Biomedical subjects

S Kohsaka

Publications and source records attributed to S Kohsaka.

At least 55 records · Page 3Linked to original sources

PubMed1999

Brainstem triggers absence seizures in human generalized epilepsy.

Simultaneous analysis of brainstem auditory evoked potentials (BAEPs) with reference to electroencephalography (EEG) was designed to examine the brainstem function corresponding to the EEG event. With this method, we investigated the brainstem function pre- and during the paroxysmal discharge in human absence seizures classified as primary generalized epilepsy (PGE). Two types of functional change in the lower brainstem were revealed as parameters of wave-III components (amplitude and area) of BAEPs without significant change in the upper brainstem. One was long-range biphasic fluctuation (acceleration followed by abrupt deceleration with the maximum -6.4+/-3.2 s before the seizure onset), and the other was rhythmic oscillation with 3 Hz. The latter, synchronized with the cortical spike-and-wave complex, imposed on the descending slope of the former. One important point is that both preceded the onset of cortical paroxysmal discharge. The results reappraise the classical hypothesis of "centrencephalic system" on seizure generating mechanism in human PGE. The results prove the primary triggering role of the lower brainstem that is independent of sleep-related synchronizations. The method is applicable to other types of EEG event for the investigation of brainstem involvement.

Adolescent↗

PubMed1999

Expression of receptor tyrosine kinase RYK in developing rat central nervous system.

Receptor tyrosine kinase RYK is a mammalian homologue of Drosophila Lio, which is involved in learning and memory and in axon guidance. We cloned a rat ryk gene and characterized its expression pattern in the central nervous system. Northern blot analysis of the whole brain revealed that the RYK mRNA was abundant during the period from 13 to 18 embryonic days (E13-18) and it decreased by E20. In the postnatal brain, the RYK signal was higher in postnatal one week (P1W) cerebrum and in P2W cerebellum than in later stages. In situ hybridization revealed that RYK was expressed throughout the central nervous system, mainly in the ventricular zone on E11 and E13. On E18 and E20, the remarkable level of RYK mRNA was detected in the ventricular zone as well as in the cortical plate of the forebrain. These two regions overlapped the immunoreactive areas of nestin and MAP2, a neural stem cell marker and a mature neural marker, respectively. Moreover, the double-labeling analysis showed that the same cells expressed both RYK and nestin in the ventricular zone. In the postnatal brain, RYK was predominantly expressed in neurons of various regions. These observations suggest that RYK plays a contributory role as a multifunctional molecule in the differentiation and maturation of neuronal cells in the central nervous system.

Animals↗

PubMed1999

Neuroprotective action of a novel compound--M50463--in primary cultured neurons.

The neuroprotective effects of a novel synthetic compound, M50463, have been determined by using embryonic rat neocortical neurons in various culture conditions. M50463 was initially characterized as a potent specific ligand for a voltage-dependent sodium channel by radioligand binding studies. In fact, M50463 inhibited neuronal cell death induced by veratrine and inhibited an increase of the intracellular calcium level in neurons evoked by veratrine. In addition to such expected effects, M50463 had the ability to prevent glutamate neurotoxicity, to promote the neuronal survival in serum-deprived medium and to prevent nitric oxide-induced neurotoxicity. These results suggested that M50463 is not a simple sodium channel blocker, but a neuroprotective agent which has some crucial mechanism of action on neuronal death occurring in various situations, and it is a novel, innovative candidate for neuroprotective therapy for various neurodegenerative disorders.

Acetylcysteine↗

PubMed1999

Analysis of human error in nursing care.

Analysis of reports about incidental and accidental events in nursing care were made using a reliability engineering method. Unnatural working hours, such as evening duty, night duty falling next to a holiday, two consecutive night-duty shifts, and two consecutive evening-duty shifts were major factors in the occurrence of errors. In a mixed-division ward (a ward containing patients belonging to different divisions), rule-based errors happened more frequently than in a single-division ward. Also, less experienced nursing staffs made errors more frequently than experienced nursing staffs.

