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Biomedical subjects

M Takigawa

Publications and source records attributed to M Takigawa.

At least 91 records · Page 5Linked to original sources
PubMed2000

Effects of combined administration of zonisamide and valproic acid or phenytoin to nitric oxide production, monoamines and zonisamide concentrations in the brain of seizure-susceptible EL mice.

This study was undertaken to elucidate the anticonvulsive effects of zonisamide (ZNS: 25, 50, and 75 mg/kg, intraperitoneal [i.p.]), which was coadministered with valproic acid (VPA: 25, 50, and 100 mg/kg, i.p.), or phenytoin (PHT: 10, 25, and 50 mg/kg, i.p.) to ZNS concentration, nitric oxide metabolites (NOx levels), and monoamines in the brain of the EL mouse, a strain highly susceptible to seizures. NOx levels were obtained from measuring of combined level of nitrite plus nitrate. Coadministration of ZNS with VPA or PHT suppressed convulsive seizures more effectively than with treatment of ZNS alone. Both serum and brain concentrations of ZNS tended to increase as the dose of VPA or PHT was increased. While coadministrations of ZNS (75 mg/kg) and VPA or PHT at any dose did not change brain and serum NOx levels, those altered brain monoamine contents. These results suggested that anticonvulsive effect of coadministrations of ZNS and VPA or PHT were caused by changes of monoamines rather than changes of NO metabolites.

Animals↗
PubMed2000

Regulation of human COL2A1 gene expression in chondrocytes. Identification of C-Krox-responsive elements and modulation by phenotype alteration.

To identify control motifs involved in human type II collagen gene transcription in both differentiated and dedifferentiated rabbit articular chondrocytes, transient transfection experiments were performed. A 715-base pair (bp) region of the first intron (+2127/+2842), including a 153-bp sequence so far uncharacterized (+2689/+2842), was found to mediate enhancer activity. In dedifferentiated chondrocytes, this enhancer activity was shown to be less effective than in primary cultures but still present. We then demonstrated that a zinc finger protein, C-Krox, activates COL2A1 gene transcription in differentiated chondrocytes through the enhancer region, whereas in subcultured cells, it inhibited the gene activity via a 266-bp promoter. Multicopies of the C-Krox binding site were found to mediate transactivation in both primary cultures and passaged cells, whereas C-Krox overexpression inhibited transcription in dedifferentiated chondrocytes. Additionally, we showed that C-Krox binds to several cis sequences that mediate its transcriptional effects. During chondrocyte dedifferentiation, the protein levels and binding activity of C-Krox were reduced, whereas those of NF-kappaB were increased. This was not associated with variations of mRNA levels, suggesting that post-transcriptional regulatory mechanisms could be involved in C-Krox expression. These results suggest that C-Krox plays a major role in type II collagen expression and the chondrocyte phenotype modulation.

Animals↗
PubMed2000

Identity of urinary trypsin inhibitor-binding protein to link protein.

Urinary trypsin inhibitor (UTI), a Kunitz-type protease inhibitor, directly binds to some types of cells via cell-associated UTI-binding proteins (UTI-BPs). Here we report that the 40-kDa protein (UTI-BP(40)) was purified from the cultured human chondrosarcoma cell line HCS-2/8 by UTI affinity chromatography. Purified UTI-BP(40) was digested with trypsin, and the amino acid sequences of the peptide fragments were determined. The sequences of six tryptic fragments of UTI-BP(40) were identical to subsequences present in human link protein (LP). Authentic bovine LP and UTI-BP(40) displayed identical electrophoretic and chromatographic behavior. The UTI-binding properties of UTI-BP(40) and LP were indistinguishable. Direct binding and competition studies strongly demonstrated that the NH(2)-terminal fragment is the UTI-binding part of the LP molecule, that the COOH-terminal UTI fragment (HI-8) failed to bind the NH(2)-terminal subdomain of the LP molecule, and that LP and UTI-BP(40) exhibited significant hyaluronic acid binding. These results demonstrate that UTI-BP(40) is identical to LP and that the NH(2)-terminal domain of UTI is involved in the interaction with the NH(2)-terminal fragment of LP, which is bound to hyaluronic acid in the extracellular matrix.

