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ABSTRACT: We investigated how the pea (Pisum sativum cv. Harunoka) root, upon return to an Al-free condition, recovers from injury caused by exposure to Al. The growing region
of the root during and after treatment with Al was examined by marking the root at intervals with India ink. Al-induced cell
death was detected by staining with Evans blue. Root growth in 40μM Al solution relative to that in Al-free solution (RRG) was approximately 45% from 6h to12h after the start of the treatment.
However, values of RRG from 12h to 24h in Al-free solution for recovery or in the same Al solution were about 75% and 35%,
respectively, indicating recovery from Al-induced growth inhibition. Images of the root characterized by zonal staining with
Evans blue were observed in the sub-apical region (more than 1mm from the tip) in Al-stressed roots. However, the interval
of the stained zone was widened in the root after recovery from Al-induced growth inhibition, though it was narrower and more
densely stained with time in the Al-stressed roots. During the recovery, the root apex may resume elongation in a specified
region without Al-induced death or injury in cells detected by Evans blue.
KeywordsAl stress-Inhibition-Injury-Pea-Recovery-Root apex
Plant and Soil 04/2012; 333(1):49-58. · 2.73 Impact Factor
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ABSTRACT: We studied the effects of natural essential oil on neurite outgrowth in PC12m3 neuronal cells to elucidate the mechanism underlying the action of the oils used in aromatherapy. Neurite outgrowth can be induced by nerve growth factor (NGF), where ERK and p38 MAPK among MAPK pathways play important roles in activating intracellular signal transduction. In this study, we investigated whether d-limonene, the major component of essential oils from oranges, can promote neurite outgrowth in PC12m3 cells, in which neurite outgrowth can be induced by various physical stimulations. We also examined by which pathways, the ERK, p38 MAPK or JNK pathway, d-limonene acts on PC12m3 cells. Our results showed that neurite outgrowth can be induced when the cells are treated with d-limonene. After treatment with d-limonene, we observed that p38 MAPK is strongly activated in PC12m3 cells, while ERK is weakly activated. In contrast, JNK shows little activity. A study using an inhibitor of p38 MAPK revealed that neurite outgrowth in PC12m3 cells is induced via the activation of p38 MAPK by d-limonene. The results thus indicate that d-limonene may promote neural cell differentiation mainly via activation of the p38 MAPK pathway.
Acta medica Okayama 04/2012; 66(2):111-8. · 0.84 Impact Factor
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ABSTRACT: We investigated how the pea (Pisum sativum cv. Harunoka) root, upon return to an Al-free condition, recovers from injury caused by exposure to Al. Elongation and re-elongation of the root during the recovery process from Al injury occurred only in the apical 5-mm region of the pea root. With the model system of the pea root for recovery from Al injury, images of the root characterized by zonal staining with Evans blue showed the existence of two regions in the root apex consisting of rupture and zonary stained regions. Ruptures enlarged by increase in their depth but without widening of the intervals among zonary stained regions in the roots treated with Al continuously. On the other hand, intervals of the zonary stained regions were widened due to re-elongation of the root and were narrow in the rupture region in the recovery root.
Plant signaling & behavior 01/2011; 6(1):98-100.
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ABSTRACT: Among the 3 mitogen-activated protein kinases--ERK, p38 MAPK and JNK--JNK has been suggested to participate in apoptosis, whereas p38 MAPK is thought to be part of the differentiation response. There are many common inducers of JNK and p38 MAPK, but the mechanisms underlying the differential response to apoptosis and differentiation are poorly understood. We found that heatshock activated p38 MAPK at 3 min after exposure to a temperature of 44 degrees C in stress-hypersensitive PC12m3 mutant cells, while it activated JNK at 20 min after the same heat treatment. However, heatshock activated p38 MAPK 5min after heat treatment and JNK 10 min after heat treatment in PC12 parental cells. The extent of phosphorylation of p38 MAPK induced by heat shock in PC12m3 cells was significantly greater than that in PC12 parental cells, and a high level of heat-shock-induced neurite outgrowth was observed only in PC12m3 cells. On the other hand, heat-shock-induced JNK activation appeared more quickly and apoptosis started earlier in PC12 parental cells. These findings indicate that short stress induces p38 MAPK and longer stress induces JNK, and that the response of these kinases to heat shock differs depending on cell type.
