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Saburo Sakoda

Publications and source records attributed to Saburo Sakoda.

At least 37 records · Page 2Linked to original sources
PubMed2004

Somatic DNA recombination yielding circular DNA and deletion of a genomic region in embryonic brain.

In this study, a mouse genomic region is identified that undergoes DNA rearrangement and yields circular DNA in brain during embryogenesis. External region-directed inverse polymerase chain reaction on circular DNA extracted from late embryonic brain tissue repeatedly detected DNA of this region containing recombination joints. Wide-range genomic PCR and digestion-circularization PCR analysis showed this region underwent recombination accompanied with deletion of intervening sequences, including the circularized regions. This region was mapped by fluorescence in situ hybridization to C1 on mouse chromosome 16, where no gene and no physiological DNA rearrangement had been identified. DNA sequence in the region has segmental homology to an orthologous region on human chromosome 3q.13. These observations demonstrated somatic DNA recombination yielding genomic deletions in brain during embryogenesis.

Animals↗
PubMed2004

Modification of cysteine residue in transthyretin and a synthetic peptide: analyses by electrospray ionization mass spectrometry.

Cysteine and cystine in protein are modified to various derivatives in vitro and in vivo. By electrospray ionization mass spectrometry (ESI-MS), we previously found derivatives of serum transthyretin (TTR) in which cysteine residue at position 10 was changed to glycine residue and sulfocysteine residue. The change, cysteine to glycine, was unique and the origin of the sulfonic acid group was controversial. In the present paper, we show the molecular masses of various TTR derivatives including these two, and the modification process was studied using a synthetic peptide with the same sequence as cysteine containing part of TTR, i.e., SKCPLMVK. After incubation of the peptide at pH 8.3, various derivatives were generated, which showed changes of cysteine residue to glycine, dehydroalanine, S-thiocysteine, and S-sulfocysteine residues, which were confirmed by molecular mass and collision-induced dissociation spectra. Dehydroalanine may react with other amino acids and contribute to form cross-linking fibril, causing amyloidosis.

Cysteine↗
PubMed2004

Hepatoma-derived growth factor is a neurotrophic factor harbored in the nucleus.

Hepatoma-derived growth factor (HDGF) is a heparin-binding proliferating factor originally isolated from conditioned medium of the hepatoma-derived cell line HuH-7. HDGF has greatest homology in an amino acid sequence with high mobility group 1 (HMG1), which has been characterized as a DNA-binding, inflammatory, and potent neurite outgrowth molecule. HDGF is reported to be widely expressed and act as a growth factor in many kinds of cells. However, it has not been investigated in the nervous system. Here, we show by Western blot analysis that HDGF is present in the mouse brain from the embryonic period until adulthood. In situ hybridization and immunohistochemical analyses revealed that HDGF was expressed mainly in neurons, and HDGF protein was localized to the nucleus. HDGF and high mobility group 1 were secreted under physiological conditions and released extracellularly in necrotic conditions. Furthermore, we showed that exogenously supplied HDGF had a neurotrophic effect and was able to partially prevent the cell death of neurons in which endogenous HDGF was suppressed. Therefore, we propose that HDGF is a novel type of neurotrophic factor, on account of its localization in the nucleus and its potential to function in an autocrine manner under both physiological and pathological conditions throughout life.

Animals↗
PubMed2004

Identification of two novel mutations in the Cu/Zn superoxide dismutase gene with familial amyotrophic lateral sclerosis: mass spectrometric and genomic analyses.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder affecting motor neurons. The majority of patients are sporadic cases, while 5-10% of the patients have a family history of ALS (fALS). Mutations in the gene that encodes cytoplasmic Cu/Zn superoxide dismutase (SOD1) have been identified in about 25% of fALS cases. Although the precise pathogenesis of ALS is still unknown, experimental studies including animal models suggest that fALS is caused by the toxic gain-of-function of the SOD1 mutant. We have analyzed not only SOD1 gene mutation by genomic sequencing, but also SOD1 mutant protein by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). We analyzed 33 fALS patients and found 10 mutations in SOD1 gene, in which two were novel: Asp101His substitution in exon 4 and Gly141Glu substitution in exon 5. Here, we present their mass spectrometric protein analyses and clinical features.

Age of Onset↗
PubMed2004

Quantitative magnetic detection of finger movements in patients with Parkinson's disease.