Cardiology Service, Hospital↗

PubMed1999

Early axonal and glial pathology in fetal sheep brains with leukomalacia induced by repeated umbilical cord occlusion.

We conducted a chronic preparation experiment involving near term fetal sheep to evaluate the contribution of umbilical cord occlusion to fetal brain injury. In experimental groups (n = 11), complete cord occlusion for 3 min followed by 5 min release, repeated 5 times were performed at 3 days after initial surgery. Instrumental cases without cord occlusion (n = 3) and uninstrumental twins (n = 6) were also examined as controls. Multiple necrotic foci predominantly in the periventricular white matter were found in the fetal brains examined at 1-3 days after cord occlusion. To estimate the contribution of early axonal and glial reaction to brain injury the following immunohistochemical study was performed. In the lesions, coagulation necrosis, axonal swelling and microglial activation were demonstrated with amyloid precursor protein or ionized calcium binding adapter molecule 1 immunohistochemistry. The induction of tumor necrosis factor alpha and inducible nitric oxide synthase were also detected immunohistochemically in the microglia at 1 and 3 days after cord occlusion. In contrast, the reaction of glial fibrillary acidic protein positive astrocytes was faint at 1 day after occlusion, but the induction of cyclooxygenase-2 was observed. These findings suggest the glial reaction of cytokines and free radicals induced by fetal hypoxia may contribute to the occurrence of brain injury.

Amyloid beta-Protein Precursor↗

PubMed1999

Gender difference of slow wave sleep in middle aged and elderly subjects.

Sleep EEG of eight healthy males and eight females aged 54-72 years were recorded at their homes. The electroencephalograms were visually scored and analyzed by spectral analysis using the FFT method. There were no significant differences in sleep parameters except for a higher percentage of stage 3+4 in females. The spectral power of the delta band EEG was classified into two frequencies: 0.5-2 Hz and 2-4 Hz. The total amount of the delta band spectral power through the night was significantly larger in females. Periodic fluctuation of delta band power was observed in females along with non-rapid eye movement-rapid eye movement cycles.

Aged↗

PubMed1999

Functional changes of the brainstem triggering vertex sharp wave with spindle.

A vertex sharp wave followed by spindle (VS-spindle) is one of the hallmarks of human stage 2 sleep. We recorded sleep electroencephalograms (EEG) simultaneously with brainstem auditory evoked potentials (BAEP) from nine healthy male subjects. To investigate the generating mechanism of the VS-spindle, sequentially changing BAEP were analyzed around the VS-spindle. The results revealed the preceding changes of wave-V amplitude to the onset of the VS-spindle. When the generator site of wave-V in the brainstem was considered, the results suggest that the functional changes in the dorsal area of the midbrain-pontine junction participate in the organization of the VS-spindle.

Adolescent↗

PubMed1999

Amino-terminal region of secreted form of amyloid precursor protein stimulates proliferation of neural stem cells.

Beta-amyloid precursor protein (APP) has been reported to be expressed in the CNS from the early stages of development. However, the functional role of APP during early development remains unclear. In the present study, we found that the secreted form of APP (sAPP) significantly enhanced proliferation of neural stem cells. Cells were prepared from 13-day embryonic rat neocortex, which was dissected with a Pasteur pipette to make cell clusters. After 12 h of cultivation in the medium without serum, cells around the centre of the cluster were still nestin-positive proliferative cells, i.e. neural stem cells. To determine whether the proliferation of cells was regulated by sAPP, cultures were treated with recombinant sAPP695, the secreted form of human APP695 produced by yeast. Both DNA synthesis and expression of proliferating cell nuclear antigen markedly increased after 5 h of sAPP695 addition. The enhancement of DNA synthesis by sAPP695 stimulation was blocked by the 22C11 monoclonal antibody specific for the amino-terminal region of sAPP. Then, we examined the effect of the amino-terminal fragment of sAPP and the epitope peptide of 22C11 antibody, and found that both of them also promoted DNA synthesis, suggesting that the amino-terminal region of sAPP is responsible for the biological activity. Our findings indicate the possibility that sAPP enhances proliferation of neural stem cells in vivo and plays an important role during the early CNS development.