Acta medica Okayama 02/2010; 64(1):55-62. · 0.84 Impact Factor
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ABSTRACT: The aim of this study was to determine the optimal heat treatment conditions for enhancement of pressed silk-mediated 3D-like proliferation of normal human dermal fibroblasts, as well as to determine the responses to heat shock of cells and intracellular signaling pathways. The beginning of 3D-like pattern formation of cells was observed in the second week after the start of the experiment. The mean rates of beginning of 3D-like pattern formation by cells heat-treated at 40 masculineC and 43 masculineC for 10 min were significantly higher (3.2- and 8.6-fold, respectively) than that of untreated cells. We found that apoptosis had occurred in 7.5% and 50.0% of the cells at one week after heat treatment for 10 min at 43 masculineC and 45 masculineC, respectively. Western blot analysis demonstrated that phosphorylation of p38 MAPK and that of Hsp27 were markedly increased by heat treatment at 43 masculineC for 10 min. The results of an experiment using a p38 MAPK inhibitor and Hsp27 inhibitor suggest that activation of p38 MAPK by heat shock is associated with 3D-like cell proliferation and that Hsp27 contributes to the inhibition of apoptosis. The results of this study should be useful for further studies aimed at elucidation of the physiologic mechanisms underlying thermotherapy.
International Journal of Molecular Sciences 11/2009; 10(11):4963-76. · 2.60 Impact Factor
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Yoshio Kano,
Noboru Horie,
Shima Doi,
Fumika Aramaki,
Hidefumi Maeda,
Fukumi Hiragami,
Kenji Kawamura,
Hirotoshi Motoda,
Yoshihisa Koike,
Junichi Akiyama,
Sueo Eguchi,
Ken Hashimoto
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ABSTRACT: We investigated whether artepillin C, a major component of Brazilian propolis, acts as a neurotrophic-like factor in rat PC12m3 cells, in which nerve growth factor (NGF)-induced neurite outgrowth is impaired. When cultures of PC12m3 cells were treated with artepillin C at a concentration of 20 microM, the frequency of neurite outgrowth induced by artepillin C was approximately 7-fold greater than that induced by NGF alone. Artepillin C induced-neurite outgrowth of PC12m3 cells was inhibited by the ERK inhibitor U0126 and by the p38 MAPK inhibitor SB203580. Although artepillin C-induced p38 MAPK activity was detected in PC12m3 cells, phosphorylation of ERK induced by artepillin C was not observed. On the other hand, artepillin C caused rapid activation of ERK and the time course of the activation was similar to that induced by NGF treatment in PC12 parental cells. However, NGF-induced neurite outgrowth was inhibited by artepillin C treatment. Interestingly, inhibition of ERK by U0126 completely prevented artepillin C-induced p38 MAPK phosphorylation of PC12m3 cells. These findings suggest that artepillin C-induced activation of p38 MAPK through the ERK signaling pathway is responsible for the neurite outgrowth of PC12m3 cells.
Neurochemical Research 04/2008; 33(9):1795-803. · 2.24 Impact Factor
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ABSTRACT: The increasing use of mobile phone communication has raised concerns about possible health hazard effects of microwave irradiation. We investigated damage and differentiation caused by microwave irradiation on drug-hypersensitive PC12 cell line (PC12m3). These cells showed enhancement of neurite outgrowth to various stimulants. The frequency of neurite outgrowth induced by 2.45 GHz (200 W) of microwave irradiation was approximately 10-fold greater than that of non-irradiated control cells. Incubation of PC12m3 cells with SB203580, a specific inhibitor of p38 MAPK, resulted in marked inhibition of the microwave radiation-induced neurite outgrowth. Also, activation of the transcription factor CREB induced by microwave irradiation was inhibited by SB203580. Heat shock treatment at 45 degrees C had a strong toxic effect on PC12m3 cells, whereas microwave treatment had no toxic effect on PC12m3 cells. These findings indicate that p38 MAPK is responsible for the survival of PC12m3 cells and might induce neurite outgrowth via a CREB signaling pathway when subjected to microwave irradiation.
