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ABSTRACT: ObjectiveTo explore the influence of EBP50 (ezrin-radixin-moesin-binding phospho-protein-50) on microfilament cytoskeleton content
and distribution in cultured Hela cells, and to investigate the relationship between the changes in microfilament cytoskeleton
localization and EBP50 after PDGF (platelet-derived growth factor) stimulation, and to further clarify the molecular mechanism
by which EBP50 suppresses tumor cell proliferation and migration.
MethodspBK-CMV-HAEBP50 wild type recombinant plasmid and pBK-CMV-HA empty vector were transfected into Hela cells. G418 at 350 mg/L
was used to screen for cell clones stably expressing EBP50. Western blot was carried out to detect EBP50 expression. Similarities
and differences in microfilament cytoskeleton content and distribution in Hela cells transfected with pBK-CMV-HA-EBP50 wild
type recombinant plasmid and pBK-CMV-HA empty vector were also treated with PDGF (10 ng/mL and 20 ng/mL, 37 °C, 15 min) and
stained by rhodamine-labeled phalloidin to observe the distribution of microfilament cytoskeleton in the two groups. EBP50
protein distribution in PDGF-stimulated Hela cells was detected by immunofluorescence.
ResultsWestern blot results confirmed that the EBP50 cDNA fragment could express EBP50 in cultured Hela cell lines and that cell
lines stably expressing EBP50 were successfully obtained. Western blot and fluorescence results showed that in the cell line
transfected with empty vector, the microfilament cytoskeleton was thick, loose, multidirectional and displayed crossing arrangements.
The content of microfilament cytoskeleton in the cell line transfected with pBK-CMV-HA-EBP50 was different from that found
in the cell line transfected with empty vector. EBP50 expression enhanced microfilament cytoskeleton polymerization into compact
thin filaments. Under the stimulation of PDGF, EBP50 migrated to the cell membrane from the cytosol together with microfilament
cytoskeleton and co-localized there.
ConclusionEBP50 can change the distribution of microfilament cytoskeleton in cultured Hela cells and can also bind the microfilament
cytoskeleton to the cell membrane under the stimulation of PDGF. EBP50 may play a role in the proliferation and migration
of tumor cells by influencing the distribution and localization of microfilament cytoskeleton.
The Chinese-German Journal of Clinical Oncology 04/2012; 8(5):282-285.
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ABSTRACT: β-Adrenergic receptors can activate extracellular signal-regulated kinases (ERKs) via different mechanisms. In this study,
we investigated the molecular mechanism of β1-adrenergic receptor (β1AR)-mediated ERK activation in African green monkey kidney
COS-7 cells. Treatment of cells with isoproterenol (ISO), a β1AR selective agonist, induced phosphorylation of ERK1/2 in a
dose-dependent manner. ISO-stimulated ERK phosphorylation was not influenced by the Gβγ inhibitor, βAR kinase carboxyl terminal
(βARKct) or by the Gi inhibitor, pertussis toxin (PTX), but it was clearly abolished via inhibition of protein kinase A (PKA)
with H89, or of mitogen-activated protein kinase kinase (MEK1) with PD98059, revealing that the Gαs subunit is involved in
ERK regulation through the PKA/MEK1 pathway. We also tested the effect of the adenylate cyclase activator forskolin on ERK
activation, and the result was identical to that of ISO stimulation. Moreover, pretreatment with the epidermal growth factor
receptor (EGFR) tyrosine kinase inhibitor AG1478 or with the Src tyrosine kinase inhibitor PP2 did not affect ERK activation.
These observations suggest a mechanism of β1AR-mediated ERK activity that involves the Gαs subunit, but not EGFR or Src tyrosine
kinase.
Amino Acids 04/2012; 38(1):75-84. · 3.25 Impact Factor
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ABSTRACT: Ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) suppresses breast cancer cell proliferation, potentially through its regulatory effect on epidermal growth factor receptor (EGFR) signaling, although the mechanism by which this occurs remains unknown. Thus in our studies, we aimed to determine the effect of EBP50 expression on EGF-induced cell proliferation and activation of EGFR signaling in the breast cancer cell lines, MDA-MB-231 and MCF-7. In MDA-MB-231 cells, which express low levels of EBP50, EBP50 overexpression inhibited EGF-induced cell proliferation, ERK1/2 and AKT phosphorylation. In MCF-7 cells, which express high levels of EBP50, EBP50 knockdown promoted EGF-induced cell proliferation, ERK1/2 and AKT phosphorylation. Knockdown of EBP50 in EBP50-overexpressed MDA-MB-231 cells abrogated the inhibitory effect of EBP50 on EGF-stimulated ERK1/2 phosphorylation and restoration of EBP50 expression in EBP50-knockdown MCF-7 cells rescued the inhibition of EBP50 on EGF-stimulated ERK1/2 phosphorylation, further confirming that the activation of EGF-induced downstream molecules could be specifically inhibited by EBP50 expression. Since EGFR signaling was triggered by EGF ligands via EGFR phosphorylation, we further detected the phosphorylation status of EGFR in the presence or absence of EBP50 expression. Overexpression of EBP50 in MDA-MB-231 cells inhibited EGF-stimulated EGFR phosphorylation, whereas knockdown of EBP50 in MCF-7 cells enhanced EGF-stimulated EGFR phosphorylation. Meanwhile, total expression levels of EGFR were unaffected during EGF stimulation. Taken together, our data shows that EBP50 can suppress EGF-induced proliferation of breast cancer cells by inhibiting EGFR phosphorylation and blocking EGFR downstream signaling in breast cancer cells. These results provide further insight into the molecular mechanism by which EBP50 regulates the development and progression of breast cancer.