Amyloid beta-Protein Precursor↗

PubMed1999

PAX6 expression in the developing human eye.

AIMS: To investigate the changes in PAX6 expression in the developing human eye. METHODS: Six developing human eyes from 6 to 22 weeks' gestation were evaluated. Frozen sections were immunohistochemically stained with monoclonal antibody to chick Pax6 (amino acids 1-223). To verify antibody specificity, western blot analysis was carried out using cell lysates from P19 cells transfected with the human PAX6 gene. RESULTS: Western blot analysis demonstrated that the antibody reacted to human PAX6 protein. Positive immunostainings for PAX6 were seen in the surface ectoderm, lens vesicle, inner and outer layers of the optic cup, and optic stalk at 6 weeks, and in the corneal epithelia and conjunctiva, lens, and non-pigmented ciliary epithelia from 8 to 22 weeks. In the retina, positive cells were seen in the entire retina from 8 to 10 weeks, and were restricted to the ganglion cell layer and the inner and outer portions of the inner nuclear layer after 21 weeks. CONCLUSIONS: PAX6 is expressed on the surface and neuroectoderms at an early stage, then in the differentiating cells in the cornea, lens, ciliary body, and retina through development. PAX6 may play a role in determining cell fate in the morphogenesis of various human ocular tissue.

Antibody Specificity↗

PubMed1998

The microglia/macrophage response in the neonatal rat facial nucleus following axotomy.

Microglia represent a population of brain macrophage precursor cells which are intrinsic to the CNS parenchyma. Transection of the facial nerve in the newborn rat causes death of the affected motor neurons which is accompanied by massive activation of local microglia. Many of these cells develop into macrophages as can be shown by immunocytochemistry for OX-42 and ED1. Using the new polyclonal microglial marker ionized calcium binding adapter molecule 1, iba1, in combination with immunocytochemical double-labeling for the proliferating cell nuclear antigen (PCNA), or [3H]thymidine autoradiography, and confocal microscopy, qualitative as well as quantitative differences can be demonstrated between the newborn and the adult axotomized rat facial nucleus. While microglial cells are the only cell population which responds to axotomy by cell division in the adult facial nucleus, GFAP positive reactive astrocytes can be shown to undergo mitosis following axotomy in the newborn rat. Furthermore, ED1 immunoreactivity, early expression of MHC class II molecules and morphological transformation of microglia into macrophages can only be observed under conditions of neuronal degeneration, i.e., in the neonatal rat facial nucleus. Thus, the combination of cellular markers described here should be useful for studies employing the neonatal rat facial nucleus as an in vivo assay system to test the efficacy of neurotrophic factors.

Age Factors↗

PubMed1998

Novel domain-specific actions of amyloid precursor protein on developing synapses.

The effect of the secretory form of amyloid precursor protein (sAPP) on synaptic transmission was examined by using developing neuromuscular synapses in Xenopus cell cultures. The frequency of spontaneous postsynaptic currents (SSCs) was reduced by the addition of sAPP, whereas the amplitude of impulse-evoked postsynaptic currents (ESCs) was increased by sAPP. These opposing effects on spontaneous versus evoked release were separated by using the specific domain of APP. The C-terminal fragment of sAPP (CAPP) only reduced SSC frequency and did not affect ESCs. By contrast, the N-terminal fragment of sAPP (NAPP) did not affect SSC frequency but did increase ESC amplitude. The reduction of SSC frequency by sAPP appears to be mediated by activation of potassium channels through a cGMP-dependent pathway, whereas the increase of ESC amplitude is mediated by a different pathway involving activation of protein kinase(s). These results suggest the potential role of sAPP as a modulator of synaptic activity by two specific domains.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1998

Microglia-specific localisation of a novel calcium binding protein, Iba1.

