Biomedical subjects

H Hatanaka

Publications and source records attributed to H Hatanaka.

At least 145 records · Page 8Linked to original sources

PubMed1996

The survival of striatal cholinergic neurons cultured from postnatal 2-week-old rats is promoted by neurotrophins.

Although the expression of nerve growth factor (NGF) in the rat striatum is the highest at 2 postnatal weeks (P2w), the action of NGF at that age has not been studied in detail. We examined the effects of several neurotrophic factors, including NGF, on striatal cholinergic neurons cultured from P2w rats. We also examined the effects of a cyclic AMP (cAMP) analog and high K(+)-evoked depolarization. NGF specifically promoted the survival of choline acetyltransferase (ChAT)-positive neurons, and consequently increased the ChAT activity per well, whereas it did not induce the ChAT activity per cholinergic neuron. NGF-responsiveness was the highest in striatal cultures from P2w rats, but it was almost lost in cultures from P4w rats. Brain-derived neurotrophic factor (BDNF), neurotrophin-4/5 (NT-4/5), and a cAMP analog had survival-promoting effects on striatal total neurons including cholinergic neurons. On the other hand, high K+ hardly promoted the survival of striatal cholinergic neurons in cultures from P2w rats, although it increased the viable number of total striatal neurons. High K+ did not increase the ChAT activity in any tested cultures from postnatal 3- to 28-day-old rats. These results demonstrated that NGF prevented the death of striatal cholinergic neurons in cultures from P2w rats, but not from P4w rats, and that high K+ could not rescue these deaths. We propose that cholinergic neurons in the striatum are programmed to die at P2w, and that this programmed cell death can be restored by neurotrophins, but not by depolarization.

Age Factors↗

PubMed1996

PC12h-R cell, a subclone of PC12 cells, shows EGF-induced neuronal differentiation and sustained signaling.

Unlike nerve growth factor (NGF), epidermal growth factor (EGF) does not induce neuronal differentiation but promotes proliferation of the rat pheochromocytoma PC12 cells. We found that PC12h-R, a subclone of PC12 cells, differentiated into neuron-like cells in response to EGF as well as to NGF. PC12h-R cells treated with EGF extended neurites, attenuated cell proliferation, and increased the levels of tyrosine hydroxylase protein synthesis and of acetylcholinesterase activity as those treated with NGF. The EGF-induced differentiation of PC12h-R cells was not mediated by the indirect activation of p140trkA by EGF. In addition, EGF induced the sustained tyrosine phosphorylation of the EGF receptor, mitogen-activated protein (MAP) kinases, and 46 and 52 kDa proteins, and the prolonged activation of MAP kinases in PC12h-R cells compared with the parent PC12h, which does not show EGF-induced differentiation. The response of PC12h-R cells to EGF was not simply due to an increase in the level of EGF receptor protein. These results indicated that the duration of EGF-induced signaling might determine the cellular response of PC12 cells between cell proliferation and neuronal differentiation.

Adrenal Gland Neoplasms↗

PubMed1996

p53 involves cytosine arabinoside-induced apoptosis in cultured cerebellar granule neurons.

The tumor suppressor p53 gene plays a key role in controlling the cell cycle checkpoint and in apoptosis following the exposure of normal cells to DNA damage. To investigate the role of p53 in cytosine arabinoside (Ara C)-induced cell death of CNS neurons, we examined the effect of Ara C on the survival of cultured cerebellar granule neurons from normal wild-type and p53 null mutant mice. When the neurons from wild-type mice were cultured with Ara C, they gradually died after 24 h in culture. In contrast, the neurons from p53 null mutant mice showed a resistance to the Ara C neurotoxicity. These results indicate that p53 involves Ara C-induced apoptosis in cultured cerebellar granule neurons, in which DNA damage may initiate the apoptotic death program of the neurons.

Animals↗

PubMed1996

EGF-induced sustained tyrosine phosphorylation and decreased rate of down-regulation of EGF receptor in PC12h-R cells which show neuronal differentiation in response to EGF.