To develop a new measurement tool for quantitatively detecting the finger movement of a patient with Parkinson's disease (PD), we designed a magnetic sensing system consisting of a magnetic induction coil, a sensing coil, and a circuit unit. The sensing coil detects the inducted magnetic field that varies with the distance between the two coils, and the detected signals are demodulated in the circuit unit in order to obtain the variation voltage from the oscillation frequency. To obtain a coefficient for converting voltage to distance, we measured the output voltages for seven fixed finger positions of 12 normal volunteers. The voltage differences corresponding to the finger movement in 20 PD patients, six age-matched controls, and 12 normal volunteers were then recorded for 30s. To investigate the velocity and acceleration of the finger movement, we calculated their waveforms from the measured displacement waveform. We also detected the main frequency of the tapping rhythm by using a fast Fourier transform (FFT). The averaged amplitude of each waveform decreased with the disorder in the Hoehn-Yahr (HY) stage, while the averaged tapping frequency of PD patients did not have any correlation with this stage. It can be concluded that this magnetic sensing system can assess finger movement quantitatively.

Adult↗
PubMed2004

Benefit of valproic acid in suppressing disease progression of ALS model mice.

Valproic acid (VPA) has long been used as an antiepileptic drug and recently as a mood stabilizer, and evidence is increasing that VPA exerts neuroprotective effects through changes in a variety of intracellular signalling pathways including upregulation of Bcl-2 protein with an antiapoptotic property and inhibiting glycogen synthase kinase 3-beta, which is considered to promote cell survival. Although the neuroprotective effects of VPA have been demonstrated in a murine model of human immunodeficiency virus-1 encephalitis, there have been no reports on the effect of VPA in chronic progressing neurodegenerative disease models including amyotrophic lateral sclerosis (ALS). ALS is a devastating disease selectively affecting motoneurons, and its disease model mice bear a close resemblance to ALS symptomatically and pathologically. First, we used an organotypic slice culture using mouse spinal cord, and showed that VPA protected spinal motoneurons against death from glutamate toxicity in vitro. Then, we treated ALS model mice with VPA at the dose effective level for epileptic model mice after 45 days of age (pre-onset treatment) or the day of the disease onset (post-onset treatment). We found a significant prolongation of the disease duration in ALS model mice in both methods of treatment. Considering the long usage of VPA for epileptic patients with good tolerance and safety, these data strongly support the clinical application of VPA for ALS treatment.

Age of Onset↗
PubMed2004

Induction of cyclooxygenase-2 in reactive glial cells by the CD40 pathway: relevance to amyotrophic lateral sclerosis.

An inflammatory process in association with reactive gliosis has been suggested to play an important role in the pathogenesis of amyotrophic lateral sclerosis (ALS). One of the key findings is a marked increase in the level of cyclooxygenase-2 (COX-2), a therapeutic target of ALS. We investigated the expression of CD40 in the spinal cord of a transgenic mouse model of ALS (G93A mice), and its relevance to COX-2 upregulation. CD40 was predominantly expressed in neurons in normal spinal cord and upregulated in reactive glial cells in spinal cord injury. In the spinal cord of G93A mice, the expression of CD40 was increased in both reactive microglia and astrocytes, where COX-2 was especially increased. The level of COX-2 was upregulated in microglia and astrocytes by CD40 stimulation in vitro. CD40 stimulation in primary spinal cord cultures caused motor neuron loss that was protected by selective COX-2 inhibitor. These results suggest that CD40, which is upregulated in reactive glial cells in ALS, participates in motor neuron loss via induction of COX-2.

Amyotrophic Lateral Sclerosis↗
PubMed2004

Ibudilast, a nonselective phosphodiesterase inhibitor, regulates Th1/Th2 balance and NKT cell subset in multiple sclerosis.

We investigated the immunoregulatory effects of ibudilast, a nonselective phosphodiesterase inhibitor, at a clinically applicable dose (60 mg/day p.o. for four weeks) in multiple sclerosis (MS) patients. Sensitive real-time PCR for quantifying cytokine mRNA in the blood CD4+ cells revealed that the ibudilast monotherapy significantly reduced tumour necrosis factor-alpha and interferon (IFN)-gamma mRNA and the IFN-gamma/interleukin-4 mRNA ratio, suggesting a shift in the cytokine profile from Th1 toward Th2 dominancy. In a flow cytometric analysis, natural killer T cells, which have been reported to relate to Th2 responses in MS and its animal model (experimental autoimmune encephalomyelitis), increased significantly after the therapy. None of the significant immunological changes were seen in healthy subjects or untreated MS patients. Ibudilast may be a promising therapy for MS and its clinical effects warrant further study.