Amino Acids 04/2012; 43(5):2027-35. · 3.25 Impact Factor
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ABSTRACT: To study the neuro-endocrine adjustment effects of Herba Epimedii and Fructus Ligustrilucidi on the asthmatic rats.
Rat asthma model was duplicated by OVA (ovalbumin) through sensitizing and challenging. Fifty male rats were randomly divided into normal group, model group, adjustment group of Herba Epimedii and Fructus Ligustrilucidi, Peibenfang group and Asimei capsule group. Investigating levels of ET (Endothelin), NO, iNOS (inducible NOS ), and cNOS (constitutive NOS) in blood serum and BALF (bronchoalveolar lavage fluid ), CORT (corticotrophin) in serum, ACTH (adrenocorticotropin hormone) in plasma, CHR (corticotropin release hormone) in hypothalamus, protein expression of GCR (glucocorticoid receptor) in lung tissue.
The adjustment of Herba Epimedii and Fructus Ligustrilucidi could inhibit ET and NO content in BALF (all P < 0.05), decrease the level of iNOS in serum and BALF (P < 0.01 or P < 0.05), and increase the level of cNOS in serum and BALF (P < 0.01 or P < 0.05), raise the concentration of serum CORT (P < 0.01), enhance the protein expression of GCR in lung tissue (P < 0.05).
The preventive and therapeutic effect of Herba Epimedii and Fructus Ligustrilucidi on asthma relates to their adjustment effect on ET/NO and HPA axis.
Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China journal of Chinese materia medica 06/2010; 35(12):1590-3.
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ABSTRACT: The beta-2 adrenergic receptor (beta2AR) has a carboxyl terminus motif that can interact with PSD-95/discs-large/ZO1 homology (PDZ) domain-containing proteins. In this paper, we identified membrane-associated guanylate kinase inverted-3 (MAGI-3) as a novel binding partner of beta2AR. The carboxyl terminus of beta2AR binds with high affinity to the fifth PDZ domain of MAGI-3, with the last four amino acids (D-S-L-L) of the receptor being the key determinants of the interaction. In cells, the association of full-length beta2AR with MAGI-3 occurs constitutively and is enhanced by agonist stimulation of the receptor. Our data also demonstrated that beta2AR-stimulated extracellular signal-regulated kinase-1/2 (ERK1/2) activation was substantially retarded by MAGI-3 expression. These data suggest that MAGI-3 regulates beta2AR-mediated ERK activation through the physical interaction between beta2AR and MAGI-3.
FEBS letters 03/2010; 584(11):2207-12. · 3.54 Impact Factor
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ABSTRACT: The Multiple T-maze (MTM) and the Barnes maze (BM) are land mazes used for the evaluation of spatial memory. The observation that mice are performing differently in individual mazes made us test the hypothesis that differences in cognitive performances in the two land mazes would be accompanied by differences in hippocampal protein levels. C57BL/6J mice were tested in the BM and in the MTM, hippocampi were extirpated 6 h following the probe trials each, and proteins were extracted for gel-based proteomic analysis. Mice learned the task in both paradigms. Levels of hippocampal proteins from several pathways including signaling, chaperone, and metabolic cascades were significantly different between the two spatial memory tasks. Protein levels were linked to spatial memory specifically as yoked controls were used.
Journal of Proteome Research 09/2009; 8(10):4479-86. · 5.11 Impact Factor
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ABSTRACT: beta-Adrenergic receptors can activate extracellular signal-regulated kinases (ERKs) via different mechanisms. In this study, we investigated the molecular mechanism of beta1-adrenergic receptor (beta1AR)-mediated ERK activation in African green monkey kidney COS-7 cells. Treatment of cells with isoproterenol (ISO), a beta1AR selective agonist, induced phosphorylation of ERK1/2 in a dose-dependent manner. ISO-stimulated ERK phosphorylation was not influenced by the Gbetagamma inhibitor, betaAR kinase carboxyl terminal (betaARKct) or by the Gi inhibitor, pertussis toxin (PTX), but it was clearly abolished via inhibition of protein kinase A (PKA) with H89, or of mitogen-activated protein kinase kinase (MEK1) with PD98059, revealing that the Galphas subunit is involved in ERK regulation through the PKA/MEK1 pathway. We also tested the effect of the adenylate cyclase activator forskolin on ERK activation, and the result was identical to that of ISO stimulation. Moreover, pretreatment with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478 or with the Src tyrosine kinase inhibitor PP2 did not affect ERK activation. These observations suggest a mechanism of beta1AR-mediated ERK activity that involves the Galphas subunit, but not EGFR or Src tyrosine kinase.
Amino Acids 12/2008; 38(1):75-84. · 3.25 Impact Factor
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ABSTRACT: Rhizobia, bacteria that fix atmospheric nitrogen, are important agricultural resources. In order to establish the evolutionary relationships among rhizobia isolated from different geographic regions and different plant hosts for systematic studies, we evaluated the use of physical structure of the rhizobial genomes as a phylogenetic marker to categorize these bacteria. In this work, we analyzed the features of genome structures of 64 rhizobial strains. These rhizobial strains were divided into 21 phylogenetic clusters according to the features of genome structures evaluated by the endonuclease I-CeuI. These clusters were supported by 16S rRNA comparisons and genomic sequences of four rhizobial strains, but they are largely different from those based on the current taxonomic scheme (except 16S rRNA).
Science in China Series C Life Sciences 07/2004; 47(3):268-78. · 1.61 Impact Factor