Aggrecans↗
PubMed2000

Reciprocal information flow between prefrontal cortex and ventral tegmental area in an animal model of schizophrenia.

The medial prefrontal cortex (PFC) is anatomically and functionally connected with the ventral tegmental area (VTA), the neuronal source of mesocorticolimbic system that is pathophysiologically related to schizophrenia-like symptoms. Methamphetamine (MAP) was applied to examine the functional relationship between PFC and VTA in an animal model of schizophrenia. Hyperactivity and stereotyped behavior were observed accompanied by a distinctive direction of information flow. In hyperactivity, information flow in the direction from PFC to VTA was dominant. Contrarily, dominant information flow from VTA to PFC was found in stereotyped behavior. These results indicate that dysfunctional interaction between PFC and VTA is the neuronal basis of MAP-induced schizophrenia-like psychosis. The information flow and its direction can be useful tool to explain the neurogenesis of these abnormal behaviors.

Animals↗
PubMed2000

Novel intracellular effects of human connective tissue growth factor expressed in Cos-7 cells.

To clarify the multiple functionality of connective tissue growth factor (CTGF), we examined the effects of nascent CTGF within the cell by transient expression. In Cos-7 cells, expression of human CTGF induced an altered cell morphology. It was associated with an increased cellular DNA content and loose attachment, indicating the cells were in G2/M phase. Overexpression of CTGF did not induce cell growth, whereas recombinant CTGF efficiently stimulated the proliferation extracellularly. These results indicate that intracellular CTGF may act as an antimitotic agent, thus it should also be noted that nascent CTGF was found to accumulate around the central mitotic machinery.

Animals↗
PubMed2000

Methamphetamine induces cytosolic Ca2+ oscillations in the VTA dopamine neurons.

Methamphetamine (METH) induces a schizophrenia-like psychosis. The dopamine neurons in the ventral tegmental area (VTA) have been implicated in schizophrenia and drug abuse. The present study investigated direct effects of METH on VTA dopamine neurons. We treated adult SD rats with METH (5 mg/kg/day) or saline for 7 days, isolated single VTA neurons, and monitored neuronal activities by measuring cytosolic Ca2+ concentration ([Ca2+]i) in immunocytochemically identified dopamine neurons. Acutely administered METH increased [Ca2+]i in dopamine neurons from METH- and saline-treated rats and induced oscillations of [Ca2+]i in dopamine neurons only from METH-treated rats. The METH-induced [Ca2+]i oscillations were inhibited by Ca(2+)-free conditions and Ca2+ channel blockers. The results indicate that acute METH increases [Ca2+]i in VTA dopamine neurons and that subchronic METH treatment sensitizes them to this drug, resulting in induction of [Ca2+]i oscillations. The activation of VTA dopamine neurons may be related to psycho-stimulant effects of METH.

Amphetamine-Related Disorders↗
PubMed2000

Percutaneous peptide immunization via corneum barrier-disrupted murine skin for experimental tumor immunoprophylaxis.

H-2K(b)-restricted tumor epitope peptides, including tyrosinase-related protein 2 residues 181-188 (TRP-2) and connexin 37 residues 52-59 (MUT1), were applied to permeability barrier-disrupted C57BL/6 (B6) mouse skin from which the stratum corneum of the epidermis had been removed by tape-stripping. This procedure primed tumor-specific cytotoxic T lymphocytes (CTLs) in the lymph nodes and spleen, protected mice against subsequent challenge with corresponding tumor cells, and suppressed the growth of established tumors. Preventive and therapeutic effectiveness was correlated with the frequency of tumor-specific CTL precursors. MHC class II Ia(b+) cells separated from tape-stripped skin, compared with those from intact skin, exhibited a strong antigen-presenting capacity for CTL, suggesting that CTL expansion after peptide application is primarily mediated by epidermal Langerhans cells. Thus, percutaneous peptide immunization via barrier-disrupted skin provides a simple and noninvasive means of inducing potent anti-tumor immunity which may be exploited for cancer immunotherapy.

