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H Hatanaka

Publications and source records attributed to H Hatanaka.

At least 109 records · Page 6Linked to original sources
PubMed1998

[Neuroprotection by neurotrophic factors in apoptosis].

During development, excess neurons are produced about half of which die. The time of cell death (apoptosis) is limited to the period of formation of synapses with the target cells, and the neurons which fail to obtain sufficient amounts of trophic factor(s) released from the target cells are eliminated. This selection system is considered to be a mechanism to ensure formation of a physiologically relevant neuronal network. Mature neurons which correctly execute their functions, however, undergo apoptosis in response to exogenous toxic stimuli. Such stimuli may be responsible for neurodegenerative diseases. The mechanism underlying cell death has been analyzed using in vitro model systems. In the present communication, we used cultured rat cerebellar granule neurons, in which low potassium concentration (LK+) in the medium induces apoptosis, and this apoptosis is prevented by high concentration of potassium (HK+), BDNF. One of the lipid-modifying kinases, phosphatidylinositol 3-kinase (PI3-K), is also activated by trophic factors including neurotrophins. BDNF and high K+ prevented low K(+)-induced apoptosis via PI3-K. BDNF also promotes the survival of basal forebrain cholinergic neurons cultured from postnatal 2-week-old (P2w) rats. The mechanism of neuronal apoptosis induced by oxidative stress using CNS neurons and PC12 cells was investigated, and we found that generation of reactive oxygen species (ROS) is highly associated with apoptosis. High oxygen induced neuronal apoptosis, which was blocked by protein or RNA synthesis inhibitors. Neurotrophic factors and Bcl-2 prevented this apoptotic cell death. Exposure to hydrogen peroxide, lipid hydroperoxide or serum deprivation triggered apoptosis associated with increased generation of ROS as determined using a ROS-specific fluorescent probe. In cultured cerebellar granule neurons from 15-day-old wild-type and p53-deficient mice, we examine the role of p53 in regulating the life and death of CNS neurons. When exposure of gamma-ray or bleomycin to neurons died in p53 dependent manner. These neuronal deaths were partially prevented by actinomycin D or cycloheximide. The pycnotic nuclei observed in these dying neurons indicated that cell death occurs via apoptosis. Although there are many evidences that p53 is involved in apoptosis in proliferating cells, it is interesting that p53 is also involved in apoptosis in postmitotic neurons as shown in this study.

Animals↗
PubMed1998

Increased expression of human histocompatibility leukocyte antigen-G in colorectal cancer cells.

Human histocompatibility leukocyte antigen (HLA)-G is a nonclassical major histocompatibility complex class I molecule. HLA-G is known to provide tolerance from recognition by natural killer cells. We studied HLA-G expression in 39 human colorectal cancers and 23 extra-neoplastic colon tissue samples by RT-PCR. The expression of HLA-G mRNA was significantly more frequent in colorectal cancer (34 of 39 cases) than in the extraneoplastic tissue (10 of 23 specimens; chi2 test, p = 0.0003). HLA-G expression was also confirmed on the cancer cells immunohistochemically. These results suggested that HLA-G on colorectal cancer cells may be correlated with escape from immunological surveillance during colon cancer development.

Colon↗
PubMed1998

Expression of cell-associated isoform of vascular endothelial growth factor 189 and its prognostic relevance in non-small cell lung cancer.

Vascular endothelial growth factor (VEGF) has four isoforms (VEGF121, VEGF165, VEGF189, VEGF206). We examined the expression patterns of VEGF isoforms in non-small cell lung cancer (NSCLC) by RT-PCR. The cell-associated isoform VEGF189 was more frequently expressed in NSCLC (90.5%) than in extraneoplastic lung tissue (57.6%, p=0.00004). Immunohistochemical examination confirmed VEGF protein expression in 33 of 76 NSCLC expressing VEGF189 (V189p+ group). The V189p+ group showed significantly poorer prognosis than those without VEGF189 mRNA and protein (V189p- group, p=0.01722). These results suggest that expression of the cell-associated isoform VEGF189 is closely associated with progression of NSCLC.

Carcinoma, Non-Small-Cell Lung↗
PubMed1998

Brain-specific angiogenesis inhibitor 1 expression is inversely correlated with vascularity and distant metastasis of colorectal cancer.