PC12h-R cell, a subclone of PC12 cells, exhibited a neuron-like phenotype, including neurite outgrowth and increased acetylcholinesterase activity, in response to epidermal growth factor (EGF) as well as nerve growth factor (NGF). We examined the mechanism by which EGF induced the neuronal differentiation in PC12h-R cells. The EGF-induced neuronal differentiation of PC12h-R cells was not blocked by K252a, whereas that induced by NGF was. EGF induced sustained tyrosine phosphorylation of the EGF receptor in PC12h-R cells, but not in the parent PC12h cells, which do not show neuronal differentiation in response to EGF. In addition, the rate of EGF-induced down-regulation of the EGF receptor in PC12h-R cells was decreased compared with that in PC12h cells. Furthermore, we found that the duration of EGF-induced tyrosine phosphorylation of the EGF receptor in PC12h-R cells was similar to that of NGF-induced tyrosine phosphorylation of p140trkA in PC12h cells. The EGF-induced phosphorylation of the EGF receptor in PC12h cells was less sustained than that of p140trkA by NGF in PC12h cells. These findings suggested that the EGF-induced neuronal differentiation of PC12h-R cells is due to the sustained activation of the EGF receptor, resulting from the decreased down-regulation of the EGF receptor and that the duration of the receptor tyrosine kinase activity determines the cellular responses of PC12 cells. We concluded that sustained activation of the receptor tyrosine kinase induces neuronal differentiation, although transient activation promotes proliferation of PC12 cells.

Acetylcholinesterase↗

PubMed1996

BDNF increases the expression of neuropeptide Y mRNA and promotes differentiation/maturation of neuropeptide Y-positive cultured cortical neurons from embryonic and postnatal rats.

The effects of neurotrophic factor on the expression of neuropeptide Y (NPY) mRNA and on morphology of NPY-immunoreactive neurons were investigated. Brain-derived neurotrophic factor (BDNF) increased the expression of NPY mRNA in cultured cortical neurons from both embryonic and postnatal rats. BDNF also increased the number of NPY neurons. Furthermore, multipolar neurites from NPY neurons were observed in cultures treated with BDNF, whereas only monopolar and bipolar neurites were observed in control cultures. These results suggest that BDNF not only increases the expression of NPY mRNA but also promotes the differentiation/maturation of NPY ergic neurons both in number and morphology. NPY expression was strongly increased by neurotrophin-4/5 similarly to BDNF and neurotrophin-3 evoked a slight increase. In contrast, basic fibroblast growth factor, cilliary neurotrophic factor and interferon-gamma had no effect on NPY expression.

Animals↗

PubMed1996

The cavity in the hydrophobic core of Myb DNA-binding domain is reserved for DNA recognition and trans-activation.

The DNA-binding domain of Myb consists of three imperfect repeats, R1, R2 and R3, each containing a helix-turn-helix motif variation. Among these repeats, R2 has distinct characteristics with high thermal instability. The NMR structure analysis found a cavity inside the hydrophobic core of R2 but not in R1 or R3. Here, we show that R2 has slow conformational fluctuations, and that a cavity-filling mutation which stabilizes the R2 structure significantly reduces specific Myb DNA-binding activity and trans-activation. Structural observations of the free and DNA-complexed stages suggest that the implied inherent conformational flexibility of R2, associated with the presence of the cavity, could be important for DNA recognition by Myb.

Amino Acid Sequence↗

PubMed1996

Free radical-independent protection by nerve growth factor and Bcl-2 of PC12 cells from hydrogen peroxide-triggered apoptosis.

To investigate the mechanism of oxidative stress induced death of PC12 cells, we performed confocal and flow cytometric analysis with a reactive oxygen species (ROS)-specific fluorogen, 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) (C-DCDHF-DA). Hydrogen peroxide significantly decreased the number of viable PC12 cells after 24 h. Hydrogen peroxide caused membrane blebbing, nuclear condensation and DNA fragmentation, indicating that the PC12 cells died due to apoptosis. The hydrogen peroxide-triggered apoptosis of PC12 cells was associated with enhanced ROS production in a dose-dependent manner by measuring with C-DCDHF-DA. Nerve growth factor (NGF) and Bcl-2 inhibited the hydrogen peroxide-induced apoptosis of PC12 cells. Neither of them, however, reduced the ROS production in PC12 cells. These data suggest that NGF or Bcl-2 protects PC12 cells from hydrogen peroxide-triggered apoptosis independently from ROS production.

Animals↗

PubMed1996

Involvement of p53 in DNA strand break-induced apoptosis in postmitotic CNS neurons.

