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ABSTRACT: PIP2;1 is an integral membrane protein that facilitates water transport across plasma membranes. To address the dynamics of Arabidopsis thaliana PIP2;1 at the single-molecule level as well as their role in PIP2;1 regulation, we tracked green fluorescent protein-PIP2;1 molecules by variable-angle evanescent wave microscopy and fluorescence correlation spectroscopy (FCS). Single-particle tracking analysis revealed that PIP2;1 presented four diffusion modes with large dispersion of diffusion coefficients, suggesting that partitioning and dynamics of PIP2;1 are heterogeneous and, more importantly, that PIP2;1 can move into or out of membrane microdomains. In response to salt stress, the diffusion coefficients and percentage of restricted diffusion increased, implying that PIP2;1 internalization was enhanced. This was further supported by the decrease in PIP2;1 density on plasma membranes by FCS. We additionally demonstrated that PIP2;1 internalization involves a combination of two pathways: a tyrphostin A23-sensitive clathrin-dependent pathway and a methyl-β-cyclodextrin-sensitive, membrane raft-associated pathway. The latter was efficiently stimulated under NaCl conditions. Taken together, our findings demonstrate that PIP2;1 molecules are heterogeneously distributed on the plasma membrane and that clathrin and membrane raft pathways cooperate to mediate the subcellular trafficking of PIP2;1, suggesting that the dynamic partitioning and recycling pathways might be involved in the multiple modes of regulating water permeability.
The Plant Cell 10/2011; 23(10):3780-97. · 8.99 Impact Factor
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ABSTRACT: The intermediate filament nestin is transiently expressed in neural stem/progenitor cells during the development of central nervous system. Recently, increasing evidence has shown that upregulation of nestin is related to malignancy of several cancers, especially glioblastoma. However, the function of nestin in carcinogenesis remains unclear. In this study, we investigated the role of nestin in glioblastoma carcinogenesis by comparing subclones of rat C6 glioblastoma cells that were either high or low for nestin expression. We found that while nestin expression did not influence the in vitro proliferation of glioblastoma cells, subclones characterized by high levels of nestin formed tumors in vivo at significantly faster rates than subclones with low expression. Importantly, C6 subclones that expressed nestin at low levels in vitro were also found to give rise to tumors highly positive for the protein, suggesting that induction of nestin plays an important role in glioblastoma carcinogenesis. Derivation of nestin positive tumors from nestin negative human U87 glioblastoma cells in immunodeficient mice further confirmed that a switch to positive expression of nestin is fundamental to the course of glioblastoma development. Blocking the expression of nestin in glioblastoma tumors via intratumor injection of shRNA significantly slowed tumor growth and volume. These results demonstrated that nestin plays a crucial role in development of glioblastoma and may potentially be targeted for treatment of the disease.
International Journal of Cancer 01/2011; 128(2):343-51. · 5.44 Impact Factor
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ABSTRACT: In this report, we describe the spontaneous malignant transformation of long-term cultured human fetal striatum neural stem cells (hsNSCs, passage 17). After subcutaneous transplantation of long-term cultured hsNSCs into immunodeficient nude mice, 2 out of 15 mice formed xenografts which expressed neuroendocrine tumor markers CgA and NSE. T1 cells, a cell line that we derived from one of the two subcutaneous xenografts, have undergone continuous expansion in vitro. These T1 cells showed stem cell-like features and expressed neural stem cell markers nestin and CD133. The T1 cells were involved in abnormal karyotype, genomic instability and fast proliferation. Importantly, after long-term in vitro culture, the T1 cells did not result in subcutaneous xenografts, but induced intracranial tumor formation, indicating that they adjusted themselves to the intracranial microenvironment. We further found that the T1 cells exhibited an overexpressed level of EGFR, and the CD133 positive T1 cells showed a truncation mutation in the exons 2-7 of the EGFR (EGFRvIII) gene. These results suggest that continuous expansion of neural stem cells in culture may lead to malignant spontaneous transformation. This phenomenon may be functionally related to EGFR by EGFRvIII gene mutation.
International journal of biological sciences 01/2011; 7(6):892-901. · 2.70 Impact Factor
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ABSTRACT: In regions of adult neurogenesis, neural progenitor cells (NPCs) are found in close proximity to blood vessels within a so-called 'vascular niche'. Neurogenesis is linked to angiogenesis via certain growth factors. We propose that angiopoietin-1 (Ang1), which is similar to VEGF, has a unique role in neurogenesis independent of its role in angiogenesis. In this study, primary cultures of NPCs were transduced with recombinant adenoviruses expressing Ang1 and induced to differentiate with dibutyryl cyclic AMP (dbcAMP). Neuronal differentiation was evaluated by quantitative PCR, immunofluorescence microscopy and Western blot analysis. The results show that ectopic expression of Ang1 promotes neuronal differentiation and neurite outgrowth in NPCs, while this effect was blocked by the presence of anti-Tie2 receptor antibody or the PI3-K inhibitor, LY294002. Our results suggest that Ang1, identified originally as an angiogenic factor, can also stimulate in vitro neurogenesis in NPCs through the Akt pathway.