Brain-specific angiogenesis inhibitor (BAI) 1 was recently isolated as a novel p53 inducible gene. BAI1 has been suggested to play a significant role in angiostasis. We studied the expression of BAI1 in 49 colorectal cancer specimens by RT-PCR. BAI1 expression was significantly reduced in colorectal cancers as compared to the extraneoplastic tissues (X(2) test, p=0.041). BAI1 expression was inversely correlated with vascular invasion and metastasis (Fisher's exact test, p 0.045). Moreover, vascularity in the colorectal cancer was inversely correlated with BAI1 gene expression (Mann-Whitney U-test, p=0.0003). These observations suggested that BAI1 expression might inhibit angiogenesis and metastasis of colorectal cancer.

Angiogenesis Inhibitors↗
PubMed1998

[Apoptosis-inducing and -preventing signal transduction pathways in cultured cerebellar granule neurons].

1. Cultured cerebellar granule neurons maintained in medium containing 26 mM potassium (high K+ or HK+) undergo cell death when switched to medium with 5 mM potassium (low K+ or LK+). This low K(+)-induced cell death has typical features of apoptosis. The intracellular signaling pathway of low K(+)-induced apoptosis has been investigated. 2. Cerebellar granule neurons become committed to undergo apoptosis between 2 and 5 h after K+ deprivation, judging from the inability of high K+ to rescue them after this time. Although the levels of most mRNAs decrease markedly concomitant with commitment, expression of c-jun mRNA increases 2-3 h after K+ deprivation. Among the family of caspases, a caspase-3-like protease is activated within 4 h of lowering the K+ concentration. A caspase-1-like protease is also activated within 2 h of K+ deprivation. 3. Inhibition of phosphatidylinositol 3-kinase (PI3-K) activity by LY294002 or wortmannin also induces apoptosis in cerebellar granule neurons. The intracellular signaling pathway of LY294002-induced apoptosis has been investigated. The activity of c-Jun N-terminal kinase (JNK) increases 8 h after addition of LY294002 to high K+ medium or low K+ medium containing BDNF. Expression of c-Jun protein also increases almost simultaneously. 4. The low K(+)-induced apoptosis of cerebellar granule neurons is prevented by high K+ (membrane depolarization by high K+), BDNF, IGF-1, bFGF or cAMP. The intracellular signaling pathways by which these agents prevent low K(+)-induced apoptosis have been investigated. Agents other than cAMP prevent apoptosis through PI3-K and a Ser/Thr kinase, Akt/PKB. The survival-promoting effect of cAMP does not depend on the PI3-K-Akt pathway.

Apoptosis↗
PubMed1998

Thrombospondin 2 gene expression is correlated with decreased vascularity in non-small cell lung cancer.

Stromal vascularity is thought to be a major factor involved in the progression of carcinoma. However, the crucial mechanisms of vascularization in the stroma are not well understood. Vascularity could be regulated by various cytokines produced by neoplastic or stromal cells in carcinoma. Thrombospondin (TSP) has an inhibitory role against vascularization in vitro, although the biological significance of TSP has not been characterized in vivo. We examined expression of TSP1 and TSP2 genes in 78 non-small cell lung cancers (NSCLCs) and 33 extraneoplastic lung tissue samples by reverse transcription-PCR. TSP1 expression was detected in 66.7% (52 of 78) of NSCLCs and in 69.7% (23 of 33) of extraneoplastic lung tissue specimens. TSP2 expression was seen in 48.7% (38 of 78) of NSCLCs, whereas 72.7% (24 of 33) of extraneoplastic lung tissue samples showed TSP2 gene expression. TSP2 expression was significantly decreased in NSCLC as compared with extraneoplastic lung tissue (chi2 test, P=0.019). Vascularity in the NSCLC was inversely correlated with TSP2 gene expression (Mann-Whitney U test, P=0.009). Patients with adenocarcinoma positive for TSP2 gene expression (22 of 49) showed significantly better prognosis than those without TSP2 (27 of 49; Cox-Mantel test, P=0.034). TSP1 expression showed no apparent correlation with these factors. These results suggested that TSP2 had an inhibitory role against vascularization and progression of NSCLC.

