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Find video protocols related to scientific articles indexed in Pubmed.
Efficacy and Safety of Ologen Implant Versus Mitomycin C in Primary Trabeculectomy: A Meta-analysis of Randomized Clinical Trials.
J. Glaucoma
PUBLISHED: 09-30-2014
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To assess the efficacy and safety of trabeculectomy with Ologen implant versus trabeculectomy with mitomycin C (MMC) for treatment of glaucoma.
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Chromatographic fingerprint analysis of metabolites in natural and artificial agarwood using gas chromatography-mass spectrometry combined with chemometric methods.
J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.
PUBLISHED: 08-04-2014
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Agarwood is a resinous material formed in wounded Aquilaria sinensis in China, which is widely used as an effective traditional Chinese medicine (TCM). This study is aimed to use gas chromatography-mass spectrometry combined with chemometric methods to create reliable criteria for accurate identification of natural agarwood and artificial agarwood, as well as for quality evaluation of artificial agarwood. Natural agarwood and artificial agarwood (stimulated by formic acid or formic acid plus fungal inoculation) were used as standards and controls for the gas chromatography-mass spectrometry (GC-MS) and multivariate analysis. The identification criteria developed were applied to commercial agarwood. A reliable criteria including correlation coefficient of GC-MS fingerprint of natural agarwood and 22 markers of metabolism in natural and artificial agarwood was constructed. Compared with chemically stimulated agarwood (formic acid) and in terms of the 22 markers, artificial agarwood obtained by formic acid stimulation and fungal inoculation were much closer to natural agarwood. The study demonstrates that the chemical components of artificial agarwood obtained by comprehensive stimulated method (formic acid plus fungal inoculation) are much closer to the natural agarwood than those obtained by chemically stimulated method (formic acid), as times goes by. A reliable criteria containing correlation coefficient of GC-MS fingerprint of natural agarwood and 22 metabolism markers can be used to evaluate the quality of the agarwood. As an application case, three samples were identified as natural agarwood from the 25 commercial agarwood by using the evaluation method.
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Comparison of secondhand smoke exposure measures during pregnancy in the development of a clinical prediction model for small-for-gestational-age among non-smoking Chinese pregnant women.
Tob Control
PUBLISHED: 07-24-2014
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To compare predictive values of small-for-gestational-age (SGA) by different measures for secondhand smoke (SHS) exposure during pregnancy and to develop and validate a prediction model for SGA using SHS exposure along with sociodemographic and pregnancy factors.
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A mini-electrochemical system integrated micropipet tip and pencil graphite electrode for detection of anticancer drug sensitivity in vitro.
Biosens Bioelectron
PUBLISHED: 07-20-2014
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Developing a reliable and cost-effective miniaturized electroanalysis tool is of vital importance for cell electrochemical analysis. In this work, a novel mini-electrochemical system has been constructed for trace detection of cell samples. The mini-electrochemical system was constructed by integrating a pencil graphite modified by threonine (PT/PGE) as working electrode, an Ag/AgCl (Sat'd) as reference electrode, platinum wire as counter electrode and a micropipet tip as electrochemical cell. The mini-electrochemical system not only saved dramatically usage of samples from 500?L in traditional electrochemical system to 10?L, but also possessed an adjustable active surface area by changing the length of PT/PGE immersed into the cell suspension from 3mm to 15mm, and the linear equation was ipa=2.25l-2.64 (R(2)=0.990). The system was successfully used in detection of MCF-7 cells, and a nonlinear exponent relationship between peak current and the cell number range from 3.0×l0(3) to 7.0×l0(6)cellsmL(-1) was established firstly with the index equation ipa=59.557e (-C/1.709)-71.486 (R(2)=0.954). Finally, the system was used for evaluating the sensitivity of cyclophosphamide on MCF-7 cell, and the result was corresponded well with that of MTT assay. The proposed system is sufficiently simple, cheap and easy operated, and could be applied in electrochemical detection of other biological samples.
