Excitatory effect of ATP on acutely dissociated ventromedial hypothalamic neurons of the rat.

The ATP-induced increase in the cytosolic Ca(2+) concentration ([Ca]i) and current in acutely dissociated ventromedial hypothalamic rats neurons were investigated using fura-2 microfluorometry and the nystatin-perforated patch recording method, respectively. The ATP-induced [Ca]i increase was mimicked by dimethyl-thio-ATP and ATPgammaS, and was inhibited by P2 purinoreceptor antagonists. The ATP-induced [Ca]i increase was markedly reduced by removal of external Na(+) or Ca(2+), and by addition of various Ca(2+) channel antagonists. ATP induced a transient inward current exhibiting a strong inward rectification at membrane potentials more positive than -20 mV. The ATP-induced current at a holding potential of -70 mV was concentration-dependent with a half-maximum effective concentration of 26 microM. Increasing the external Ca(2+) concentration to 10 mM shifted the dose-response relationship to the right. ATP induced only a small current and a small increase in [Ca]i, even at 10 mM Ca(2+), when external Na(+) was removed, suggesting the relatively low permeability to Ca(2+) of purinoceptor channels. These results suggest that ATP activates non-selective cation channels by acting on P2X purinoceptors on dissociated ventromedial hypothalamic neurons, which in turn increases [Ca]i by increasing Ca(2+) influx through voltage-dependent Ca(2+) channels.

Activation of ATP receptor increases the cytosolic Ca(2+) concentration in nucleus accumbens neurons of rat brain.

ATP increased the cytosolic Ca(2+) concentration ([Ca](i)) in nucleus accumbens neurons acutely dissociated from rat brain. The ATP response was dependent on external Ca(2+) and Na(+), and was blocked by voltage-dependent Ca(2+) channel blockers. The results suggest that the ATP-induced depolarization increases Ca(2+) influx resulting in the increase in [Ca](i).

Intraventricular administration of the neurotrophic factor midkine ameliorates hippocampal delayed neuronal death following transient forebrain ischemia in gerbils.

Midkine (MK) is a growth factor with neurotrophic activities, and is expressed during the early stages of experimental cerebral infarction in rats in the zone surrounding the infarct. To evaluate in vivo activity of MK in preventing neuronal death, MK produced in yeast (Pichia pastoris) was administered into the brain ventricle immediately before occlusion of the bilateral common carotid artery of Mongolian gerbils. MK administration at the dose of 0.5-2 microg immediately before occlusion was found to ameliorate delayed neuronal death in the hippocampal CA1 region caused by transient ischemia 7 days after the insult. The hippocampal neurons of the MK-administered gerbils tended to degenerate 14 and 21 days after the insult, but their numbers remained higher than those in saline-administered controls; however, the hippocampal neurons were degenerated 28 days after the insult. MK administration at 2 h after occlusion did not ameliorate the neuronal death. These findings suggested that the therapeutic time window was narrow. The two to four times repeated administration of 2 microg MK immediately before and at 1, 2, or 3 weeks after the occlusion were not significantly different for the hippocampal neuronal death at 28 days after the insult compared with a single injection, but were significantly effective compared with vehicle administration alone. These findings suggested that the therapeutic time window was relatively narrow. The potent neuroprotective activity of MK observed in vivo suggested that MK might be useful as a therapeutic reagent for prevention of neuronal death in neurodegenerative diseases.

Activation of macrophages/microglia with the calcium-binding proteins MRP14 and MRP8 is related to the lesional activities in the spinal cord of HTLV-I associated myelopathy.

Macrophages and microglia may play an important role in the pathogenesis of chronic inflammatory process in HTLV-I associated myelopathy (HAM) and tropical spastic paraparesis (TSP). However, the etiology and cellular mechanism of chronic inflammation are poorly understood in HAM/TSP. To help to define the roles of macrophages and microglia we analyzed the various patterns of macrophage and microglia activation in the central nervous system (CNS) of HAM/TSP using several monoclonal antibodies recognizing the different states of activation. The results indicate that a large number of macrophages and microglia express both MRP14 and MRP8 in active-chronic inflammatory lesions of the patients with a short duration of illness (2.5 years). In the patient whose duration of illness was 4.5 years, perivascular and parenchymal macrophages and microglia were reactive for MRP8 but not for MRP14. In contrast, MRP14 and MRP8 were negative on the perivascular and parenchymal macrophages and microglia in inactive-chronic lesions and in controls. This study suggests that (a) activated macrophages and microglia as well as CD4+ T lymphocytes and CD8+ cytotoxic T lymphocytes are main components of the inflammatory process in the CNS in HAM/TSP, (b) activation of macrophages and microglia is related to the amount of HTLV-I proviral DNA in situ.

