary hypertension (PH); Group 2, ventricular septal defect (VSD) without PH; Group 3, VSD with PH; Group 4, pulmonary valvular stenosis; and Group 5 as a control group. Then, we examined the relationships between mean pulmonary artery pressure (MPAP) and mean right ventricular pressure (MRVP), and also between mean systemic arterial pressure (MSAP) and mean left ventricular pressure (MLVP) among the five groups. Furthermore, we defined new indicators to express the ease of blood flow through each ventricle. They were referred to as a conductance of the right ventricle (CDR) and a conductance of the left ventricle (CDL), respectively. Then they were compared among the five groups. The values of MPAP/MRVP and MSAP/MLVP were kept constant to be about 1.3 and 1.7, respectively. Furthermore, CDR was different between each group according to the property of the pulmonary vascular bed, whereas CDL took almost the same value among the five groups. The ventricle works as a converter of atrial blood flow so that it can achieve efficient blood transport.

A rapid microassay for dichloroacetate in serum by gel-permeation chromatography.

We have developed a novel, rapid microassay for dichloroacetate in the serum. The serum sample is directly injected into a gel-permeation high-performance liquid chromatography apparatus. The peak of dichloroacetate appears after a giant protein peak. The method requires a very small amount of serum (10 microliters), and the analysis time is short (20 min). Using this micro method, we measured the serum concentrations of dichloroacetate in healthy adult volunteers and paediatric patients with congenital lactic acidosis. Although the effect of dichloroacetate on the neurological manifestations of congenital lactic acidosis has not been proved to be beneficial, the potential usefulness of dichloroacetate in refractory lactic acidosis in cardiac and respiratory failure has been recognized, and human as well as animal studies have been undertaken in many laboratories. To prevent possible side effects of dichloroacetate, it has been recommended that the minimal effective dose be used. Our microassay method is useful for both human and animal experiments, even after administration of minimal doses.

Liver function studies in children with acute lymphocytic leukemia after cessation of therapy.

We investigated liver function in 27 children with acute lymphocytic leukemia (ALL) after cessation of therapy. Induction therapy consisted of prednisolone+vincristine (VP regimen) alone (16 patients) or with addition of daunorubicin (4 patients) or L-asparaginase (7 patients). Patients treated with VP regimen received short courses of VP regimen every 12 weeks for the first year of maintenance. Twenty-five patients remained in first complete remission and had completed 3-year maintenance therapy with methotrexate (MTX) and 6-mercaptopurine (6-MP) 1-7 years prior to this study. Twenty-three patients had received transfusions of packed red blood cells or fresh whole blood (1-11 units; median: 2 units) but none had evidence of either hepatitis B or hepatitis C. Alanine aminotransferase (ALT), which was measured every 3 months during maintenance therapy, had values more than three times the upper limit of the normal range in 25% of the measurements in more than half of the patients. However, by 3 months after the completion of maintenance therapy, ALT had normalized in all patients and remained normal in all but two patients until the time of this study. Serum bilirubin, serum albumin, and prothrombin time were all within normal limits. Fasting and 2-hour postprandial total serum bile acids were high in 5 of 13 patients and in 6 of 13 patients, respectively. The ratio of cholic acids+deoxycholic acids to chenodeoxycholic acids+lithocholic acids was below 1 in all but two patients, whereas this ratio was above 1 in all controls. Our bile acid profile results indicate the necessity of careful long-term follow-up of survivors of ALL treated with hepatotoxic chemotherapy during childhood.

Infantile bilateral striatal necrosis: chronic and acute manifestations in a single case.

We report a girl who exhibited a slowly progressive extrapyramidal disorder with onset in early infancy. CT examinations demonstrated progressive atrophy of the cerebrum. At the age of 6 years, acute exacerbation occurred with impairment of consciousness and autonomic functions. During the acute episode, CT revealed lesions in the bilateral striatum and globus pallidus.

Mutations of the p53 gene are involved in Ewing's sarcomas but not in neuroblastomas.

We have investigated the frequency of p53 gene mutations in Ewing's sarcoma (ES) and neuroblastoma (NB) by using polymerase chain reaction-single strand conformation polymorphism analysis for genomic DNA or complementary DNA generated from total RNA. Mutations of the p53 gene were found in six of seven ES cell lines: a missense mutation of TGC (Cys)-->TAC (Try) at codon 141 in one, a missense mutation of CGT (Arg)-->TGT (Cys) at codon 273 in one, a missense mutation of TGC (Cys)-->TTC (Phe) at codon 176 in three, and one base deletion of CGC-->CG at codon 283 in one. Further analysis of 14 ES and related primary tumors showed mutations of the p53 gene in only two: one base insertion of CCG-->CCCG at codon 152 in one and a missense mutation of GGC (Gly)-->GTC (Val) at codon 154 in the other. Both of the two tumors were obtained from patients with an advanced stage disease. Three of the eight ESs with mutations of the p53 gene showed the same missense mutation at codon 176, suggesting the mutational hot spot of the p53 gene in ESs. In contrast to ES, none of 6 NB cell lines or 48 NB tumors including advanced-stage ones with or without N-myc amplification showed any aberration of the p53 gene. Our findings suggest that mutations of the p53 gene in ES might represent late genetic events related to tumor progression, and that aberrations of the p53 gene might not be involved in the development or the progression of NB.