Epidemiological study of acute encephalitis in Tottori Prefecture, Japan.

BACKGROUND AND PURPOSE: To conduct an epidemiological survey of acute encephalitis focusing on non-herpetic acute limbic encephalitis (NHALE) in Tottori Prefecture, western area of Japan. METHODS: A questionnaire survey on the annual number of patients aged 16 years or more with acute encephalitis from 2001 to 2005 was undertaken in 2006. RESULTS: During the study period, 49 patients were diagnosed with acute encephalitis. The subtype of acute encephalitis was as follows: 10 patients with herpes simplex encephalitis (HSE), 12 patients with NHALE, 4 patients with paraneoplastic encephalitis, 2 patients with encephalitis associated with collagen disease, one patient with viral encephalitis other than HSE, 20 patients with encephalitis with unknown causes. The service-based incidence rate of acute encephalitis was 19.0 per million person-years. The incidence rate of NHALE subtype was 4.7 per million person-years. CONCLUSIONS: Our epidemiological survey indicated an estimated 550 patients would develop NHALE per year in Japan, suggesting that NHALE may not be a rare disorder.

Mutational analysis of carbamoylphosphate synthetase I deficiency in three Japanese patients.

We describe the results of mutational analysis of the carbamoylphosphate synthetase I (CPSI) gene in three nonconsanguineous patients with CPSI deficiency. Compound heterozygotes of 3422T/G (V1141G) plus 3784C/T (R1262X), 1528delG (510-514 ARQLX) plus 2752T/C (S918P), and 2549G/A (R850H) plus 2797delT (L933X) were identified through genomic analysis; however, the 2797delT (L933X) mutation was not detected in cDNA analysis using biopsied liver, suggesting that mRNA expression rom this mutant allele is absent or markedly low.

Three-dimensional distribution of astrocytes in zebrafish spinal cord.

We prepared a monoclonal antibody (A-22) that recognizes a 60-kDa protein in the zebrafish brain. The antigen is distributed throughout the brain but is not found outside it. The antibody recognizes star-shaped cells with long processes in the spinal cord. All A-22-positive cells are also GFAP-immunopositive, but there are GFAP-positive cells that are A-22-negative. The cells are connected to small veins and to the surface of the spinal cord. Immunopositive cells are generally homogeneous in size and shape and are found not only in the spinal cord but also in several areas of the brain. These results indicate that the stained cell is an astrocyte. Most of these cells (88%) are distributed in the gray matter of the spinal cord; the remainder (12%) are found in the white matter. Most of the cells in the gray matter are found in the ventral and dorsal horns, but some are also present in the central area along the ventricle. Glial cell bodies form an array along the longitudinal axis and are connected to each other by thick projections. The cellular array is not visible in coronal sections. In contrast, thin processes from the cells extend to the surfaces of veins, to neurons, and to the periphery of the spinal cord. We estimate that there are about 13,500 A-22-positive astrocytes in the spinal cord; however, this represents only 26% of the total number of astrocytes in the spinal cord (approximately 52,000).

Zonal distribution of Purkinje cells in the zebrafish cerebellum: analysis by means of a specific monoclonal antibody.

We have isolated a monoclonal antibody that recognizes a 42-kDa protein from adult zebrafish brain. The antibody stains the typical drop-shaped perikaryon of Purkinje cells and their dendrites. The cerebellum of teleosts has complex features. It is composed of three parts; the valvula cerebelli (Va), the corpus cerebelli (CCe), and the crista cerebellaris (CC). In higher vertebrates, the molecular layer is always found as the most outer layer of the cerebellum, but in teleosts, some of the granular cells are located on the surface of the Va. In higher vertebrates, the boundary between the granular and molecular layers always contains Purkinje cells, but this does not occur in teleosts. The Purkinje cells are found only in a part of the boundary in Va. We have found that the layer containing Purkinje cells forms a continuous zone in the cerebellum in the zebrafish. The complex structure of the cerebellum is more easily understood with the aid of the concept of a "Purkinje zone". The Purkinje zone starts at the caudal end of Val (lateral division of Va), turns at the edge of Va toward Vam (medial division of Va), connects to CCe, and ends at the bottom of CCe. The dendrites are found only on one side of the zone. The dendrites of the Purkinje cells in Vam are planar and are packed regularly, similar to those of higher vertebrates. However, the dendrites in Val and the posterior part of CCe are not planar and are irregularly packed.