Biochemical and Biophysical Research Communications 12/2008; 378(2):296-301. · 2.48 Impact Factor
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Chuanfei Yu,
Wenling Han,
Taiping Shi,
Bingfeng Lv, Qihua He,
Yanfei Zhang,
Ting Li,
Yingmei Zhang,
Quansheng Song,
Lu Wang,
Dalong Ma
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ABSTRACT: Cell migration plays a critical role during the development of most organisms and the process of malignant tumor metastasis. In the present study, we investigated the role of PTPIP51 (protein tyrosine phosphatase interacting protein 51) in cell motility. Overexpression of PTPIP51 induced cell elongation, increased cell migration, adhesion, and spreading, while downregulation of PTPIP51 had the opposite effects. We demonstrated here, that PTPIP51 could regulate ERK activity on Raf level, since MEK inhibitor and dominant-negative Raf-1 but not Ras could inhibit the ERK activation induced by PTPIP51. Further studies proved that PTPIP51 could interact with Raf-1 through 14-3-3, suggesting that PTPIP51 is a regulator of the Raf-MEK-ERK cascade through modulation of Raf-1 by 14-3-3. In addition, two redundant 14-3-3 binding domains in the PTPIP51 protein have been identified by deletion/mutation studies. We conclude that PTPIP51 regulates cell morphology and cell motility via interaction with Raf-1 through 14-3-3, and that PTPIP51 binds to 14-3-3 through two redundant binding domains.
Cellular signalling 09/2008; 20(12):2208-20. · 4.09 Impact Factor
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Haihao Zhu,
Yanrui Yang,
Hua Zhang,
Yan Han,
Yafang Li,
Ying Zhang,
Dongmin Yin, Qihua He,
Zhiqi Zhao,
Peter M Blumberg,
Jisheng Han,
Yun Wang
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ABSTRACT: In previous studies we demonstrated that protein kinase D1 (PKD1/PKCmu) could directly phosphorylate the transient receptor potential V1 (TRPV1) at its N-terminal region and enhance the function of TRPV1 in CHO cells stably transfected with TRPV1. In the current study we assessed the involvement of PKD1 in pain modulation and explored the possible interaction between PKD1 and TRPV1 in rat inflammatory heat hypersensitivity. PKD1 was translocated to cytoplasmic membrane fraction and was trans-phosphorylated only in membrane fraction but not in cytoplasmic fraction of dorsal root ganglia (DRG) at 2 and 6h after Complete Freund's Adjuvant (CFA) treatment. Pre i.t. injection of PKD1 antisense for 4 d or post-i.t. injection for 4 d both alleviated CFA-induced thermal hypersensitivity. Likewise, overexpression of PKD1 in DRG significantly enhanced, while dominant negative PKD1 (DN-PKD1) partly attenuated, heat hypersensitivity. Both PKD1 and TRPV1 were translocated to the cytoplasmic membrane in DRG 6 h after CFA treatment and, at that time, PKD1 interacted with TRPV1 by co-immunoprecipitation in DRG. Electrophysiological measurements indicated that DRG with overexpression of PKD1 were more sensitive to low dose capsaicin than those expressing DN-PKD1. The average magnitude of the peak inward current evoked by capsaicin was greater in the DRG overexpressing PKD1 than in those expressing DN-PKD1. Furthermore, overexpressed PKD1 could up regulate, whereas PKD1 antisense could knock down TRPV1 content in DRG through posttranscriptional regulation manner. We concluded that PKD1 in DRG, through interaction with TRPV1, is involved in developing and maintaining inflammatory heat hypersensitivity.
Pain 08/2008; 137(3):574-88. · 5.78 Impact Factor
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Qihua He,
Zhaofei Liu,
Bing Jia,
Xiaoxia Li,
Jiyun Shi,
Jun Zhang,
Feng Lan,
Zhi Yang,
Yinan Liu,
Li Shen,
Fan Wang
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ABSTRACT: This study describes gamma-imaging of the secondary tumors from the transplanted human fetal striatum neural stem cells-derived primary tumor cells in nude mice. The subcutaneous primary tumors were detected to express integrin alphavbeta3, and the corresponding cells were isolated and enriched in vitro, then transplanted to the nude mice. The technetium-99m-labeled Arg-Gly-Asp peptide, with high affinity to integrin alphavbeta3, was prepared for biodistribution and gamma-imaging. The secondary tumors were readily visualized at 1-h postinjection, and the tumor uptake of radiotracer was similar to that of positive control animals transplanted with U87MG human glioma cells. The tumor specificity of radiotracer was demonstrated by blocking experiment. We concluded that gamma-imaging is a promising approach in imaging the tumorigenesis of transplanted stem cells in vivo.