Recently it has been shown that mRNA of Iba1 (ionized calcium binding adaptor molecule 1), which was a novel calcium binding protein cDNA-cloned by our group, is specifically expressed in microglia in cultures of rat brain cells [Imai et al. Biophys. Biochem. Res. Commun., 224 (1996) 855-862]. In the present study, immunocytochemical and immunohistochemical examinations demonstrated that Iba1 protein is expressed in microglia alone both in cultured brain cells and in the brain, respectively. In a mixed cell culture of embryonic rat brain, immunocytochemically positive for Iba1 protein were the microglia but it was not detectable in neurons, astroglia, or oligodendroglia. Immunohistochemical staining of adult rat brain sections showed Iba1 protein to be specifically localised in ramified microglia. In addition, immunohistochemical staining and immunoblot analysis of activated microglia in the facial nucleus after facial nerve axotomy shows that expression of Iba1 protein was upregulated and peaked at 7 days. These results indicated that localisation of Iba1 protein is restricted to microglia both in vitro and in vivo, and that Iba1 protein plays a role in regulating the function of microglia, especially in the activated microglia.

Animals↗

PubMed1998

Involvement of amyloid precursor protein in functional synapse formation in cultured hippocampal neurons.

Amyloid precursor protein (APP) is known to be widely expressed in neuronal cells, and enriched in the central and peripheral synaptic sites. Although it has been proposed that APP functions in synaptogenesis, no direct evidence has yet been reported. In this study we investigated the involvement of APP in functional synapse formation by monitoring spontaneous oscillations of intracellular Ca2+ concentration ([Ca2+]i) in cultured hippocampal neurons. As more and more neurons form synapses with each other during the culture period, increasing numbers of neuronal cells show synchronized spontaneous oscillations of [Ca2+]i. The number of neurons that showed synchronized spontaneous oscillations of [Ca2+]i was significantly lower when cultured in the presence of monoclonal antibody 22C11 against the N-terminal portion of APP. Moreover, incubation with excess amounts of the secretory form of APP or the N-terminal fragment of APP also inhibited the increase in number of neurons with synchronized spontaneous oscillations of [Ca2+]i. The addition of monoclonal antibody 22C11 or secretory form of APP did not, however, affect MAP-2-positive neurite outgrowth. These findings suggest that APP play a role in functional synapse formation during CNS development.

Amyloid beta-Protein Precursor↗

PubMed1998

Neurotrophins regulate the function of cultured microglia.

Although the physiological role of neurotrophins in neuronal development and survival has been extensively investigated, their role in glial cell physiology remains to be elucidated. In the present study, we investigated the effects of neurotrophins on cultured microglia from newborn rat brain. All of the neurotrophins tested nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4), increased the secretion of plasminogen and urokinase type-plasminogen activator and specific activity of acid phosphatase, but suppressed the release of constitutively-produced and lipopolysaccharide-stimulated nitric oxide (NO) from microglia. The reverse transcription-polymerase chain reaction, immunocytochemical staining, and Western blotting revealed that cultured microglia express Trk A, B, and C, and low-affinity NGF receptor, LNGFRp75. Neurotrophin was found to phosphorylate Trk A and B, and the neurotrophin-induced enhancement of plasminogen-secretion was suppressed by protein kinase inhibitor, K252a. Furthermore, neurotrophins caused an activation of transcription factor, NF-kappaB. These results indicate that the neurotrophin family regulate the function of microglia through Trk and/or LNGFRp75-mediated signal transduction.

5'-Nucleotidase↗

PubMed1998

Secreted form of beta-amyloid precursor protein activates protein kinase C and phospholipase Cgamma1 in cultured embryonic rat neocortical cells.