Neuroscience Letters 03/2008; 432(1):35-9. · 2.11 Impact Factor
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ABSTRACT: The wheat ALMT1 gene encodes an aluminum (Al)-activated malate transport protein which confers Al-resistance. We investigated the membrane topology of this plasma-membrane localized protein with immunocytochemical techniques. Several green fluorescent protein (GFP)-fused and histidine (His)-tagged chimeras of ALMT1 were prepared based on a computer-predicted secondary structure and transiently expressed in cultured mammalian cells. Antibodies raised to polypeptide epitopes of ALMT1 were used in conjunction with the antibody to the His-tags to determine the topology of ALMT1. This study shows that the ALMT1 protein contains six transmembrane domains with the amino and carboxyl termini located on the extracellular side of the plasma membrane.
Plant signaling & behavior 12/2007; 2(6):467-72.
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ABSTRACT: The aim of the present study was to determine both the minimal and the optimal conditions under which heat treatment is effective for enhancing 3D (three-dimensional)-like cell proliferation. C3H10T1/2 mouse fibroblasts were cultured with hydroxyapatite granules for 10 weeks after heat treatment at 40, 41.5, 43, 44 or 45 degrees C for 2, 10, 15, 20, 30, 45, 60, 90, 180 and 360 min. Then minimal and optimal conditions of temperature and duration of heat treatment for induction of 3D-like proliferation of cells were determined. The minimal conditions of heat treatment to induce 3D-like proliferation were 43 degrees C for 2 min and the optimal conditions were 43 degrees C for 10 min. The mean rates of formation of 3D-like proliferation patterns by cells heat-treated at 43 degrees C for 2 and 10 min were significantly higher (1.7- and 3.7-fold respectively) than that by untreated cells (P<0.05). We also observed significantly greater effects of heat treatment on 3D-like proliferation at 40 degrees C for 90 or 180 min and at 41.5 degrees C for 15 min and 44 degrees C for 10 min. We found that apoptosis had occurred in 7.5 and 87.0% of the cells at 1 week after heat treatment at 43 degrees C for 10 min and 30 min respectively. Western-blot analysis demonstrated that phosphorylation of p38 MAPK (mitogen-activated protein kinase) was markedly increased by heat treatment at 43 degrees C for 10 min. These findings suggest that activation of p38 MAPK by heat shock is associated with 3D-like cell proliferation. The results of the present study should be useful for further studies aimed at elucidation of the physiological mechanisms underlying thermotherapy and hyperthermia.
Biotechnology and Applied Biochemistry 05/2007; 47(Pt 1):49-57. · 1.53 Impact Factor
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ABSTRACT: The periodontal ligament (PDL) is a highly specialized tissue connecting the cementum with the tooth socket bone and affects the life span of the tooth. However, little is known about the precise characteristics and regenerative mechanism of PDL cells because of the absence of specific markers and cell lines. Therefore, we aimed to establish three immortalized human PDL fibroblast cell lines by using simian virus40 T-antigen (SV40T-Ag) and human telomerase reverse transcriptase (hTERT) transfection, expecting these cells to have the characteristics of primary cells. The transfected cells were named STPLF. The expression of SV40T-Ag and hTERT in all STPLF lines was verified by using the semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method, stretch PCR analysis, or Western blotting analysis. All STPLF showed stable proliferation at more than 120 population doublings (PD), whereas primary human PDL fibroblasts (HPLF) stopped at 10-20 PD. Characterization by RT-PCR analysis revealed that all STPLF genes mimicked the expression of their respective original HPLF genes. STPLF expressed runt-related transcription factor-2, osterix, alkaline phosphatase, osteopontin, osteocalcin, periostin, receptor activator of NF-kappa B ligand, osteoprotegerin, epidermal growth factor receptor, alpha-smooth muscle actin, and type XII collagen. STPLF stimulated with 50 micro g/ml ascorbic acid and 2 mM beta-glycerophosphate for 4 weeks produced more calcified deposits than did HPLF cultured with the same reagents. These results suggest that each STPLF line retained the characteristics of the respective original HPLF, that STPLF gained increased calcification activity, and that STPLF are helpful tools for studying the biology and regenerative mechanisms of human PDL.