Alpha-like activity in terminal stage of Creutzfeldt-Jakob disease.

We report a case of Creutzfeldt-Jakob disease showing various changes in electroencephalogram (EEG) throughout the course of the disease. The patient's EEG patterns showed periodic synchronous discharge in the intermediate stage of the disease, delta activity in the advanced stage and alpha-like activity in the terminal stage. The mechanism generating alpha-like activity may resemble, at least in part, that of an alpha coma.

Ex vivo culture of isolated zebrafish whole brain.

We have succeeded in culturing whole zebrafish brains ex vivo for 1 week. While isolated cells and tissue slices have previously been employed for neurobiological studies, these techniques are limited, because while local networks may be preserved, their original context in the whole brain is lost. Culture of the whole brain would facilitate the study of cells and systems within an intact brain infrastructure. Our culture method entailed isolating the whole brain and placing it on a sterile and porous membrane, after which it was maintained with a conditioned medium in a six-well plate in a CO2 incubator at 28.5 degrees C. Whole brains cultured by this simple method were relatively unaltered in terms of their morphology, cytoarchitecture, immunohistochemistry and ability to transport horse radish peroxidase (HRP). This method of cultivation may be very useful for neurobiological research.

A novel monoclonal antibody recognizes a previously unknown subdivision of the habenulo-interpeduncular system in zebrafish.

The habenulo-interpeduncular system is an evolutionarily conserved structure found in the brain of almost all vertebrates. We prepared a monoclonal antibody (6G11) which very specifically recognizes only a part of this system. 6G11 is a monoclonal antibody prepared from a neuronal membrane protein in adult zebrafish brain. In western blot analysis of the adult zebrafish brain, the antibody recognized a 95 kDa protein, and the class of the antibody was determined to be IgM. The 6G11 antigen was not detected in zebrafish muscle, intestine, testis or ovary. A group of neurons stained by the 6G11 antibody was located in the caudomedial part of the zebrafish habenula. The 6G11-immunopositive neurons extended their axons into the fasciculus retroflexus (FR). One group of immunopositive neurons projected toward the interpeduncular nucleus (IPN), especially to the intermediate and the central subnucleus (type 1 neuron). The other group projected to the ventral midline at the level of the raphe nucleus; these axons passed ipsilaterally beside the IPN and converged in the ventral midline under the raphe nucleus (type 2 neuron). Both type 1 and type 2 fibers are relatively minor components of the FR. Little has previously been known about this topological pattern in any species. The 6G11 monoclonal antibody could be a useful tool for expanding knowledge of the habenulo-interpeduncular system.

[A case of late-onset carbamoyl phosphate synthetase I deficiency, presenting periodic psychotic episodes coinciding with menstrual periods].

Carbamoyl phosphate synthetase I deficiency (CPSID) is a rare metabolic disorder affecting the first enzymatic step of urea cycle. We report clinical manifestations of a female case of late-onset CPSID in Japan. An 18-year-old girl was admitted to emergency room due to acute comatose state. Her parents had no apparent consanguineous history. She had suffered from intermittent psychotic episodes (excitation, aggressive behavior and insomnia) with nausea and vomiting from the age of 13, mostly coinciding with menstrual period. She had minor learning disability without major neurological deficits and convulsions. Her mental status was estimated as normal in her intermenstrual period. She had been diagnosed as having hysteria and premenstrual syndrome. Her neurological findings on admission showed deep coma and hypotonic tetraparesis. Plasma ammonia level was markedly elevated (684 micrograms/dl) without significant liver dysfunction. Blood urea nitrogen decreased to 6 mg/dl. Continuous venovenous filtration with subsequential administration of sodium benzoate and l-arginine was started to eliminate blood ammonia. Although the plasma ammonia level decreased to 300 mu/dl in next 10 hours, severe cerebral edema was observed in head computed tomography subsequently, followed by marked cerebral atrophy. Finally, her consciousness status became almost alert a month after the onset, but her mental status was severely retarded. CPSI activity of her biopsied liver markedly decreased and she was diagnosed as having CPS ID. CPSI cDNA analysis of her biopsied liver demonstrated a V1149G mutation. Genomic DNA analysis showed that she was heterozygous in V1149G mutation. The mutation allele was derived from her father. The causative factor for absence or very low level of maternal CPSI mRNA will require further analysis.