The tumour suppressor p53 gene serves as a critical regulator of the cell cycle and of apoptosis following the exposure of normal cells to DNA damage. To examine the role of p53 in postmitotic CNS neurons, we cultured cerebellar neurons from normal wild-type mice and mutant p53-null mice under various conditions inducing neuronal death. When cerebellar neurons from 15- to 16-day postnatal wild-type mice were treated with ionizing radiation or DNA-damaging agents, massive neuron death occurred after 24-72 h. In contrast, neurons from p53-/- mice evidently resisted gamma-irradiation and some DNA-damaging agents, such as etoposide and bleomycin. On the other hand, low-K+ medium-induced apoptosis of cerebellar neurons was not affected by p53 status. Neither cell cycle progression nor DNA synthesis occurred during cell death induced by gamma-irradiation and low-K+ medium, as well as in normal cultures of p53+/+ and p53-/- neurons. These results suggest that p53 is required for the apoptotic death of postmitotic cerebellar neurons induced by DNA strand breaks.

Animals↗

PubMed1996

Brain-derived neurotrophic factor blocks long-term depression in rat visual cortex.

1. Brain-derived neurotrophic factor (BDNF) has been reported to play a role in long-term potentiation (LTP) in hippocampus, but whether it is involved also in long-term depression (LTD) is not yet known. In this study, we tested whether BDNF and its gene family, nerve growth factor (NGF), have any effect on synaptic transmission and LTD in visual cortical slices of young rats. 2. An application of BDNF at the concentration of 20 ng/ml did not significantly change layer II/III field responses evoked by layer IV stimulation at 0.1 Hz, although at 200 ng/ml it enhanced responses. BDNF at 20 ng/ml prevented LTD of field responses from being induced by low-frequency stimulation (1 Hz for 15 min) of layer IV. NGF did not have such effects in the same concentration range as that of BDNF. 3. The action of BDNF was antagonized by K252a, an inhibitor of receptor tyrosine kinases. When K252a alone was applied to slices, LTD of stronger magnitude than in control slices was induced by low-frequency stimulation. 4. These results suggest that endogeneous BDNF may prevent synapses from being depressed by low-frequency inputs in the developing visual cortex.

Animals↗

PubMed1996

A role of cofilin/destrin in reorganization of actin cytoskeleton in response to stresses and cell stimuli.

1. Cofilin is an essential actin-regulating protein widely distributed in all eucaryotes. The structure and function of cofilin are conserved during evolution. 2. Cofilin depolymerizes F-actin in vitro at alkaline pH and severs F-actin in vitro at pH lower than 7.3. Overexpression of cofilin in viable cells induced bundles of actin filaments suggesting that the severing activity rather than the actin-depolymerizing or monomeric actin-sequestering activity is physiologically significant in vivo. 3. The actin bundle formation induced by overexpression of cofilin is accompanied with an increase in cell motility of Dictyostelium cells. 4. In higher vertebrates, the actin-binding activity of cofilin is negatively regulated by phosphorylation on its Ser-3 residue. The actin-binding activity is essential for yeast cells to grow. 5. Stresses and various cell stimuli activate cofilin by inducing dephosphorylation of cofilin in resting vertebrate cells. 6. Cofilin has an nuclear localization signal sequence and translocates into the nucleus together with actin in response to various stresses. Functional roles of cofilin/actin in the nucleus remain to be elucidated. 7. Tertiary structure of destrin (cofilin) resembles that of gelsolin segment 1 and well explains its functions such as Ca(2+)-independent actin binding activity.

Actin Depolymerizing Factors↗

PubMed1995

Selective acid vulnerability of dopaminergic neurons and its recovery by brain-derived neurotrophic factor.

Among the pathogenetic phenomena of Parkinson's disease, the character of the selective degeneration of nigrostriatal system with severe gliosis is not fully understood. Here, we have shown that dopaminergic neurons may be exclusively sensitive to elevated acidity elicited after the addition of glial mitogenic factors such as epidermal growth factor and basic fibroblast growth factor or after the direct treatment with hydrochloric acid. The acid sensitivity was specific to dopaminergic neurons. The neurons other than dopaminergic neurons in culture from the ventral mesencephalon were not sensitive to acidity and the neurons from several brain areas were the same as above, except for the hippocampal neurons which had slight acid vulnerability. Choline acetyltransferase assay studies demonstrated that the cholinergic neuronal population in the septum and corpus striatum had no acid sensitivity. The vulnerability of dopaminergic neurons either elicited by glial mitogenic factor or derived from the direct acid exposure was inhibited by the addition of brain-derived neurotrophic factor (BDNF), but not by neurotrophin-3 or nerve growth factor. These findings suggest that dopaminergic neurons have selective acid vulnerability on which BDNF has a pronounced protective effect.