Midkine exists in astrocytes in the early stage of cerebral infarction.

Midkine (MK), a heparin-binding neurotrophic factor, is expressed in the early stage of experimental cerebral infarction in the zone surrounding the infarct. Double immunostaining with anti-MK and anti-glial fibrillary acidic protein showed existence of MK in astrocytic cytoplasm on postoperative day 2. Immunoelectron microscopic analysis revealed the presence of MK in the swollen astrocytic processes on postoperative day 4.

Immunological abnormality in patients with lysinuric protein intolerance.

Lysinuric protein intolerance (LPI) is a rare hereditary disorder manifesting hyperammonemia induced by low levels of basic amino acids, these low levels being due to the impaired transport of these acids in the intestinal mucosa and the renal tubules. Low serum arginine levels and probably the consequently low in vivo levels of nitric oxide (NO), which against acts as a physiological and immunological mediator/modulator, are thought to influence the immunological status in patients with LPI. Accordingly, this study was conducted to. We found that patients with LPI had leukocytopenia, high serum IgG levels, a high ratio of CD44B4-positive lymphocytes (helper inducer) to CD42H4-positive lymphocytes (suppressor inducer), low levels of leukocyte phagocytic, cytotoxic, and natural killer cell activity, and increased spontaneous proliferation of lymphocytes. These results were probably the consequence of persistent low NO levels in vivo.

Genetic diagnostic test of hepatocellular carcinoma by telomerase catalytic subunit mRNA.

This study investigated the relationship between telomerase activity and telomere length and between telomerase reverse transcriptase (hTERT) mRNA and telomere length. Both cancerous and non-cancerous tissues were studied in individuals with hepatic carcinoma. In this study, the telomere length in HCC livers had a wide range, no clear significant correlation was found between hTERT mRNA and telomere length. Telomerase activity was more strongly correlated with hTERT mRNA than with telomere length. The correlation between hTERT mRNA and telomerase activity shown here indicates that hTERT mRNA has potential for cancer diagnosis.

Phenotypic Duchenne muscular dystrophy with C-terminal domain.

We report a patient with X-linked muscular dystrophy who had rapidly progressive muscle weakness and became wheelchair-bound at age 10 years. Clinically, he was diagnosed as having Duchenne muscular dystrophy; however, he was diagnosed as having Becker muscular dystrophy by dystrophin tests using a C-terminal monoclonal antibody. No immunolabelling was observed with a monoclonal antibody against the N-terminal domain. Multiplex polymerase chain reaction analysis revealed the deletion of exons 3-19. The data suggest that the deletion of the N-terminal domain of dystrophin can cause a severe phenotype even when the C-terminus of the protein is well preserved.

Human T-cell lymphotropic virus type 1 can infect primary rat retinal glial cells and induce gene expression of inflammatory cytokines.

PURPOSE: To examine whether or not retinal glial cells can be infected by human T-cell lymphotropic virus type 1 (HTLV-1) and test the possibility that HTLV-1-infected retinal glial cells are involved in the pathogenesis of HTLV-1 uveitis (HU). METHODS: We tested infection of HTLV-1 by a standard coculturing method using WKAH rat retinal glial cells and irradiated MT-2, a human T cell line that produces HTLV-1. Infection was confirmed by detecting the integrated HTLV-1 provirus, using polymerase chain reaction (PCR), viral gene expression, using reverse transcriptase-PCR (RT-PCR) and HTLV-1 p19 ELISA, and by identifying the HTLV-1-infected glial cells by immunofluorescence cytochemistry and in situ hybridization. Changes in cytokine gene expression were studied by RT-PCR. RESULTS: Using a semiquantitative PCR of HTLV-1 provirus sequence, we found that 2.6% of the retinal glial cells were infected at 3 days after infection, followed by a gradual decrease in the percentage with an extended period of culture up to 4 weeks. This time course of infection was also verified by RT-PCR and ELISA studies that detect viral mRNA expression and protein production, respectively. Expression of HTLV-1 gag protein and tax mRNA was detected in a part of glial cells by indirect immunofluorescence cytochemistry and in situ hybridization, respectively. RT-PCR analysis of cytokine gene expression revealed that gene expression of IL-6, CINC-1 (Gro, KC), and TNF-alpha were induced in these cells, with a peak at 3 weeks after infection. CONCLUSION: These results provided supportive evidence for the theory that the infection of retinal glial cells by HTLV-1 and subsequent production of inflammatory cytokines could be one contributing factor for the development of the unique clinical features of HU. A better understanding of the specific roles of the inflammatory cytokines in the pathogenesis of HU would be beneficial in the treatment and control of this disease.