A monoclonal antibody stains radial glia in the adult zebrafish (Danio rerio) CNS.

We have prepared a monoclonal antibody, denoted as C-4, which specifically recognizes astroglia with radial forms in the adult zebrafish brain. This report suggests that there are at least two different types of astroglia in the mature teleost brain, only one of which is recognized by C-4. Further, we have found that the C-4-binding astroglia are comprised of three morphologically distinct cellular types. Immunoblot analysis revealed that the antibody recognized only one protein band of approximately 30 kDa in the membrane fraction of the adult zebrafish brain. In the spinal cord, stained glial cells appeared to occur in the same location as ependymocytes. The processes and cell bodies of extra-ependymal cells, many adjacent to ependymocytes and a few near the pial surface, were also stained by the antibody. In the cerebellum, long processes stained by C-4 were found in the molecular layer, and these connected the cerebellum surface to the Purkinje-like cell layer. Long processes were also stained in the mesencephalon, but these were thicker than those in the spinal cord and they linked the two ventricles. The optic tectum, olfactory bulb and cranial nerves, including the optic nerves, were, however, completely devoid of the C-4 antigen. Double-immunofluorescence with antibodies against glial fibrillary acidic protein (GFAP) and C-4 demonstrated that C-4-positive cells were also GFAP-positive, although there was also a subset of GFAP-positive cells which were C-4-negative. The C-4 antibody is thus a useful tool for studying subtypes of GFAP-positive astroglia.

Monoclonal antibody stains oligodendrocytes and Schwann cells in zebrafish (Danio rerio).

We prepared a monoclonal antibody that recognizes oligodendrocytes and Schwann cells in zebrafish. On immunoblots, the antibody mainly recognized three protein bands of 34 kDa in a membrane fraction from adult zebrafish brain. Medaka fish (Oryzias latipes) also possessed the same protein bands in a membrane fraction. The antibody did not stain neurons, but stained cells in fiber tracts and cranial and spinal nerves. In order to determine the nature of these cells, the staining pattern of the monoclonal antibody was compared with that of a myelin basic protein antiserum. Both antibodies stained oligodendrocytes and Schwann cells in fixed sections from the adult zebrafish. Both antigens were also co-localized in cultured glial cells. Taken together, these results indicate that the new monoclonal antibody recognizes myelinating glial cells in zebrafish and will be useful for the analysis of piscine glia.

Study of the pharmacological effect of the bile salt, sodium scymnol sulfate, from Rhizoprionodon acutus. III. Protective effect of scymnol against vascular endothelial cell damage in a rat peripheral arterial occlusion model.

The prophylactic action of scymnol in a rat peripheral arterial occlusion model, involving injection of 5% lactic acid into the femoral artery, was investigated. Increases in serum lactate dehydrogenase (LDH), glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) activities and in plasma levels of thrombin and antithrombin III complex (TAT) were observed in this model 3 h after injection of lactic acid. Changes in LDH activity were characterized by increases in isozymes LDH4 and LDH5 and an elevated LDH4/LDH5 ratio. The ratio of the LDH4 to LDH5 increments was similar to that seen in a rat endothelial cell culture. Oral preadministration of scymnol had a preventive effect on the development of lesions in this model. It significantly reduced the LDH4 and LDH5 activity, the LDH4/LDH5 ratio and the TAT levels dose-dependently over the range 1, 3 or 10 mg/kg, compared with the values in control rats. However, its administration after lactic acid injection, or to sham-operated rats was ineffective, even at a dose of 10 mg/kg. The effects of scymnol were also compared with those of ticlopidine and argatroban. The findings show that scymnol may be useful in preventing thrombotic peripheral arterial occlusive disorders and that it potently protects endothelial cells against lactic acidosis in this model.