Acids↗

PubMed1995

Role of nerve growth factor in the expression of trkA mRNA in cultured embryonic rat basal forebrain cholinergic neurons.

Using a quantitative reverse transcription-polymerase chain reaction (RT-PCR), we studied the regulation of trkA mRNA expression in serum-free, cultured basal forebrain neurons from 17-day fetal rats. Besides increasing choline acetyltransferase (ChAT) activities, nerve growth factor (NGF) strikingly induced trkA gene expression in a time- and NGF concentration-dependent manner. Therefore, NGF might play a critical role in trkA gene expression during the development of basal forebrain cholinergic neurons. Furthermore, to investigate whether this up-regulation is connected with the trophic effects on basal forebrain cholinergic neurons, we examined the effects of some other neurotrophic agents (BDNF, NT-3, bFGF, CNTF, and 40 mM KCI) upon ChAT activity and trkA gene expression. Some neurotrophic factors increased ChAT activities to the same degree as NGF, whereas they did not stimulate trkA mRNA expression so potently. NT-3 plus K252b promotes the tyrosine phosphorylation of TrkA in PC12 cells and increases ChAT activity in cultured basal forebrain cholinergic neurons like NGF (Knusel et al., J Neurochem 59: 715-722, 1992). We found that NT-3 plus K252b upregulated the level of trkA mRNA. These results suggested that the expression of trkA mRNA is regulated directly by its specific ligand NGF, rather than neurotrophic effects upon basal forebrain cholinergic neurons and that the up-regulation is connected to a molecular event initiated by the binding of NGF to the TrkA receptor.

Animals↗

PubMed1995

Flow cytometric analysis of serum deprivation-induced apoptosis of PC12 cells, with special reference to role of bcl-2.

To investigate the mechanism of serum deprivation-induced apoptosis in PC12 cells, we performed flow cytometry with the viable cell-specific fluorogen, fluorescein diacetate (FDA). Intact PC12 cells were positive when stained with FDA, and those cells were identical with the major population which possess larger forward and side scattered light. Apoptotic PC12 cells decreased the forward and side scatter light associated with the loss of FDA-positive cells. Nerve growth factor (NGF) and the bcl-2 protein protected the cells from serum deprivation-induced apoptosis. These data suggested that the process of apoptosis in PC12 cells can be analyzed with flow cytometry.

Animals↗

PubMed1995

A 13-Mer peptide of a brain injury-derived protein supports neuronal survival and rescues neurons from injury caused by glutamate.

Neuronal survival is mediated by several kinds of proteins. Among these, neurotrophic factors play important roles in the nervous system by supporting neuronal activity and survival. It has been suggested recently that certain factors promote neuronal survival in the case of brain injury. To examine this possibility, we purified a novel neurotrophic factor from Gelfoam that was implanted at the site of injury caused in neonatal rats. During amino acid sequence analysis, we found that a fragmental peptide of this neurotrophic protein consisting of 13 amino acids showed neurotrophic activity. This 13-mer peptide promoted survival of septal cholinergic and mesencephalic dopaminergic neurons in culture and rescued hippocampal neurons from injury caused by glutamate in culture. This peptide rescued neurons from cell death caused by glutamate, even when added 4.5 h after glutamate exposure.

Amino Acid Sequence↗

PubMed1995

Three-dimensional solution structure of bombyxin-II an insulin-like peptide of the silkmoth Bombyx mori: structural comparison with insulin and relaxin.

The three-dimensional solution structure of bombyxin-II, an insulin-like two-chain peptide produced by the brain of the silkworm Bombyx mori, has been determined by simulated annealing calculations based on 535 distance constraints and 24 torsion-angle constraints derived from NMR data and three distance constraints of the disulfide bonds. To our knowledge, this is the first three-dimensional structure determined for an invertebrate insulin-related peptide. The root-mean-square deviations between the best 10 structures and the mean structure are 0.58(+/- 0.15) A for the backbone heavy atoms (N, C alpha, C) and 1.03(+/- 0.18) A for all non-hydrogen atom if less well-defined N and C termini (A1, A20, B(-2) to B4 and B23 to B25) are excluded. The overall main-chain structure of bombyxin-II is similar to that of insulin. However, there are significant conformational and functional differences in their B-chain C-terminal parts. The B-chain C-terminal part of bombyxin-II adopts an extension of the B-chain central helix like that of relaxin and is not required for bombyxin activity, while the corresponding part of insulin adopts a sharp turn and a beta-strand and is essential for insulin activity. This structure demonstrates that bombyxin-II is more closely related to relaxin than to insulin, and suggests that insulin might have evolved the additional receptor-recognition site in the B-chain C-terminal beta-strand to distinguish itself from bombyxin and relaxin. The structure of bombyxin-II thus provides novel insights into the receptor recognition and divergent molecular evolution of insulin-superfamily peptides.