Cell and Tissue Research 05/2006; 324(1):117-25. · 3.11 Impact Factor
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ABSTRACT: Regulation of the biocompatibility of compositional hydroxyapatite (HA) with cells is affected by various environmental factors. The aim of this study was to determine whether the p38 mitogen-activated protein kinase (MAPK) pathway has a key role in enhancement of HA-mediated three-dimensional (3D)-like proliferation of mouse fibroblasts after heat treatment. C3H10T1/2 mouse fibroblasts were cultured with HA granules for 10 weeks after heat treatment at 44 degrees C for 5, 10, 20, and 30 min. The mean rate of formation of 3D-like proliferation patterns by cells heat treated for 20 min was only 2.1-fold higher than that by untreated cells, but the mean rates of formation of 3D-like proliferation patterns by cells heat treated for 5 and 10 min were significantly higher (3.7- and 3.3-fold higher, respectively) than that by untreated cells (p < 0.01). Western blot analysis demonstrated that phosphorylation of p38 MAPK was markedly increased by heat treatment at 44 degrees C for 5 and 10 min. In addition, the activation of heat shock-induced p38 MAPK was markedly reduced by treatment at 44 degrees C for 30 min. We concluded that 3D-like proliferation of heat-treated cells was induced by activation of p38 MAPK. The results of this study should be useful for further studies aimed at elucidation of regulation of the biocompatibility of compositional HA with cells.
Journal of Biomedical Materials Research Part A 09/2005; 74(4):705-11. · 2.63 Impact Factor
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ABSTRACT: We investigated the role of the p38 mitogen-activated protein kinase (MAPK) pathway in heat-shock-induced neurite outgrowth of PC12 mutant cells in which nerve growth factor (NGF)-induced neurite outgrowth is impaired. When cultures of the PC12 mutant (PC12m3) cells were exposed to heat stress at 44 degrees C for 10 min, activity of p38 MAPK increased and neurite outgrowth was greatly enhanced. The neurite extension was inhibited by the p38 MAPK inhibitor BS203580. Longer heat treatment of PC12m3 cells provoked cell death, which was enhanced by SB203580. These findings suggest that heat-induced activation of p38 MAPK is responsible for the neurite outgrowth and survival of PC12m3 cells.
Brain Research 12/2004; 1026(2):302-6. · 2.73 Impact Factor
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ABSTRACT: Various undifferentiated embryonic stem (ES) cells can grow on mouse embryonic fibroblast (MEF) feeders. However, the risk of zoonosis from animal feeders to human ES cells generally excludes the clinical use of these human ES cells. We have found that human placenta is a useful source of feeder cells for the undifferentiated growth of primate ES cells. As on MEF feeders, primate ES cells cultured on human amniotic epithelial (HAE) feeder cells and human chorionic plate (HCP) cells had undifferentiated growth. The cultured primate ES cells expressed Oct-4, alkaline phosphatase, and SSEA-4. The primate ES cells on HAE feeder cells produced typical immature teratomas in vivo after injection into severe combined immunodeficient mice. Human placenta is quite novel and important because it would provide a relatively available source of feeders for the growth of human ES cells for therapeutic purposes that are also free of ethical complications.
Stem Cells 02/2004; 22(4):433-40. · 7.78 Impact Factor
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ABSTRACT: Hydroxyapatite (HAP) ceramics are widely used as implant materials for periodontal bone defects because of their excellent biocompatibility. We demonstrated that physical stimulation, that is, (1). mechanical stimuli or (2). laser irradiation, causes HAP-mediated C3H10T1/2 mouse fibroblasts to form three-dimensional tissue-like structures. Trypsinized 10T1/2 cells were cultured simultaneously with 200 HAP granules on a rotator for 7 days in mechanical stimulation experiments. The cells were later transferred to a regular incubator. Cell reactions were observed by phase-contrast microscopy. The formation of three-dimensional structures around the HAP granules was observed in the third week of cultivation after stimulation. In laser irradiation experiments, trypsinized cells were irradiated with 1, 5, and 16 J/cm(2) at a wavelength of 1000 nm and cultured with 200 HAP granules for 10 weeks. The formation of three-dimensional structures, like those observed in the mechanical stimulation experiments, was observed in the third week after irradiation. The formation of these structures was most frequent at 1 J/cm(2), and the frequency of formation of these structures gradually decreased as the irradiation dose was increased. These results indicate that physical stimuli may stimulate cell proliferation, leading to the repair of damaged tissue. These results also indicate that mouse fibroblasts do not form these three-dimensional structures without HAP and that HAP alone is not sufficient to stimulate the formation of three-dimensional structures.