Neuroreport 08/2008; 19(10):1009-14. · 1.66 Impact Factor
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Caining Jin,
Ying Wang,
Wenling Han,
Yingmei Zhang, Qihua He,
Dan Li,
Caihua Yin,
Linjie Tian,
Dazhen Liu,
Quanshen Song,
Dalong Ma
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ABSTRACT: The mitochondria-mediated apoptotic pathway is regulated by members of the Bcl-2 family. Epidermal growth factor (EGF) induces Bad phosphorylation at Ser112 via mitogen-activated protein kinase (MAPK), impairing its binding to Bcl-2 and Bcl-xL and interfering with their anti-apoptotic functions. In the current study, we utilized Western blot, immunofluorescence, flow cytometry, and confocal microscopy to examine the effects of CMTM8 overexpression on apoptosis. Our data indicated levels of Bad-S112 phosphorylation were lower in CMTM8-transfected cells compared to pCDB-transfected cells. Caspase-dependent and independent mediated apoptosis, induced by CMTM8 overexpression, was facilitated by the mitochondria and inhibited by knockdown of Bad or overexpression of Bcl-xL. Previous research in our laboratory also demonstrated CMTM8 attenuated EGFR-mediated signaling pathways by decreasing ERK1/2 phosphorylation levels. These data implicate CMTM8 as a negative regulator of EGF-induced signaling, with potential use as a novel therapeutic gene for EGFR-targeted anticancer gene therapy.
Journal of Cellular Physiology 05/2007; 211(1):112-20. · 3.87 Impact Factor
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Xiaoping Chen,
Zebin Mao,
Shuhong Liu,
Hong Liu,
Xuan Wang,
Haitao Wu,
Yan Wu,
Tong Zhao,
Wenhong Fan,
Yong Li,
David T Yew,
Pawel M Kindler,
Linsong Li, Qihua He,
Lingjia Qian,
Xiaomin Wang,
Ming Fan
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ABSTRACT: Ciliary neurotrophic factor (CNTF) is primarily known for its important cellular effects within the nervous system. However, recent studies indicate that its receptor can be highly expressed in denervated skeletal muscle. Here, we investigated the direct effect of CNTF on skeletal myoblasts of adult human. Surprisingly, we found that CNTF induced the myogenic lineage-committed myoblasts at a clonal level to dedifferentiate into multipotent progenitor cells--they not only could proliferate for over 20 passages with the expression absence of myogenic specific factors Myf5 and MyoD, but they were also capable of differentiating into new phenotypes, mainly neurons, glial cells, smooth muscle cells, and adipocytes. These "progenitor cells" retained their myogenic memory and were capable of redifferentiating into myotubes. Furthermore, CNTF could activate the p44/p42 MAPK and down-regulate the expression of myogenic regulatory factors (MRFs). Finally, PD98059, a specific inhibitor of p44/p42 MAPK pathway, was able to abolish the effects of CNTF on both myoblast fate and MRF expression. Our results demonstrate the myogenic lineage-committed human myoblasts can dedifferentiate at a clonal level and CNTF is a novel regulator of skeletal myoblast dedifferentiation via p44/p42 MAPK pathway.
Molecular Biology of the Cell 08/2005; 16(7):3140-51. · 4.94 Impact Factor
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ABSTRACT: Neural progenitor cells have shown the effectiveness in the treatment of Parkinson's disease, but the therapeutic efficacy remains variable. One of important factors that determine the efficacy is the necessity of pre-differentiation of progenitor cells into dopaminergic neurons before transplantation. This study therefore investigated the therapeutic efficacy of mesencephalon-derived human neural progenitor cells with or without the pre-differentiation in alleviating a rat model of Parkinson's disease. We found that a combination of 50 ng/ml fibroblast growth factor 8, 10 ng/ml glial cell line-derived neurotrophic factor and 10 microM forskolin facilitated the differentiation of human fetal mesencephalic progenitor cells into dopaminergic neurons in vitro. More importantly, after transplanted into the striatum of parkinsonian rats, only pre-differentiated grafts resulted in an elevated production of dopamine in the transplanted site and the amelioration of behavioral impairments of the parkinsonian rats. Unlike pre-differentiated progenitors, grafted intact progenitors rarely differentiated into dopaminergic neurons in vivo and emigrated actively away from the transplanted site. These data demonstrates the importance of pre-differentiation of human progenitor cells before transplantation in enhancing therapeutic potency for Parkinson's disease.