Administration, Cutaneous↗
PubMed2000

Effects of CTGF/Hcs24, a hypertrophic chondrocyte-specific gene product, on the proliferation and differentiation of osteoblastic cells in vitro.

Connective tissue growth factor/hypertrophic chondrocyte-specific gene product Hcs24 (CTGF/Hcs24) promotes the proliferation and differentiation of chondrocytes and endothelial cells which are involved in endochondral ossification (Shimo et al., 1998, J Biochem 124:130-140; Shimo et al., 1999, J Biochem 126:137-145; Nakanishi et al., 2000, Endocrinology 141:264-273). To further clarify the role of CTGF/Hcs24 in endochondral ossification, here we investigated the effects of CTGF/Hcs24 on the proliferation and differentiation of osteoblastic cell lines in vitro. A binding study using (125)I-labeled recombinant CTGF/Hcs24 (rCTGF/Hcs24) disclosed two classes of specific binding sites on a human osteosarcoma cell line, Saos-2. The apparent dissociation constant (Kd) value of each binding site was 17.2 and 391 nM, respectively. A cross-linking study revealed the formation of (125)I-rCTGF/Hcs24-receptor complex with an apparent molecular weight of 280 kDa. The intensity of (125)I-rCTGF/Hcs24-receptor complex decreased on the addition of increasing concentrations of unlabeled rCTGF/Hcs24, but not platelet-derived growth factor-BB homodimer or basic fibroblast growth factor. These findings suggest that osteoblastic cells have specific receptor molecules for CTGF/Hcs24. rCTGF/Hcs24 promoted the proliferation of Saos-2 cells and a mouse osteoblast cell line MC3T3-E1 in a dose- and time-dependent manner. rCTGF/Hcs24 also increased mRNA expression of type I collagen, alkaline phosphatase, osteopontin, and osteocalcin in both Saos-2 cells and MC3T3-E1 cells. Moreover, rCTGF/Hcs24 increased alkaline phosphatase activity in both cells. It also stimulated collagen synthesis in MC3T3-E1 cells. Furthermore, rCTGF/Hcs24 stimulated the matrix mineralization on MC3T3-E1 cells and its stimulatory effect was comparable to that of bone morphogenetic protein-2. These findings indicate that CTGF/Hcs24 is a novel, potent stimulator for the proliferation and differentiation of osteoblasts in addition to chondrocytes and endothelial cells. Because of these functions, we are re-defining CTGF/Hcs24 as a major factor to promote endochondral ossification to be called "ecogenin: endochondral ossification genetic factor."

3T3 Cells↗
PubMed2000

The effect of alcohol on convulsions and nitric oxide levels in seizure-susceptible el mice.

To evaluate the influences of ethanol intake on convulsive seizures and brain nitric oxide (NO) production, EL mice, a strain highly susceptible to seizures, were given a 10% ethanol solution ad libitum. In mice consuming ethanol for 4, 8, and 12 weeks, seizures were not suppressed by zonisamide (75 mg/kg ip). Brain NO metabolite levels in mice after 12 weeks of consumption were significantly lower than those in control mice and those consuming ethanol for 4 weeks. Numbers of NADPH diaphorase-positive neurons in the hippocampal formation and parietal cortex of mice consuming for 4 and 12 weeks were significantly higher than in controls. These results suggested that increasing of numbers of NADPH diaphorase-positive neurons in the hippocampal formation and parietal cortex were assumed to develop in compensation for reduction in whole-brain NO metabolite levels of EL mice exposed to ethanol.

Journal Article↗
PubMed2000

Topographic analysis of dimension estimates of EEG and filtered rhythms in epileptic patients with complex partial seizures.