Nucleotide sequence analysis of HTLV-I isolate from a Korean patient with HAM/TSP.

Limited nucleotide sequences of human T-cell lymphotropic virus type I (HTLV-1) provirus isolated from the first case of a Korean patient with HTLV-I associated myelopathy and tropical spastic paraparesis (HAM/TSP) were analysed and compared with other isolates from different regions of the world. The sequences of the env, LTR regions (536bp, 690bp respectively) showed 98.7%, 99.3% homologies with the prototype HTLV-I, ATK-1, isolated from a Japanese Adult T-cell leukemia (ATL) patient. A comparison between other isolates from different geographical origins revealed that the Korean HTLV-I isolate is more closely related to Japanese isolates than to those from other geographical origins.

[A case of Klippel-Trenaunay-Weber syndrome--special reference to myelopathy].

A 49-year-old woman with Klippel-Trenaunay-Weber syndrome (KTW) is reported. She had characteristic features of KTW; cutaneous angiomas on her back, left knee and both plantae, varicosities on both legs, and hypertrophy of the left leg. In addition she had rare complications of KTW; asymmetric skull bone, hemicranial hypertrophy, kyphosoliosis, idiopathic hypoparathyroidism, and abnormal length of the dactyl. Her hemicranial hypertrophy was examined by CT scan, MRI and angiography. Her right hemicranial volume was enlarged and her left lateral ventricle was enlarged. The T2-weighted MRI imaging demonstrated high signal intensity beside the right lateral ventricle and in the right basal ganglia. Cerebrovascular anomalies were not revealed. She also had neurological manifestations of KTW that are rarely seen in adult cases; mental retardation, and myelopathy. The anti-HTLV-I antibodies in serum and cerebrospinal fluid were positive, so it was thought for her to have HTLV-I associated myelopathy (HAM). She was treated with peroral prednisolone and alpha-interferon (intramuscular), which improved her ability to walk. It is known that kyphosis sometimes makes the disorder of spinal cord. Although KTW is known to be associated with the spinal arteriovenous malformation, several examinations did not reveal the spinal vascular anomalies in this case. So we think the myelopathy of this case was caused mainly by the kyphoscoliosis and HAM.

[Two cases of facial diplegia with elevated serum antibodies to Borrelia burgdorferi].

Lyme disease, a tick-borne infection with spirochete named Borrelia burgdorferi, is a complex multi-organ disorder involving neurological complication. Recently, the existence of Lyme disease was also recognized in middle Japan. We described here two cases of facial diplegia with elevated serum antibodies to B. burgdorferi. Case 1 was a 48 year-old man, who developed facial diplegia on April 29, 1987, and referred to our hospital on May 1. Neurological examination showed facial diplegia in addition to right-sided hemiparesis due to head injury of twenty years ago. The cell count of the cerebrospinal fluid was 17/mm3 and the protein was 139.9 mg/dl. The serological examinations for B. burgdorferi were performed by means of immunoperoxidase test. This patient had no serum IgG antibodies to B. burgdorferi on admission. However, twelve weeks later his serum showed significant elevation of IgG antibodies (titer: X400). Tests for syphilis and leptospira were negative. Based on these observation, we considered that facial diplegia of this patient was associated with the infection of B. burgdorferi. His facial diplegia remained unchanged with oral corticosteroid treatment. Case 2 was a 64 year-old man, who developed facial diplegia on May 31, 1987, and referred to our hospital on June 3. Neurological examinations were entirely negative except for facial diplegia. The findings of the cerebrospinal fluid showed the albumin cytologic dissociation with the cell count 2/mm3 and the protein 85.4 mg/dl. Serum IgG antibodies to B. burgdorferi were present (titer: X200) on admission. However, seven weeks later his serum showed a marked elevation of IgG antibodies (titer: X1600). Tests for syphilis were negative.(ABSTRACT TRUNCATED AT 250 WORDS)

[A family of Machado-Joseph disease with a patient having frequent apnea in all day].

One male and two female cases in a family of Machado-Joseph disease were reported. Two cases showed typical symptoms that are characterized by bulging eyes, ophthalmoplegia, dystonia, ataxia, spasticity of extremities and amyotrophy, and were consistent with Type II (Rosenberg et al). But another one lacked diversity of the symptoms, showing mainly progressive cerebellar ataxia for over 10 years. We pointed out the existence of a new type of MJD case exhibiting only progressive cerebellar ataxia over a long period. A female patient had dyspnea and insomnia after 20 years in her clinical course, and central sleep apnea was revealed by respiratory monitor. But, the apnea and irregular respiration appeared in both awake and sleep stages. We described the importance of attention to the apnea as a new complication of Machado-Joseph disease.