International Journal of Developmental Neuroscience 07/2004; 22(4):175-83. · 2.42 Impact Factor
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ABSTRACT: To investigate the effect of intracellular-free Ca2+ concentration ([Ca2+]i) on injury following transient Mg(2+)-free treatment in vitro in developing cortical neurons.
Embryo cortical neurons of rats cultured for 6 d and 17 d were directly exposed to Mg(2+)-free media, or pretreated with NMDA receptor antagonists or calcium channel antagonist before being exposed to Mg(2+)-free media. MTT assay was used to study the injury of neurons. [Ca2+]i were measured using fluo-3, a fluorescent calcium-sensitive dye and laser-scanning confocal microscope, and calculated by the fluorescent intensity.
Compared with control, MTT conversion rates decreased after transient (3 h) Mg(2+)-free treatment in neurons cultured for 6 d and 17 d in vitro, (59.1 +/- 6.87)% and (51.2 +/- 5.90)%, respectively. In neurons pre- and co-treated with 10 mumol.L-1 MK-801, 50 mumol.L-1 AP-5 and 10 mumol.L-1 nifedipine, MTT conversion rates were higher than those of neurons with only Mg(2+)-free treatment. Peak values of [Ca2+]i in neurons cultured for 6 d and 17 d were 2.4 +/- 0.23 and 3.2 +/- 0.32, respectively. Peak value of neurons 17 d in vitro was significantly higher than that of neurons 6 d in vitro (P < 0.05). In neurons pre- and co-treated with MK-801, AP-5 and nifedipine, [Ca2+]i were lower than those of neurons with only Mg(2+)-free treatment.
Neuronal injury and [Ca2+]i changes following Mg(2+)-free-treatment-induced seizure were different between neurons 6 d and 17 d in vitro. It suggested that the age-dependent [Ca2+]i changes might play a role in an age-dependent manner of injury following Mg(2+)-free-treatment-induced seizure. NMDA receptor-Ca2+ pathway activation was crucial in the [Ca2+]i change and the cellular injury induced by Mg(2+)-free treatment.
Beijing da xue xue bao. Yi xue ban = Journal of Peking University. Health sciences 10/2003; 35(5):466-70.
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ABSTRACT: To investigate whether the dopamine receptor D2 can express or can be induced to express in HNG1210-E, a monoclonal, telomerase-immortalized, human neural progenitor cell line.
By means of RT-PCR, immuno-fluorescent staining, and fluo3 Ca2+ imaging to the expression of D2 mRNA and protein as well as the reaction to dopamine were demonstrated.
HNG1210-E cells could be induced to express D2 mRNA and its proteins. The induced cells also reacted to dopamine (5 mmol.L-1), which caused rapid rising of cytoplasm Ca2+.
HNG1210-E cells can be induced to express D2 mRNA and functional proteins.
Beijing da xue xue bao. Yi xue ban = Journal of Peking University. Health sciences 07/2003; 35(3):271-3.
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ABSTRACT: To study the neuroprotective effect and related mechanisms of triptolide.
After being treated with 10(-10), 10(-9) mol.L-1 triptolide and/or 10, 50 mmol.L-1 glutamate for 24 hours, and the viability of PC12 was detected by MTT conversion, and the apoptosis rate was detected by AnnexinV-FITC and PI staining. In order to understand the underlying mechanism of the neuroprotective effect of triptolide, H2DCFDA and JC1 were used to detect the reactive oxygen species (ROS) and mitochondrial membrane potential.
Glutamate could induce PC12 necrosis and apoptosis. T10 at 10(-10) or 10(-9) mol.L-1 inhibited glutamate-induced cell death, ROS formation and decrease of mitochondrial membrane potential.
T10 at 10(-10) or 10(-9) mol.L-1 can protect PC12 from the death induced by glutamate; and the underlying mechanisms may be involved in the inhibition of ROS formation and decrease of mitochondrial membrane potential.
Beijing da xue xue bao. Yi xue ban = Journal of Peking University. Health sciences 07/2003; 35(3):252-5.
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ABSTRACT: To study the significance of calcium in the apoptosis of HL-60 cells induced by VP-16, the technology of flow cytometry, confocal laser scanning microscopy and Western blot were used. The results showed that VP-16 could induced the apoptosis of HL-60 cells and transient increase of intracellular calcium concentration; EGTA [ethylene glycol-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid], that could combine the extracellular calcium, did not prevent the apoptosis of HL-60 cells. BAPTA-AM [1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxy-methyl) ester], however, a chelating agent of intracellular calcium ions, could prevent apoptosis and the release of cytochrome C from HL-60 cells. It was concluded that the calcium plays an important role in apoptosis and the release of cytochrome C.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 01/2001; 8(4):283-286.