Nonlinear dynamic properties were analyzed on the EEG and filtered rhythms recorded from healthy subjects and epileptic patients with complex partial seizures. Estimates of correlation dimensions of control EEG, interictal EEG and ictal EEG were calculated. The values were demonstrated on topograms. The delta (0.5-4 Hz), theta (4-8 Hz), alpha (8-13 Hz), beta (13-30 Hz) and gamma (30-40 Hz) components were obtained and considered as signals from the cortex. Corresponding surrogate data was produced. Firstly, the influence of sampling parameters on the calculation was tested. The dimension estimates of the signals from the frontal, temporal, parietal and occipital regions were computed and compared with the results of surrogate data. In the control subjects, the estimates between the EEG and surrogate data did not differ (P > 0.05). The interictal EEG from the frontal region and occipital region, as well as its theta component from the frontal region, and temporal region, showed obviously low dimensions (P < 0.01). The ictal EEG exhibited significantly low-dimension estimates across the scalp. All filtered rhythms from the temporal region yielded lower results than those of the surrogate data (P < 0.01). The dimension estimates of the EEG and filtered components markedly changed when the neurological state varied. For each neurological state, the dimension estimates were not uniform among the EEG and frequency components. The signal with a different frequency range and in a different neurological state showed a different dimension estimate. Furthermore, the theta and alpha components demonstrated the same estimates not only within each neurological state, but also among the different states. These results indicate that the theta and alpha components may be caused by similar dynamic processes. We conclude that the brain function underlying the ictal EEG has a simple mechanism. Several heterogeneous dynamic systems play important roles in the generation of EEG.

Adult↗
PubMed2000

Rheumatoid arthritis-related antigen 47kDa (RA-A47) is a product of colligin-2 and acts as a human HSP47.

We previously isolated RA-A47, which is recognized as an antigen of rheumatoid arthritis (RA), from a human chondrosarcoma-derived cell line (HCS-2/8). The N-terminal 21-amino-acid sequence of RA-A47 had 81% homology to the deduced amino acid sequence of the human heat-shock protein (HSP) 47 gene, the colligin gene, and 100% homology to that of the colligin-2 gene. Moreover, as is HSP47, RA-A47 was a heat-inducible collagen-binding protein. To further characterize RA-A47, we isolated ra-a47 cDNA from HCS-2/8 cells and human periodontal ligament fibroblast (HPLF) cells. The isolated ra-a47 cDNAs from both cells were almost the same as that of colligin-2. C504 and G505 in the cDNA sequences of both cells and C598 in the cDNA of HCS-2/8 were different from the corresponding bases of colligin-2 cDNA. These differences were also observed in genomic DNA. colligin cDNA was not isolated. To show that the isolated cDNA encodes RA-A47 protein, it was expressed in Cos-7 cells. The produced protein was 47kDa and was recognized both with RA sera and antirat HSP47 antibody, indicating that it is RA-A47 and has structural similarity to HSP47. These results taken together with our previous findings show that RA-A47 is the putative colligin-2 gene product and behaves as a human HSP47. Although colligin has been considered the human HSP47 gene, failure to detect the colligin gene and its mRNA suggests that colligin does not exist in human cells and that the HSP47 gene is identical with colligin-2, which encodes RA-A47.

Amino Acid Sequence↗
PubMed2000

Age-related alterations of nitric oxide production in the brains of seizure-susceptible EL mice.