[A case of Crow-Fukase syndrome which developed seven years following myelopathy of unknown origin].

A rare case of Crow-Fukase syndrome was reported, which developed 7 years following myelopathy of unknown origin. In September 1983, a 31-year-old man came to our department for progressive gait disturbance and numbness in both lower extremities. Examination on admission showed hyperreflexia with clonus and moderated muscle weakness of legs, and paresthesia below Th9 level, but myelography and CT indicated no abnormality. Thereafter, he was doing as well as walkable with a cane, but in 1989 he developed hypertrichosis, skin pigmentation, leg edema and gynecomastia with aggravation of numbness of lower extremity and was rehospitalized in October. Detailed examination on the present admission indicated compression of the conus medullaris due to an osteosclerotic lesion of vertebral body L1, which did not correspond to the possible site of pyramidal sign. Thus, the origin of the myelopathy remained unknown. After hospitalization, polyneuropathy newly developed, making him confined to his bed. Thus, the diagnosis was established as Crow-Fukase syndrome. Subtotal extirpation of L1 vertebral body and steroid therapy (PSL 10 mg/day) caused improvements of polyneuropathy and numbness of lower extremity, making him walkable again and return to his social work 3 months later. Pathohistological examination of the osteosclerotic lesion proved partial plasmacytoma. In MRI findings of the osteosclerotic lesion, the osteosclerotic and plasmacytomatous regions were outlined as low and high signals, respectively for both T1 and T2-weighted. MRI examination was very useful for grasping the tumorous extent within the osteosclerotic lesion as observed with Crow-Fukase syndrome and for determining the extent of indication for surgery and radiotherapy.

[A case of rapidly progressive HTLV-I-associated myelopathy (HAM)].

We report a 65-year-old woman with HAM who showed rapid progression of the clinical symptoms. The initial symptom was lumbago and she became unable to walk within 4 months after the onset of the lumbago. When seen on admission, she had flaccid paraplegia and areflexia was seen in the lower extremities with positive Babinski and Chaddock reflexes. She had numbness below the level of the navel, vibratory sensation was decreased in both lower limbs, and there was a hyperesthesic zone at the tenth thoracic vertebral level. She had a difficulty in urination and defecation. Laboratory examination revealed elevated anti-HTLV-I antibody titers both in serum (4,096x by PA method) and in cerebrospinal fluid (CSF) (4,096x). The levels of IgG and neopterin in CSF were also increased to 16.6 mg/dl (normal: < 5 mg/dl) and 360.3 pmol/ml (normal: < 30 pmol/ml), respectively. HTLV-I messenger RNA positive cells were detected in 0.1% to 0.01% of cells in CSF by in situ hybridization using an oligonucleotide probe labelled with alkaline phosphatase. Spinal cord MRI detected neither spinal cord compression nor vascular diseases. She was treated with 1,000 mg methylprednisolone for 3 days intravenously, followed by 60 mg oral prednisolone therapy. In several days after receiving the treatments, her muscle tonus became spastic and deep tendon reflexes in the legs became brisk. The hyperesthesia at the tenth thoracic vertebral level and numbness below the level of the navel were also gradually improved. Subsequently, her clinical features were consistant with those of the typical HAM. Therefore, the patient was diagnosed as rapidly progressive HAM. The initial phase of rapidly progressive HAM patients had been described only from clinical history. These patients had common characteristic clinical features, such as older age at onset, relatively severe motor dysfunction, high titers of anti-HTLV-I antibody in CSF, and increased levels of neopterin and IgG in CSF, when compared with those of other HAM patients. The clinical course and laboratory findings in the present patient were compatible with those in the previous cases reported as rapidly progressive HAM. This patient showed flaccid paraplegia and areflexia which have rarely been seen in HAM patients. However, these symptoms were changed to spastic and hyperactive after prednisolone therapy. We speculate that inflammation in the spinal cord in this patient was severe enough to spread to the dorsal root, and disturbed the afferent pathway from the peripheral to the central nervous system. This inflammatory reaction might be suppressed by prednisolone to facilitate the recovery of the afferent pathway, which led to the typical clinical symptoms of HAM.

[Clinical significance of serum cardiac myosin light chain I in patients with Duchenne muscular dystrophy].