Adult↗
PubMed2004

[Organotypic spinal cord culture using mice].

Amyotrophic lateral scleorsis (ALS) is a progressive neurodegenerative disease, which predominantly affects both upper and lower motor neurons. ALS is usually fatal within a few years after clinical onset. An organotypic slice culture of rat spinal cord, in which glutamate toxicity induces slow loss of spinal motoneurons, has been used for preclinical drug screening for ALS. In this report, we modified the conventional slice culture to put mouse spinal cords to use, as an alternative in vitro model of ALS. L-trans pyrrolidine 2, 4-dicarboxylic acid (PDC), an inhibitor of glutamate uptake, induced slow loss of spinal motoneurons in anterior horns, whereas small neurons in posterior horns were relatively preserved. This technique using mouse allows us to use transgenic and knockout mice. In addition to glutamate toxicity, many other mechanisms including oxidative stress and neurofilamentous disorganization are also considered to be involved in development of this devastating disease. Thus, a better in vitro model of ALS is strongly anticipated. At present, we are making efforts to apply this technique to establish a better in vitro model using spinal cord from transgenic ALS model mice, where spontaneous loss of spinal motoneurons will be observed.

Amyotrophic Lateral Sclerosis↗
PubMed2004

A pitfall in MRI.

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False Negative Reactions↗
PubMed2003

Microglial cell cycle-associated proteins control microglial proliferation in vivo and in vitro and are regulated by GM-CSF and density-dependent inhibition.

Hyperplasia of microglia is one characteristic of reactive gliosis, which is observed in various pathologic conditions in the central nervous system (CNS). To investigate the regulation mechanisms of microglial proliferation, the effect of granulocyte macrophage colony-stimulating factor (GM-CSF) on the expression of cell cycle-associated proteins was examined in the microglial cell line GMI-M6-3. After GM-CSF administration, cyclins D1, E, and A and cyclin-dependent kinase inhibitor p21Cip1 were increased, and another cyclin-dependent kinase inhibitor, p27Kip1, was decreased with morphologic transformation into ameboid form. By contrast, downregulation of these cyclins and p21Cip1, and strong upregulation of p27Kip1 accompanied by ramification were observed with GM-CSF deprivation. We also found that GMI-M6-3 exhibited homotypic contact inhibition of proliferation without any morphologic transformation. The increase of p27Kip1 and the decrease of cyclin A were suggested to play an important role in microglial contact inhibition. In addition, the direct effect of p27Kip1 to inhibit microglial proliferation was demonstrated both in vitro and in vivo by overexpression of p27Kip1.

Animals↗
PubMed2003

Clinical and pathological studies of familial amyotrophic lateral sclerosis (FALS) with SOD1 H46R mutation in large Japanese families.

We clarified the clinical and pathological aspects of familial amyotrophic lateral sclerosis (FALS) with SOD1 H46R heterozygous mutation in the Miyakonojo Basin, a region in southern Japan where the prevalence of ALS is 11.4 per 10(5) of the population. We studied 17 patients, including one autopsy case, in three FALS families with the mutation. The average age at disease onset in the families was 44.3+/-8.7 years, and the mean disease duration was 12+/-7.6 years, with a range of 6 to 30 years. Ten of 17 patients were unable to walk by the mean age of 56.4+/-12.2 years. The initial symptom was muscle weakness in the distal leg muscle in all patients. The autopsy findings of one FALS patient showed atrophy of lateral and anterior funiculi, decreased numbers of anterior horn cells, preserved posterior funiculus and absence of neuronal inclusion bodies. Percentages of mutant SOD1 protein measured by mass spectrometry were 14% in erythrocytes, 43% in the spinal cord, 47% in the iliopsoas muscle and 60% in the diaphragm. In this study, we confirmed that FALS with SOD1 H46R mutation showed uniform initial symptoms and slow disease progression with intra-familial variation of disease severity and that inclusion body formation is not essential in FALS with this mutation.

Adult↗
PubMed2003

Expression and distribution of a small-conductance calcium-activated potassium channel (SK3) protein in skeletal muscles from myotonic muscular dystrophy patients and congenital myotonic mice.