We evaluated age-related changes in nitric oxide (NO) production in the brains of EL mice, a strain highly susceptible to seizures. A group of EL(s) mice were tossed up weekly to induce convulsive seizures, while in a nonstimulated EL(ns) group induction of convulsive seizures was avoided. Brain levels of nitrite plus nitrate (NOx) in EL(ns) mice were significantly higher than in nonstimulated mice at 10 days, and also higher than levels at 15 and 50 weeks in either EL(s) or EL(ns) mice. A significantly higher number of NO-producing cells were demonstrated in the hippocampus and parietal cortex by staining for nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase in EL(s) mice at the ages of 15 and 50 weeks than in EL(ns) mice at the age of 6 weeks. In EL(ns) mice, significantly fewer neurons showed NADPH-diaphorase staining in the hippocampus, striatum and parietal cortex at the age of 50 weeks than at 6 weeks. The present results suggest that whole-brain NOx levels in EL(ns) and EL(s) mice and numbers of NADPH-diaphorase-positive neurons in EL(ns) mice decreased with aging, while increasing of numbers of such neurons in EL(s) mice were assumed to develop in compensation for reduction in whole-brain NOx levels.

Aging↗
PubMed2000

Comparison of human ictal, interictal and normal non-linear component analyses.

OBJECTIVES: The non-linear properties of EEG and filtered rhythms obtained from healthy subjects and epileptic patients with complex partial seizures were analyzed to investigate whether EEG in different neurological states can be generated by the mechanism that integrates several non-linear dynamic systems. METHODS: The control EEG (from 26 healthy subjects), interictal EEG and ictal EEG (from 25 patients) were digitally filtered into delta (0.5-4 Hz), theta (4-8 Hz), alpha (8-13 Hz), beta (13-30 Hz) and gamma (30-40 Hz) components. The correlation dimension was calculated on each original signal and corresponding surrogate data. A new method was developed to accelerate the calculation of the correlation integral. Function P(m,r) was defined to visualize the meaning of the correlation dimension. The point critical to the estimation was determined by the P(m,r) function. RESULTS: The EEG in the control subjects and patients showed significantly lower correlation dimensions than the surrogate data. The delta, alpha, beta and gamma components from the control EEG exhibited similar complexity to the surrogate data, while only the alpha component from the interictal EEG presented the same dimension as the surrogate data. The correlation dimensions of the theta and alpha components remained the same when the neurological state changed from interictal EEG to ictal EEG. The complexity of the beta component was higher than the complexity of other components in both control subjects and patients. The correlation dimension of EEG was significantly correlated to the complexity of delta, theta, beta and gamma components. CONCLUSIONS: Our results suggest that EEG and filtered components in different neurological states demonstrate varied dynamic properties. The characteristics of neuronal networks can be differentiated by the dynamics of filtered components. Separating EEG into different dynamic systems may facilitate understanding of the mechanisms involved in the human EEG.

Adult↗
PubMed2000

Observation of EEG coherence after repetitive transcranial magnetic stimulation.

OBJECTIVE: The effects before and after repetitive transcranial magnetic stimulation (rTMS) on EEG activity were investigated. METHODS: Nineteen healthy subjects received two trains (10 Hz, 100% of motor threshold, 3 s/train) of rTMS to the left frontal area. Directed coherence and ordinary coherence were calculated from EEG epochs recorded before and after (1 approximately 3 and 3 approximately 5 min) rTMS. The results were compared and demonstrated on maps. RESULTS: Directed coherence between cortical areas increased after rTMS (F=5.62, P<0.005), with the intra-hemispheric change being more pronounced than the inter-hemispheric change. Connections from the stimulated site to other sites were selectively reinforced. In contrast, ordinary coherence did not change after the stimulation. rTMS did not influence the dominant frequency at which maximal coherence was calculated. Differences in the directed coherence with opposite directions after rTMS were significantly correlated with the differences before rTMS (r=0.88, 0.89, P<0.001). CONCLUSIONS: (1) rTMS can enhance the connections between cortical areas, especially connections between the stimulated cortex and other sites in the brain. (2) Comparing with connections from the parietal area to frontal area, connections from the frontal area to parietal area are obviously improved by rTMS in both hemispheres. (3) The effects of rTMS can last for several minutes. Therefore, the necessity of EEG monitoring in rTMS studies is suggested.

Adult↗
PubMed2000

Expression of neurotrophins and their receptors (TRK) during fracture healing.