Carcinoma, Non-Small-Cell Lung↗
PubMed1997

BDNF potentiates spontaneous Ca2+ oscillations in cultured hippocampal neurons.

Brain-derived neurotrophic factor (BDNF) is thought to regulate neuronal plasticity in developing and matured neurons, although the molecular mechanisms are less well characterized. We monitored changes in the intracellular calcium (Ca2+) levels induced by BDNF using a fluorescence Ca2+ indicator (Fluo-3) by means of confocal laser microscopy in rat cultured hippocampal neurons. BDNF acutely potentiated spontaneous Ca2+ oscillations in dendrites and also in the soma of several neurons, although it increased intracellular Ca2+ in only selective proportion of resting neurons without Ca2+ oscillations. The potentiation was observed both in the frequency and the amplitude of Ca2+ oscillations, completely blocked by K-252a, and significantly reduced by 2-aminophosphonovaleric acid. These findings suggest that BDNF increases glutamate release and N-methyl-D-aspartate (NMDA) channel-gated Ca2+ influx via TrkB and regulates the frequency and the amplitude of Ca2+ oscillations. BDNF may have the potential to modulate spontaneous Ca2+ oscillations to regulate neuronal plasticity in developing hippocampal neurons.

2-Amino-5-phosphonovalerate↗
PubMed1997

Solution structure of midkine, a new heparin-binding growth factor.

Midkine (MK) is a 13 kDa heparin-binding polypeptide which enhances neurite outgrowth, neuronal cell survival and plasminogen activator activity. MK is structurally divided into two domains, and most of the biological activities are located on the C-terminal domain. The solution structures of the two domains were determined by NMR. Both domains consist of three antiparallel beta-strands, but the C-terminal domain has a long flexible hairpin loop where a heparin-binding consensus sequence is located. Basic residues on the beta-sheet of the C-terminal domain form another heparin-binding site. Measurement of NMR signals in the presence of a heparin oligosaccharides verified that multiple amino acids in the two sites participated in heparin binding. The MK dimer has been shown to be the active form, giving signals to endothelial cells and probably to neuronal cells. We present a head-to-head dimer model of MK. The model was supported by the results of cross-linking experiments using transglutaminase. The dimer has a fused heparin-binding site at the dimer interface of the C-terminal domain, and the heparin-binding sites on MK fit the sulfate group clusters on heparin. These features are consistent with the proposed stronger heparin-binding activity and biological activity of the dimer.

Amino Acid Sequence↗
PubMed1997

Insulin receptor substrate (IRS)-1 and IRS-2 are tyrosine-phosphorylated and associated with phosphatidylinositol 3-kinase in response to brain-derived neurotrophic factor in cultured cerebral cortical neurons.

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophins, promotes differentiation and survival of various types of neurons in the central nervous system. BDNF binds to and activates the tyrosine kinase receptor, TrkB, initiating intracellular signaling and exerting its effects. Phosphatidylinositol 3-kinase (PI3-K), which has been implicated in promotion of neuronal survival by neurotrophic factors, is a component in the signaling pathway of BDNF. We examined how BDNF activates PI3-K in cultured cerebral cortical neurons. We found that insulin receptor substrate (IRS)-1 and -2 are involved in the BDNF signaling pathway that activates PI3-K. IRS-1 and -2 were tyrosine-phosphorylated and bound to PI3-K in response to BDNF. This BDNF-stimulated signaling via IRS-1 and -2 was inhibited by K-252a, an inhibitor of Trk tyrosine kinase. In addition, signaling via IRS-1 and -2 was markedly sustained as well as the BDNF-induced tyrosine phosphorylation of TrkB. On the other hand, we observed no association of PI3-K with TrkB in response to BDNF. These results indicate that the activation of TrkB by BDNF induces the activation of PI3-K via IRS-1 and -2 rather than by a direct interaction of TrkB with PI3-K in cultured cortical neurons.

Animals↗
PubMed1997

Changes in mitochondrial membrane potential during oxidative stress-induced apoptosis in PC12 cells.