Amino Acid Sequence↗

PubMed1995

Identification of the receptor-recognition surface of bombyxin-II, an insulin-like peptide of the silkmoth Bombyx mori: critical importance of the B-chain central part.

Bombyxin-II, a brain-secretory peptide of the silkmoth Bombyx mori, shares 40% sequence identify and the characteristics core structure with human insulin. In spite of the structural similarity, no cross-activity is observed between them. To localize the active region of bombyxin-II, we have synthesized chimeric molecules of bombyxin-II and human insulin, and examined their bombyxin activity. Two chimeric molecules, which were sequentially identical except for the B-chain central part, showed significantly different potencies in bombyxin activity. Solution structure determination of these chimeric molecules revealed that their B-chain central parts took similar main-chain conformation, but formed dissimilar patches on their molecular surfaces. Therefore, the surface patch formed by the central part of the bombyxin-II B-chain is of critical importance for recognition of the bombyxin receptor. The above results, together with other data on the structure-activity relationships of bombyxin, indicate that the receptor-recognition surface of bombyxin-II includes the A-chain N and C, termini in addition to the B-chain central part. Though bombyxin-II, human insulin and human relaxin 2 use the common surface as their receptor-recognition sites, each of the surface patches is characterized by the variety of involved side-chains. Insulin and relaxin involve additional parts for receptor recognition, particularly the B-chain C-terminal part and the extended A-chain N-terminal helix, respectively. In conclusion, these ligands have evolved their own specific mechanisms for receptor recognition while retaining the major recognition surface.

Amino Acid Sequence↗

PubMed1995

Epidermal growth factor prevents oxygen-triggered apoptosis and induces sustained signalling in cultured rat cerebral cortical neurons.

Epidermal growth factor (EGF), a conventional mitogenic factor, acts as a neurotrophic factor on several types of neurons in the central nervous system. We found that EGF prevented the death of rat cerebral cortical neurons cultured in a 50% oxygen atmosphere. This high-oxygen-triggered cell death showed features of apoptotic cell death, which was blocked by inhibitors of RNA or protein synthesis. EGF prevented the oxygen-induced death of the cultured cortical neurons in a dose-dependent manner. Basic fibroblast growth factor (bFGF) also prevented this cell death, although there was no apparent additive effect of EGF and bFGF. Among the cultured cortical neurons, we observed neurons possessing the EGF receptor and cells expressing c-Fos protein in response to EGF. The cortical neurons were cultured in the presence of cytosine arabinoside, and the number of glial fibrillary acidic protein-positive astroglial cells was < 0.5% of that of the corresponding microtubule-associated protein 2-positive neurons. Therefore, the effect of EGF on the cultured cortical neurons is thought to be due to a direct action. We also examined EGF-induced signalling in the cultured cortical neurons. We found that EGF induced the sustained tyrosine phosphorylation of the EGF receptor and sustained the activation of mitogen-activated protein kinase in the cultured cortical neurons. We suggest that EGF may exert the survival effect through the prolonged activation of the EGF signalling.

Animals↗

PubMed1995

Involvement of free radicals in MPP+ neurotoxicity against rat dopaminergic neurons in culture.

To examine the mechanisms of the neurotoxicity of 1-methyl-4-phenylpyridinium (MPP+) against dopaminergic neurons, ventral mesencephalic cells from embryonic rats were cultured and exposed to MPP+ with various antioxidants or glutamate receptor antagonists to investigate the participation of free radicals and glutamate, respectively. Such antioxidants as vitamin E, vitamin C, coenzyme Q10, and catalase, but neither allopurinol nor superoxide dismutase, alleviated the MPP(+) -induced death of dopaminergic neurons, while glutamate receptor antagonists did not alter MPP+ neurotoxicity. These findings suggest the participation of free radicals, particularly hydroxyl radicals rather than superoxides, in the process of dopaminergic neuronal death evoked by MPP+.

1-Methyl-4-phenylpyridinium↗