The secreted form of beta-amyloid precursor protein (sAPP) has been reported to exert various biological activities in cultured neurons. The signal transduction mechanisms underlying these physiological functions of sAPP remain unclear. We now report that treatment of neural cells with the secreted form of APP695 (sAPP695) leads to dose- and time-dependent increase in phosphorylation of the endogenous substrates with a molecular mass of 80, 57 and 43 kDa. Pretreatment of cells with protein kinase C (PKC) inhibitor H-7 reduced phosphorylation of the 80- and 43-kDa proteins in a dose-dependent manner. The effect of sAPP695 on the phosphorylation is mimicked by phorbol 12-myristate-13-acetate (PMA). Downregulation of PKC by prolonged treatment of cells with PMA abolished sAPP695-enhanced phosphorylation of the 80- and 43-kDa proteins, indicating PKC is involved in the sAPP695-enhanced phosphorylation of these proteins in the cells. We also suggest that the 80- and 43-kDa proteins phosphorylated by sAPP695-stimulation are the major PKC substrates myristoylated alanine-rich C-kinase substrate and growth-associated protein-43. Furthermore, we demonstrate that tyrosine phosphorylation of phospholipase Cgamma1 and formation of inositol 1,4,5-trisphosphate were increased by sAPP695-stimulation. These observations suggest that sAPP695 induces the activation of the signaling pathways through a stimulation of phosphoinositide-PKC cascade.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1998

Pharmacological detection of AMPA receptor heterogeneity by use of two allosteric potentiators in rat hippocampal cultures.

1. In order to examine whether a recently developed allosteric potentiator for AMPA receptors, 4-[2-(phenylsulphonylamino)ethylthio]-2,6-difluoro-phenoxyaceta mide (PEPA), can be utilized as an indicator of AMPA receptor heterogeneity, the action of PEPA upon the increase of intracellular free calcium ion concentration ([Ca2+]i) elicited by AMPA was investigated in rat hippocampal cultures, and the action was compared with that of cyclothiazide, a well characterized allosteric modulator of AMPA receptors. 2. PEPA dose-dependently potentiated AMPA-induced increase of [Ca2+]i. In 90% (72 out of 80) of the cells in which cyclothiazide acts, PEPA potentiated the increased [Ca2+]i induced by AMPA with pronounced cell-to-cell variation in rat hippocampal cultures. 3. The ratio of the potentiation by PEPA to the potentiation by cyclothiazide (P/C ratio) also varied with cells between 0 and 2.15. It was found that the cultured hippocampal cells consisted of multiple populations with different P/C ratios. Among them two populations exhibited characteristic P/C ratios; low (0 to 0.15; 27 out of 80 cells, 34%) and high (> or = 2.00; 1 out of 80 cells, 1%) P/C ratios. The P/C ratios of the other populations were between 0.25 and 1.20, and these cells constituted 65% (52 out of 80 cells) of the cells tested. 4. Reverse transcriptase-polymerase chain reaction analysis suggested that GluR2-flip, GluR1-flip, GluR2-flop, and GluR1-flop were abundantly expressed (in this rank order) in the cultures used. 5. In Xenopus oocytes expressing GluR1, GluR3, or these subunits plus GluR2, the potentiation of AMPA response by PEPA and by cyclothiazide varied with subunit and splice-variant combinations, and the P/C ratio was between 0.19 and 2.20. Oocytes with low P/C ratios (0.19 to 0.50) and low sensitivity to PEPA potentiation (1.9 fold to 6.41 fold) were those expressing flip variants predominantly, and oocytes with high P/C ratios (1.8 to 2.2) were those expressing flop variants predominantly. Oocytes with intermediate P/C ratios (0.51 to 1.20) were those expressing various combinations of flip and flop variants, and it was impossible to specify the relative abundance of flip and flop variants in these cells. Therefore, the P/C ratio can be used to infer subunit/splice variant expression only when the ratio is low or high. 6. These results suggest that the potentiation by PEPA alone reveals cell-to-cell heterogeneity of AMPA receptors, but a comparison of the actions of PEPA and cyclothiazide further facilitates the detection of the heterogeneity.