Tissue Engineering 05/2003; 9(2):357-64. · 4.02 Impact Factor
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ABSTRACT: We obtained a drug-hypersensitive PC12 mutant cell (PC12m3), in which neurite outgrowth was strongly stimulated by various drugs such as FK506, calcimycin and cAMP, under the condition of NGF treatment. The frequency of neurite outgrowth stimulated by FK506 was approximately 40 times greater than by NGF alone. The effects of FK506 on neurite outgrowth in PC12m3 cells were inhibited by rapamycin, an FK506 antagonist, and by calcimycin, a calcium ionophore. PC12m3 cells had a strong NGF-induced MAP kinase activity, the same as PC12 parental cells. However, FK506-induced MAP kinase activity was detected only in PC12m3 cells. The activation of MAP kinase by FK506 in PC12m3 cells was markedly inhibited by rapamicin and calcimycin. FK506-induced MAP kinase activity was also inhibited by MAP kinase inhibitor U0126. These results demonstrate that drug-hypersensitive PC12m3 cells have a novel FK506-induced MAP kinase pathway for neuritogenesis.
Neurochemical Research 01/2003; 27(12):1655-61. · 2.24 Impact Factor
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ABSTRACT: During the continuous culturing of neural PC12 cells, a drug hypersensitive PC12 mutant cell line (PC12m3) was obtained, which demonstrated high neurite outgrowth when stimulated by various drugs. When the immunosuppressant drug FK506 and nerve growth factor (NGF) were introduced to the PC12m3 cells, the frequency of neurite outgrowth increased approximately 40-fold for NGF alone. However, the effect of FK506 on neuritogenesis in PC12 parental and drug insensitive PC12m1 mutant cells was much lower than in PC12m3 cells. The sustained activation of mitogen-activated protein (MAP) kinase plays an important role in neurite outgrowth of PC12 cells. Interestingly, the drug hypersensitive PC12m3 cells exhibited the sustained activation of MAP kinase with FK506 in comparison to low or no activities in PC12 parental or drug insensitive PC12m1 cells. These results indicate that PC12m3 cells have a novel FK506-induced MAP kinase pathway for neuritogenesis.
Cell Structure and Function 11/2002; 27(5):393-8. · 2.29 Impact Factor
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ABSTRACT: During continuous culture of neural PC12 cells, we obtained a drug-hypersensitive PC12 mutant cell that showed high stimulation of neurite outgrowth by various drugs. When several Chinese medicines such as shu-jing-huo-xie-tang and Wu-Ling-San were provided to these PC12 mutant cells, the frequency of nerve growth factor (NGF)-induced neurite outgrowth increased approximately 30-fold compared to NGF alone. Neurite outgrowth induced by NGF in PC12 cells is accompanied by sustained activation of mitogen-activated protein kinase (MAPK); however, these Chinese medicines did not induce MAPK activity. The findings thus indicate that certain Chinese medicines may induce neurite outgrowth by a novel mechanism which is distinct from the NGF-activated pathway in PC12 mutant cells.
The American Journal of Chinese Medicine 02/2002; 30(2-3):287-95. · 1.98 Impact Factor
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祐子 木俣,
良男 加納,
好久 小池,
景三 沼田,
清美 立山,
久男 大西,
泰男 内藤,
雅子 加藤,
Yuko KIMATA, Yoshio Kano,
Yoshihisa Koike,
Keizo NUMATA,
Kiyomi TATEYAMA,
Hisao OHNISHI,
Yasuo NAITO,
Masako KATO
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ABSTRACT: The effects of sensory stimulation on the body have been reported by many. Gross vibration and equilibrium stimulation, such as swinging in a hammock, and finer stimulation that is applied with a vibrator, are both used in sensory-integration therapy. The sonic stimulation in music therapy uses low frequency vibration. Fifty hertz was reported to give the best results in physiological changes. The purpose of this study was to address the effects of vibration on mutant cells (PC12m3) neurite growth. The vibration was produced by a low frequency oscillator. Frequency, intensity and duration of the vibration were examined. Results showed that the neurite outgrowth rate increased significantly with vibration. The growth was high between 10-150 Hz., and peaked at 40 Hz with an oscillator intensity level over 5 and duration longer than 15 minutes. Since the stimulation in sensory-integration therapy is processed through the sensory nervous system, it may not be appropriate to discuss the vibratory stimulation used in sensory-integration therapy and the vibratory stimulation used in this study in the same context. However, the most effective frequency level obtained in this study corresponded with that of music therapy. Our highly developed and complex nervous system is built on cells, and the response of the nervous system to vibration may be based on the cell level response.