Study of the pharmacological effect of the bile salt, sodium scymnol sulfate, from Rhizoprionodon acutus. IV. Effects of naturally occurring bile alcohols, bile acids and their conjugates on lesion development and vascular endothelial cell injury in a rat peripheral arterial occlusion model.

A series of naturally occurring bile alcohols, bile acids and their conjugates has been investigated as part of our studies to develop unique anticoagulants with a potent prophylactic effect against vascular endothelial cell injury induced by lactic acidosis in vivo and in vitro. In an in vivo rat peripheral arterial occlusion model induced by lactic acid injection, oral administration of a single dose of 3 mg/kg scymnol significantly inhibited edematous swelling and development of lower limb lesions, including gangrene, and reduced changes in clotting system functions and serum lactate dehydrogenase activity. It had no effect on clotting system functions in sham-operated rats. The structure-activity relationship suggests that the [24R-(+)-5beta-cholestane-3alpha,7alpha,24,26-pento l] or [3alpha,7alpha-dihydroxy-5beta-cholanic acid] structure is important for a potent prophylactic effect following oral administration. Intravenous administration of a single dose of 0.3 mg/kg sodium (25S)-scymnol sulfate or scymnol prevented lesion progression as effectively as oral administration of scymnol. Sodium (25S)-scymnol sulfate and ursodeoxycholic acid showed clear protective effects against cultured vascular endothelial cell damage due to lactic acidosis which were dose-dependent. The above results suggest that bile steroids such as scymnol, sodium (25S)-scymnol sulfate, ursodeoxycholic acid, and chenodeoxycholic acid may play a role in protecting endothelial cells against injury caused by lactic acidosis. These compounds are candidates for novel anti-ischemic drugs that act by specifically protecting vascular endothelial cells.

Ceramide accumulation is associated with increased apoptotic cell death in cultured fibroblasts of sphingolipid activator protein-deficient mouse but not in fibroblasts of patients with Farber disease.

Ceramide is recognized as an intracellular mediator of cell growth, differentiation and apoptosis. Tumour necrosis factor, anti-fas antibody, radiation and anticancer drugs such as actinomycin D are known to induce apoptosis in several cell types through generation of ceramide by activation of the sphingomyelinase pathway or ceramide synthetase. In this study, we examined the occurrence of apoptosis in fibroblasts from patients with Farber disease and from sphingolipid activator protein-deficient (sap -/-) mouse. These cells accumulate ceramide as the result of genetic deficiency of acid ceramidase and the ceramidase activator (sap-D), respectively. Amounts of ceramide in fibroblasts from Farber patients and in fibroblasts from sap -/- mouse were increased 2.9-fold and 2.8-fold, respectively, over the level of controls. Despite the similar degree of ceramide accumulation, cells exhibiting apoptotic features were increased only in fibroblasts from the sap -/- mouse but not those from the Farber patients. Thymidine uptake of Farber fibroblasts was normal while that of sap -/- mouse fibroblasts was twice normal, consistent with the apparently normal growth and the different rates of apoptotic cell death in these two cell lines. These data suggest that intralysosomal accumulation of ceramide due to defective acid ceramidase or its activator may not play an important role as a mediator of apoptosis. The increased apoptosis in the cultured fibroblasts from the sap -/- mouse may be caused by mechanisms other than the ceramide accumulation. Although more frequent than normal, significant apoptotic cell death was not observed in sap -/- mouse brain in vivo.

Pathological study of mice with total deficiency of sphingolipid activator proteins (SAP knockout mice).