We examined the effects of various types of oxidative stress on cell survival and on mitochondrial membrane potential (delta psi m) in PC12 cells transfected with BCL-2. Several types of oxidative stress such as exposure to hydrogen peroxide, 13-L-hydroperoxylinoleic acid, and xanthine + xanthine oxidase triggered apoptotic nuclear condensation and DNA fragmentation in normal PC12 cells. These types of oxidative stress induced significant increases in level of reactive oxygen species (ROS) before cell death. By contrast, BCL-2 prevented the apoptosis induced by these oxidative stresses. However, BCL-2 did not reduce ROS levels, indicating that it functions downstream of ROS generation. We measured delta psi m as a potential target of ROS during oxidative stress-induced cell death. Hydrogen peroxide, 13-L-hydroperoxylinoleic acid, and xanthine + xanthine oxidase induced a significant loss of delta psi m simultaneously with cell death. BCL-2 prevented the decrease in delta psi m as well as apoptosis induced by oxidative stress. These observations suggest that the oxidative stress triggers apoptosis associated with both increased generation of ROS and decreases in level of delta psi m and that BCL-2 prevents cell death as well as delta psi m but not ROS production.

Animals↗
PubMed1997

Adenovirus-mediated transduction with human glial cell line-derived neurotrophic factor gene prevents 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopamine depletion in striatum of mouse brain.

As a novel trial of neuroprotective therapy of neurodegenerative diseases, we have constructed a recombinant adenovirus vector (rAdv) bearing a neurotrophic factor gene to deliver the factor to rescue neurons in vivo. In the present study, human glial cell line-derived neurotrophic factor (hGDNF) was chosen to examine the applicability of our strategy to a mouse model of Parkinson's disease. During the construction of the rAdv, we found that the strong constitutive hGDNF expression unit somehow inhibited the appearance of the rAdv. Therefore we adopted a self-contained tetracycline-regulated expression system to acquire an rAdv expressing hGDNF. By analyzing the condition medium of SH-SY5Y cells infected with our constructed virus vector, we confirmed that biologically active GDNF was successfully expressed in vitro. For an animal study, we delivered this virus vector directly to the C57 black mouse brain and then exposed the animal to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to injure the nigrostriatal dopaminergic neurons. One week after the MPTP exposure, the neuroprotective effect of the virus vector was estimated by measurement of the dopamine content in the striatum of the mouse brain. The mice that had received our constructed virus had significantly higher dopamine levels in their striatum, demonstrating that our rAdv expressing hGDNF has therapeutic potential to protect the nigrostriatal dopaminergic neurons in vivo.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1997

Brain-derived neurotrophic factor enhances long-term potentiation in rat visual cortex.

Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), members of the nerve growth factor (NGF) gene family, have been suggested to play a role in experience-dependent modification of neural networks in the developing nervous system. In this study we addressed the question of whether these neurotrophins are involved in long-term potentiation (LTP) in developing visual cortex. We recorded layer II/III field potentials and whole-cell currents evoked by test stimulation of layer IV at 0.1 Hz in visual cortical slices prepared from young rats (postnatal day 15-25) and observed effects of BDNF, NT-3, and NGF on these responses. Then we analyzed the effects of these neurotrophins on LTP induced by tetanic (Theta-burst type) stimulation of layer IV. We found that BDNF at 200 ng/ml potentiated field potentials and EPSCs in most cases and that this potentiation lasted after cessation of the BDNF application. At the concentration of 20 ng/ml, BDNF did not show such an effect, but it enhanced the magnitude of expressed LTP. On the other hand, NT-3 and NGF had none of these effects. Immunohistochemical staining of slices with antibody against BDNF showed that exogenous BDNF penetrated into the whole slice within approximately 5 min of its application. The actions of BDNF were blocked by preincubation of slices with TrkB-IgG fusion protein, a BDNF scavenger, or coapplication of K252a, an inhibitor for receptor tyrosine kinases. TrkB-IgG or K252a itself completely blocked LTP, suggesting that endogenous BDNF or another TrkB ligand plays a role in LTP in the developing visual cortex.

Action Potentials↗
PubMed1997

Involvement of phosphatidylinositol-3 kinase in prevention of low K(+)-induced apoptosis of cerebellar granule neurons.