The cardiac myosin light chain I (LCI) is one of the cardiac muscle structural proteins. A sensitive immunoradiometric assay kit for LCI by using LCI monoclonal antibodies is developed. We estimated LCI in the patients with Duchenne muscular dystrophy (DMD) and Kugelberg-Welander disease (KW). The results suggested that LCI has close relationships with the functional disturbances of skeletal muscles, especially disturbances of pulmonary ventilation. Therefore we studied properties and localizations of LCI in the skeletal muscles by Western blotting and immunohistochemical methods. In Western blotting method LCI monoclonal antibodies have a band of 27 KD proteins of skeletal muscles. LCI has also found to be localized in type 1 fibers in frozen sections of biopsied of human skeletal muscles. LCI was measured from 47 patients with DMD and 8 patients with KW. The average serum LCI levels in the patients with DMD were 11.79 ng/dl and its levels in the patients with KW were in the normal range (under 2.5 ng/dl). Among 12 patients receiving negative pressure chest respirator, the levels of LCI were also under 2.5 ng/dl. Serum LCI decreased with increasing age and reduced physical activity. The levels of LCI has obvious positive correlations with CK and myoglobin. These results suggested that the measurements of serum LCI are useful as one of the markers of disease severity and the determination of suitable time of using respirator.

[Clinical and histochemical findings in spinal muscular atrophy].

The childhood form of the spinal muscular atrophy (SMA) is classically subdivided into three groups on the basis of a combination of age of onset, milestones of development and age of survival: acute Werdning-Hoffmann (type I), intermediate Werdnig-Hoffmann (type II) and Kugelberg-Welander disease (type III). Now we examined 7 cases of type I and 9 cases of type II on clinical and histochemical ground. Of the total of 16 cases, 5 cases had a family history of the disease. (1) In type I, three were males and 4 females. The onset was within 30 days and the disease was manifest before or at delivery in 3 cases. The progression was so severe. All cases were dead by 10 months. They showed generalized hypotonia, abnormal respiration and could not sit without support. In type II, five were males and 4 females. The onset of the disease was between the age of 3 and 15 months. The progression was slow. All patients couldn't walk by themselves at all but 7 of them had abilities to sit without support. Clinically it was easy to classify type I from type II. (2) The most characteristic histochemical findings of both types were group atrophy, fiber hypertrophy, fiber type predominance and fibrosis. Though there was a slight difference between two types in histological pattern, the basis was so similar. There is controversy about the proper classification of recessive childhood SMA. Now it is suggested that the majority of both acute and chronic cases are allelic, similar to the patterns of Duchenne and Becker forms of muscular dystrophy.

Plasma amantadine concentrations in patients with Parkinson's disease.

We determined plasma amantadine concentrations in patients with Parkinson's disease (PD) in daily clinical practice and investigated the relationship between plasma concentration and adverse reactions to clarify the safe therapeutic range. Seventy-eight consecutive PD patients on stable amantadine treatment were recruited. Plasma concentration of amantadine was measured 3h after the administration of morning amantadine dose. Serum creatinine was measured to estimate renal function. The mean daily dose of amantadine was 135.1+/-62.3mg/day, and the mean plasma amantadine concentration was 812.5+/-839.5 ng/ml (range, 91-4400 ng/ml). Plasma amantadine concentration increased according to increasing renal dysfunction. Three patients exhibited adverse reactions, such as myoclonus, hallucinations, and delirium, and all of them showed plasma amantadine concentration >3000 ng/ml. None of the three cases had previously shown such side effects. PD patients who have not developed any psychiatric symptoms as adverse reactions to the treatment may develop myoclonus, hallucination, or delirium when the plasma concentration of amantadine exceeds 3000 ng/ml. It is therefore recommended to use amantadine at the plasma concentration of less than 3000 ng/ml in the treatment of Parkinson's disease, especially in elderly patients.

Long-term follow-up of patients with Duchenne muscular dystrophy receiving ventilatory support.

We, retrospectively, examined the clinical course, decline in pulmonary function, and requirements for ventilatory assistance in 54 patients with Duchenne-type muscular dystrophy (DMD) who were followed in the muscle disease ward of the National Hospital in Kagoshima, Japan, over the past 20 years. The percentage of the predicted vital capacity (%VC) declined in relation to age and stage of disease. Most patients required assisted ventilation when the %VC fell below 10%. Twenty patients were treated with a negative pressure chest respirator. Six of these died at the mean age of 23.2 years after being on the respirator for a mean period of 18 months. Fourteen patients are surviving at a mean age of 23.5 years after being on the respirator for a mean period of 39 months.