Sphingolipid activator proteins (SAPs) A to D are lysosomal factors required in degradation of sphingolipids with short hydrophilic head groups and are derived from a precursor protein. Sap-B deficiency causes a variant of metachromatic leukodystrophy and sap-C deficiency causes a variant of Gaucher disease. Human total SAP deficiency has been reported in two patients in a single family. In these cases, various inclusions were described in the liver, skin, muscle and peripheral nerves ultrastructurally, but there was no report on the pathological study of the central nervous system (CNS). With targeted disruption of the precursor protein gene, we have generated mice with total SAP deficiency. These mice developed progressive neurological symptoms around day 20 and could not survive beyond day 40. Their cardinal pathology is extensive neurovisceral storage. Neuronal storage was already detected in the dorsal root ganglia as early as postnatal day 1 and diffuse neuronal storage was detected in the CNS after day 10. This storage was immunoreactive with anti-ubiquitin antibody and ultrastructurally appeared as inclusions consisting of numerous concentric lamellar and dense granular structures in the perikarya as well as in dendrites and axons. Axonal spheroids containing electron-dense concentric lamellar bodies and neurofilaments were also conspicuous. The extent of neuronal storage, numbers of storage neurons and axonal spheroids increased with age, accompanied with hypomyelination, astrogliosis and increase of macrophages. After day 30, argyrophilic tangle-like structures, which were immunoreactive with an antibody to phosphorylated neurofilaments, were found in the perikarya of many spinal and some neocortical neurons. Inclusions with various ultrastructural features were also noted in the glial cells, choroid plexus epithelial cells, vascular endothelial cells, Schwann cells, macrophages, fibroblasts, hepatocytes, and renal tubular epithelial cells. Some inclusions in the visceral organs were closely similar to those described in human cases of total SAP deficiency. The ultrastructural features of these inclusions in SAP knockout mice appeared unique and were different from those of other known sphingolipidoses.

The cytoplasmic domain of the large myelin-associated glycoprotein isoform is needed for proper CNS but not peripheral nervous system myelination.

The myelin-associated glycoprotein (MAG) is a member of the immunoglobulin gene superfamily and is thought to play a critical role in the interaction of myelinating glial cells with the axon. Myelin from mutant mice incapable of expressing MAG displays various subtle abnormalities in the CNS and degenerates with age in the peripheral nervous system (PNS). Two distinct isoforms, large MAG (L-MAG) and small MAG (S-MAG), are produced through the alternative splicing of the primary MAG transcript. The cytoplasmic domain of L-MAG contains a unique phosphorylation site and has been shown to associate with the fyn tyrosine kinase. Moreover, L-MAG is expressed abundantly early in the myelination process, possibly indicating an important role in the initial stages of myelination. We have adapted the gene-targeting approach in embryonic stem cells to generate mutant mice that express a truncated form of the L-MAG isoform, eliminating the unique portion of its cytoplasmic domain, but that continue to express S-MAG. Similar to the total MAG knockouts, these animals do not express an overt clinical phenotype. CNS myelin of the L-MAG mutant mice displays most of the pathological abnormalities reported for the total MAG knockouts. In contrast to the null MAG mutants, however, PNS axons and myelin of older L-MAG mutant animals do not degenerate, indicating that S-MAG is sufficient to maintain PNS integrity. These observations demonstrate a differential role of the L-MAG isoform in CNS and PNS myelin.

Study of the pharmacological effect of the bile salt, sodium scymnol sulfate, from Rhizoprionodon acutus. II. Prophylactic effect of scymnol on lesion development in a rat peripheral arterial occlusion model.

The effect of scymnol on the development of lesions in a rat peripheral arterial occlusion model, involving injection of 5% lactic acid into the femoral artery, was investigated. In this model oral administration of scymnol significantly prevented edematous swelling and development of lower limb lesions, including gangrene, and also reduced changes in blood coagulation parameters, platelet aggregation and retention rate at a dose of 10 or 30 mg/kg. However, it had no effect on these clotting system functions in sham-operated rats at a dose of 10 mg/kg. The effects of scymnol were also compared with those of ticlopidine and argatroban. The findings suggest that scymnol may be clinically useful for preventing thrombotic peripheral arterial occlusive disorders. Its prophylactic action appears to be mainly due to its potent ability to protect against endothelial cell damage due to lactic acidosis.

An alternative elastase-mediated degradation of fibrinogen and fibrin observed in a patient with herpes simplex encephalitis and pneumonia.