Allosteric Regulation↗

PubMed1998

The effect of a secreted form of beta-amyloid-precursor protein on intracellular Ca2+ increase in rat cultured hippocampal neurones.

1. The effects of secreted forms of beta-amyloid-precursor proteins (APP(S)s) on the intracellular Ca2+ concentration ([Ca2+]i) were investigated in rat cultured hippocampal neurones. APP695S, a secretory form of APP695, attenuated the increase in [Ca2+]i evoked by glutamate. In addition, APP695S itself evoked an increase in [Ca2+]i in 1 or 2 day-cultured hippocampal cells, but not in 7 to 13 day-cultured cells. 2. Eighty-one percent of neurones which were immunocytochemically positive for microtubule-associated protein 2 responded to APP695S with an increase in [Ca2+]i. 3. APP695S induced a transient rise in [Ca2+]i even in the absence of extracellular Ca2+ and produced an elevation in inositol-1,4,5-trisphosphate (IP3) in a concentration-dependent manner from 100 to 500 ng ml(-1). In the presence of extracellular Ca2+, APP695S caused a transient rise in [Ca2+]i followed by a sustained phase at high [Ca2+]i, suggesting Ca2+ entry from the extracellular space. 4. The [Ca2+]i elevation was mimicked by amino terminal peptides of APPs, but not by carboxy terminal peptides. 5. These results taken together suggest that APP695S induces an increase in [Ca2+]i in hippocampal neurones through an IP3-dependent mechanism that changes according to the stage of development.

Amyloid beta-Protein Precursor↗

PubMed1998

ATP stimulation of Ca2+ -dependent plasminogen release from cultured microglia.

1. ATP (10-100 microM), but not glutamate (100 microM), stimulated the release of plasminogen from microglia in a concentration-dependent manner during a 10 min stimulation. However, neither ATP (100 microM) nor glutamate (100 microM) stimulated the release of NO. A one hour pretreatment with BAPTA-AM (200 microM), which is metabolized in the cytosol to BAPTA (an intracellular Ca2+ chelator), completely inhibited the plasminogen release evoked by ATP (100 microM). The Ca2+ ionophore A23187 induced plasminogen release in a concentration-dependent manner (0.3 microM to 10 microM). 2. ATP induced a transient increase in the intracellular calcium concentration ([Ca2+]i) in a concentration-dependent manner which was very similar to the ATP-evoked plasminogen release, whereas glutamate (100 microM) had no effect on [Ca2+]i (70 out of 70 cells) in microglial cells. A second application of ATP (100 microM) stimulated an increase in [Ca2+]i similar to that of the first application (21 out of 21 cells). 3. The ATP-evoked increase in [Ca2+]i was totally dependent on extracellular Ca2+, 2-Methylthio ATP was active (7 out of 7 cells), but alpha,beta-methylene ATP was inactive (7 out of 7 cells) at inducing an increase in [Ca2+]i. Suramin (100 microM) was shown not to inhibit the ATP-evoked increase in [Ca2+]i (20 out of 20 cells). 2'- and 3'-O-(4-Benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP), a selective agonist of P2X7 receptors, evoked a long-lasting increase in [Ca2+]i even at 1 microM, a concentration at which ATP did not evoke the increase. One hour pretreatment with adenosine 5'-triphosphate-2', 3'-dialdehyde (oxidized ATP, 100 microM), a selective antagonist of P2X7 receptors, blocked the increase in [Ca2+]i induced by ATP (10 and 100 microM). 4. These data suggest that ATP may transit information from neurones to microglia, resulting in an increase in [Ca2+]i via the ionotropic P2X7 receptor which stimulates the release of plasminogen from the microglia.

Adenosine Triphosphate↗