Cerebellar granule neurons obtained from 9-day-old rats die in an apoptotic manner when cultured in serum-free medium containing a low concentration of potassium (5 mM). A high concentration of potassium (26 mM) in the culture medium and BDNF can effectively prevent this apoptosis. The survival effects of high potassium and BDNF were additive, and the effect of high potassium was not blocked by addition of anti-BDNF antibody. These observations indicated that these survival effects were independent. To examine which molecules are involved in the survival pathway induced by BDNF or high K+, we used wortmannin, a specific inhibitor of PI-3 kinase. Wortmannin blocked the survival effects of both BDNF and high K+ on cerebellar granule neurons. Furthermore, in vitro PI-3 kinase assay showed that treatment with BDNF or high K+ induced PI-3 kinase activity, which was diminished by addition of wortmannin. These results indicate that different survival-promoting agents, BDNF and high K+, can prevent apoptosis in cerebellar granule neurons via a common enzyme, PI-3 kinase.

Androstadienes↗
PubMed1997

Solution structure of ferredoxin from the thermophilic cyanobacterium Synechococcus elongatus and its thermostability.

The three-dimensional structure of ferredoxin, purified from the thermophilic cyanobacterium Synechococcus elongatus, was determined in aqueous solution by two-dimensional proton nuclear magnetic resonance. In addition to the 946 distance constraints from nuclear Overhauser effect connectivities, we added 241 distance constraints derived from the crystal structure of Spirulina platensis ferredoxin to the 19 residues close to the [2Fe-2S] iron-sulfur center, where crosspeaks disappeared due to paramagnetic effects. The atomic root-mean-square difference of the ten converged structures from the mean structure was 0.61(+/-0.12) A for backbone atoms (N, C(alpha), C'). The main-chain structure was almost the same as the crystal structures of other mesophile ferredoxins, but comparison of the side-chain structures revealed an extension of the hydrophobic core, a unique hydrophobic patch on the surface of the large beta-sheet, and two unique charge networks in this thermostable ferredoxin structure, some of which might contribute to thermostability.

Amino Acid Sequence↗
PubMed1997

NGF and BDNF increase the immunoreactivity of vesicular acetylcholine transporter in cultured neurons from the embryonic rat septum.

The expression of vesicular acetylcholine transporter (VAChT), which transports ACh into synaptic vesicles, is coregulated with choline acetyltransferase (ChAT). Therefore, the effects of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) on the levels of VAChT in cultured neurons from the septum of embryonic rats were investigated by immunocytochemistry. NGF and BDNF increased the number of VAChT-immunoreactive neurons by approximately 1.5-fold and enhanced the immunoreactivity in each positive cell. These results suggest that the neurotrophins enhance not only synthesis but also storage of ACh in septal neurons.

Acetylcholine↗
PubMed1997

Generation of free radicals during lipid hydroperoxide-triggered apoptosis in PC12h cells.

The compound 13-L-hydroperoxylinoleic acid (LOOH) triggered the death of clonal rat pheochromocytoma PC12h cells (LD50 = about 8 microM). LOOH induced nuclear condensation and DNA fragmentation, which was prevented by cycloheximide (a protein synthesis inhibitor) and NGF, indicating that LOOH triggered apoptosis in PC12h cells. LOOH produced reactive oxygen species (ROS) in PC12h cells in a time- and dose-dependent manner, as measured by flow cytometry using the ROS-specific fluorescent indicator, 6-carboxy-2,7-dichorodihydrofluorescein diacetate, di(acetoxymethyl ester) (C-DCDHF-DA). Antioxidants such as N,N'-diphenyl-p-phenylenediamine (DPPD), vitamin E and N-acetylcysteine, and a ferric iron chelator, deferoxamine, inhibited the LOOH-triggered apoptosis and simultaneously decreased the generation of ROS, whereas an inhibitor of glutathione synthesis, buthionine sulfoximine (BSO), enhanced the apoptosis and increased the generation of ROS. These results indicate that LOOH triggers the apoptosis of PC12h cells by increasing the production of ROS. A confocal analysis with the Ca(2+)-specific fluorescent indicator, fluo-3, demonstrated that LOOH at concentrations up to 200 microM, did not increase the intracellular Ca2+ concentration. These data indicate that LOOH induces apoptosis of PC12h cells through the enhanced production of ROS, not through increasing the permeability of Ca2+.

Animals↗