A 74-year-old female developed pneumonia following herpes simplex encephalitis. Her white blood cell counts reached 28,400/microliters, about 90% of which consisted of granulocytes. The polymorphonuclear (PMN) elastase/alpha 1-antitrypsin complex levels increased and reached the maximum of 5,019 ng/ml, indicating the release of a large amount of elastase derived from the granulocytes. The mechanism of PMN elastase release was most likely to be granulocyte destruction associated with phagocytosis. The cleavage of fibrinogen and fibrin by PMN elastase, independent of plasmin, was indicated by the presence of the fragments in immunoprecipitated plasma from the patient corresponding to elastase-induced FDP D and DD fragments and the absence of fragments corresponding to plasmin-induced FDP D and DD fragments on SDS-PAGE. These findings suggested that the large amount of PMN elastase released from the excessive numbers of granulocytes in this patient with herpes simplex encephalitis and pneumonia, induced the cleavage of fibrinogen and fibrin without the participation of plasmin.

A hyperphosphorylated form of the large subunit of RNA polymerase II is associated with splicing complexes and the nuclear matrix.

A hyperphosphorylated form of the largest subunit of RNA polymerase II (pol IIo) is associated with the pre-mRNA splicing process. Pol IIo was detected in association with a subset of small nuclear ribonucleoprotein particle and Ser-Arg protein splicing factors and also with pre-mRNA splicing complexes assembled in vitro. A subpopulation of pol IIo was localized to nuclear "speckle" domains enriched in splicing factors, indicating that it may also be associated with RNA processing in vivo. Moreover, pol IIo was retained in a similar pattern following in situ extraction of cells and was quantitatively recovered in the nuclear matrix fraction. The results implicate nuclear matrix-associated hyperphosphorylated pol IIo as a possible link in the coordination of transcription and splicing processes.

Targeted disruption of the mouse sphingolipid activator protein gene: a complex phenotype, including severe leukodystrophy and wide-spread storage of multiple sphingolipids.

The four established or putative sphingolipid activator proteins derive from a large precursor protein encoded by a single gene. In addition to generating the four sphingolipid activator proteins, the precursor protein is suspected of having functions of its own, as, for example, a lipid binding/transport protein or a neurotrophic factor. The gene also appears to encode the Sertoli cell major sulfated glycoprotein. Sequence similarities have been noted with many other proteins of diverse functions. One patient and a fetus in a single family with a complete defect of this gene due to a mutation in the initiation codon exhibited complex pathological and biochemical abnormalities. Mutant mice homozygous for an inactivated gene of the sphingolipid activator protein precursor exhibit two distinct clinical phenotypes-neonatally fatal and later-onset. The latter develop rapidly progressive neurological signs around 20 days and die by 35-38 days. At 30 days, severe hypomyelination and periodic acid-Schiff-positive materials throughout the nervous system and in abnormal cells in the liver and spleen are the main pathology. Most prominently lactosylceramide, and additionally ceramide, glucosylceramide, galactosylceramide, sulfatide, and globotriaosylceramide are abnormally increased in the brain, liver, kidney, and their catabolism abnormally slow in cultured fibroblasts. Brain gangliosides are generally increased, particularly the monosialogangliosides. The clinical, pathological and biochemical phenotype closely resembles that of the human disease. This model not only allows further clarification of the physiological functions of the four individual sphingolipid activator proteins but also should be useful to explore putative functions of the precursor protein.

Crystal violet as an indicator dye for nonequilibrium pH gradient electrophoresis (NEpHGE).

I found that a blue-colored dye, crystal violet, is a useful indicator for nonequilibrium pH gradient electrophoresis (NEpHGE). Unlike isoelectrofocusing, NEpHGE basically depends on the nonequilibrium gradient; therefore, the location of the proteins differs with acrylamide concentration or voltage used. To get reproducible results, colored marker which indicates the end of the electrophoresis might be very helpful. Crystal violet is an adequate dye for this purpose. This deeply colored, nontoxic material has a positive charge and migrated slightly faster than almost all proteins on NEpHGE. This dye affected neither the formation of pH gradient nor migration of proteins. Moreover, the ratio of migration (protein/dye) in the NEpHGE gels did not change under different conditions tested. Therefore, the addition of trace amounts of this dye gives a convenient endogenous color indicator to determine the end of the NEpHGE. Other positively charged dyes could be used as such an indicator, but some dyes (for example, methylene blue) lost their color by reduction during electrophoresis. Some dyes (for example, ethidium bromide) migrate too fast; therefore, they were not suitable indicators for NEpHGE.