The SK3 channel, a small-conductance calcium-activated potassium channel, is expressed in immature fibers of skeletal muscle and becomes down regulated after innervation. We have previously shown that the level of mRNA of the SK3 channel is increased in muscle from myotonic dystrophy. In this study, we have carried out an immunohistochemical study using a polyclonal anti-SK3 antibody. SK3 protein is partly expressed at the cell membrane of normal sized fibers in myotonic dystrophy. Although SK3 channels are also expressed in muscles from polymyositis and amyotrophic lateral sclerosis, the positive staining is observed only in regenerating or denervated fibers. No expression of SK3 protein in a myotonic mouse (ADR) suggests that the increase in the SK3 channel in myotonic dystrophy is not due to hyperexcitability. These data support the hypothesis of a differentiation defect in myotonic dystrophy.

Animals↗
PubMed2003

Determination of cabergoline and L-dopa in human plasma using liquid chromatography-tandem mass spectrometry.

We determined cabergoline and L-dopa in human plasma using liquid chromatography-mass spectrometry with tandem mass spectrometry (LC-MS-MS). The deproteinized plasma samples with organic solvent or acid were analyzed directly by reversed-phase liquid chromatography. Using multiple reaction monitoring (MRM, product ions m/z 381 of m/z 452 for cabergoline and m/z 152 of m/z 198 for L-dopa) on LC-MS-MS with electrospray ionization (ESI), cabergoline and L-dopa in human plasma were determined. Calibration curves of the method showed a good linearity in the range 5-250 pg/ml for cabergoline and 1-200 ng/ml for L-dopa, respectively. The limit of determination was estimated to be approximately 2 pg/ml for cabergoline and approximately 0.1 ng/ml for L-dopa, respectively. The method was applied to the analysis of cabergoline and L-dopa in plasma samples from patients treated with these drugs. The precision of analysis showed coefficients of variation ranging from 3.8% to 10.5% at cabergoline concentration of 13.8-26.2 pg/ml and from 2.9% to 8.9% at an L-dopa concentration of 302.5-522.1 ng/ml in patient plasma. As a result, the procedure proved to be very suitable for routine analysis.

Adult↗
PubMed2003

The efficacy of trientine or ascorbate alone compared to that of the combined treatment with these two agents in familial amyotrophic lateral sclerosis model mice.

One of the hypotheses regarding the pathogenesis of familial ALS (FALS) is a copper-mediated oxidative toxicity derived from the mutant Cu, Zn-superoxide dismutase (SOD1). We have previously demonstrated the efficacy of the combined treatment with a copper chelator (trientine) and an antioxidant (ascorbate) on the disease expression of the FALS-linked mutated SOD1 transgenic mice. Here, we investigated the efficacy of trientine or ascorbate alone on FALS mice when administered before or after the onset of the disease. The mice with a high dose of trientine or ascorbate administered before the onset survived significantly longer than the control. In the combined treatment with a high dose of trientine and ascorbate initiated before the onset, survival lengthened and the motor function of the mice remained more significantly than the control. None of the treatments affected the mean age of the onset, and none of the agents administered after the onset prolonged survival. These findings suggest that better outcomes may be expected by the administration of these agents at the preonset stage of the disease, and the combination of the agents acting on different sites might be useful in preserving the motor performance in FALS.

Amyotrophic Lateral Sclerosis↗
PubMed2003

The role of CD36 in peripheral nerve remyelination after crush injury.

We previously demonstrated that the deficiency of class A macrophage scavenger receptor type I/II was involved in the delayed phagocytosis of degraded myelin by macrophages in class A macrophage scavenger receptor type I/II knockout mice after crush injury of the sciatic nerve [Naba et al. (2000) Exp. Neurol., 166, 83-89]. In order to elucidate the role of CD36, one of the scavenger receptors, here we inflicted crush injury to the sciatic nerves of CD36 knockout mice and investigated the remyelination after crush injury in comparison with that of class A macrophage scavenger receptor type I/II knockout mice. Although we previously reported a lot of onion-bulbs in class A macrophage scavenger receptor type I/II knockout mice at 3 weeks, the number of onion-bulbs was limited both in CD36 knockout mice and wild-type mice. In the morphometry, the remyelination was seriously delayed, and the infiltrating macrophages into the nerve fascicles were quite frequent in CD36 knockout mice compared with wild-type mice at 3 and 6 weeks postinjury. The immunohistochemistry with the monoclonal antibody reacted with oxidized phosphatidylcholine and oil red O staining were positive in wild-type mice, but were negative in CD36 knockout mice, suggesting that the oxidation of phosphatidylcholine and the generation of neutral lipids in macrophages were disturbed in CD36 knockout mice. We hypothesize that the delayed phagocytosis by macrophages and the defect in reuse of lipids from degraded myelin are related to seriously delayed remyelination and a small number of onion-bulbs in CD36 knockout mice.

Animals↗