To clarify the roles of neurotrophins and their receptors in bone formation, expression of neurotrophins and their receptors (TRK) in a model of mouse fracture healing was investigated. A total of 120 male ICR mice were studied. The right eighth rib of 70 mice was fractured. For sham operation as a control, the right eighth rib of 50 mice was similarly exposed but not fractured. Localization of TRKA, TRKB, and TRKC in a rectangular region of the rib together with surrounding soft tissues was investigated by immunostaining. Localizations of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) at the fracture callus were also investigated by immunostaining, and their mitochondrial RNA (mRNA) expressions were investigated by reverse transcriptase-polymerase chain reaction (RT-PCR). As a result, we observed two types of neurotrophin receptors in the bone forming area: immunostaining by anti-TRKA was observed in almost all bone forming cells, and staining with anti-TRKC was observed in osteoblast-like cells and hypertrophic chondrocytes, but no staining was observed with anti-TRKB. On the other hand, localization of NGF was observed in almost all bone forming cells, localization of BDNF was observed in osteoblast-like cells, and localization of NT-3 was observed in osteoblast-like cells and hypertrophic chondrocytes at the fracture callus. Expression levels of the mRNA of three neurotrophins in the fractured rib were increased during the process of healing, especially those of NGF and NT-3, which peaked at 2 days after the fracture. The level of BDNF mRNA increased gradually over 8 days. These findings show that neurotrophins and their receptors were expressed in bone forming cells, and suggest that they are involved in the regulation of bone formation as an autocrine and paracrine factor in vivo.

Animals↗
PubMed2000

Effects of neonatal melatonin administration on the extra-hypothalamic regions in rat brains: effects on the serotonergic system.

The effects of 100microg melatonin injection at postnatal day 5 (PD 5) on the development of the central serotonergic systems in male and female rats were investigated. The contents of serotonin (5-HT) and 5-hydroxy-3-indolacetic acid (5-HIAA) were measured in several extrahypothalamic regions at 3, 10 and 42 weeks of age. The neonatal melatonin administration increased both 5-HT and 5-HIAA levels in the striatum throughout the examined period. In the hippocampus, an increase in 5-HIAA contents by neonatal melatonin administration was found at 3 weeks but not 10 or 42 weeks of age. There were no significant differences in the effects of melatonin between male and female rats. These results indicated that exogenous melatonin administration during the early neonatal period influenced the development of the serotonergic systems in extrahypothalamic regions including the hippocampus and the striatum.

Animals↗
PubMed2000

Disordered expression of inhibitory receptors on the NK1-type natural killer (NK) leukaemic cells from patients with hypersensitivity to mosquito bites.

Recent studies have revealed the existence of a distinct type of NK cell leukaemia of the juvenile type, which presents with hypersensitivity to mosquito bites (HMB) as an essential clinical manifestation and is infected with clonal Epstein-Barr virus (EBV). This disorder is thus called HMB-EBV-NK disease and has been reported in Orientals, mostly from Japan. We investigated the profile of cytokine production and the expression of both types of NK inhibitory receptors, i.e. CD94 lectin-like dimers and killer-cell immunoglobulin-like receptors, in NK leukaemic cells from three patients with HMB-EBV-NK disease. It was found that freshly isolated NK leukaemic cells expressed mRNA for interferon-gamma (IFN-gamma) and additionally produced IL-10 upon stimulation with IL-2, indicating that the NK cells were of NK1 type. More than 98% of NK cells from the patients bore CD94 at a higher level than did normal NK cells, whereas p70 or NKAT2, belonging to immunoglobulin-like receptor, was not expressed in those NK cells. Freshly isolated leukaemic NK cells transcribed mRNA for CD94-associated molecule NKG2C at an abnormally high level, and upon stimulation with IL-2 and/or IL-12 they expressed NKG2A as well. The disordered expression of these inhibitory receptors not only provides some insights into the pathogenesis of HMB-EBV-NK disease but also can be used as phenotypic markers for the diagnosis of this type of NK cell leukaemia.

Adolescent↗