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Transduced protein transduction domain linked HSP27 protected LECs against UVB radiation-induced damage.
Exp. Eye Res.
PUBLISHED: 01-17-2014
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PTD-fusion protein technology was used to transduce heat shock protein 27 (HSP27), an anti-apoptotic protein, into human lens epithelial cells (HLECs) (SRA01/04). The protein transduction domain (PTD) of the 11-amino acid YGRKKRRQRRR was tagged at the N-terminus of HSP27. The fusion protein was purified from bacteria transformed with a pKYB-PTD-HSP27 construct. The HLECs were incubated with PTD-HSP27-FITC and the fluorescence inside HLECs was found by fluorescence microscopic examination. To test the ability of PTD-HSP27 to pass through the corneas, PTD-HSP27-FITC was dropped onto the conjunctival sacs of rabbits; fluorescent labeled PTD-HSP27 was then observed in the rabbit aqueous humor. After being incubated with the PTD-HSP27 protein and irradiated with ultraviolet-B (UVB) light, HLECs was analyzed by flow cytometry, Hoechst 33258 staining and measurement of the potential of the mitochondrial transmembrane. HLECs incubated with PTD-HSP27 had a lower apoptotic rate and a higher mitochondrial membrane potential than the control cells. PTD-HSP27 appears to be sufficient to protect HLECs against UVB-induced apoptosis.
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Independent oncogenic and therapeutic significance of phosphatase PRL-3 in FLT3-ITD-negative acute myeloid leukemia.
Cancer
PUBLISHED: 01-08-2014
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Internal tandem duplication of FMS-like tyrosine kinase (FLT3-ITD) is well known to be involved in acute myeloid leukemia (AML) progression, but FLT3-ITD-negative AML cases account for 70% to 80% of AML, and the mechanisms underlying their pathology remain unclear. This study identifies protein tyrosine phophatase PRL-3 as a key mediator of FLT3-ITD-negative AML.
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Lycium barbarum Polysaccharides Protect Human Lens Epithelial Cells against Oxidative Stress-Induced Apoptosis and Senescence.
PLoS ONE
PUBLISHED: 01-01-2014
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We aimed to investigate the protective effect of Lycium barbarum polysaccharides (LBPs) against oxidative stress-induced apoptosis and senescence in human lens epithelial cells.
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Non-genetic direct reprogramming and biomimetic platforms in a preliminary study for adipose-derived stem cells into corneal endothelia-like cells.
PLoS ONE
PUBLISHED: 01-01-2014
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Cell fate and function can be regulated and reprogrammed by intrinsic genetic program, extrinsic factors and niche microenvironment. Direct reprogramming has shown many advantages in the field of cellular reprogramming. Here we tried the possibility to generate corneal endothelia (CE) -like cells from human adipose-derived stem cells (ADSCs) by the non-genetic direct reprogramming of recombinant cell-penetrating proteins Oct4/Klf4/Sox2 (PTD-OKS) and small molecules (purmorphamine, RG108 and other reprogramming chemical reagents), as well as biomimetic platforms of simulate microgravity (SMG) bioreactor. Co-cultured with corneal cells and decellularized corneal ECM, Reprogrammed ADSCs revealed spherical growth and positively expressing Nanog for RT-PCR analysis and CD34 for immunofluorescence staining after 7 days-treatment of both purmorphamine and PTD-OKS (P-OKS) and in SMG culture. ADSCs changed to CEC polygonal morphology from spindle shape after the sequential non-genetic direct reprogramming and biomimetic platforms. At the same time, induced cells converted to weakly express CD31, AQP-1 and ZO-1. These findings demonstrated that the treatments were able to promote the stem-cell reprogramming for human ADSCs. Our study also indicates for the first time that SMG rotary cell culture system can be used as a non-genetic means to promote direct reprogramming. Our methods of reprogramming provide an alternative strategy for engineering patient-specific multipotent cells for cellular plasticity research and future autologous CEC replacement therapy that avoids complications associated with the use of human pluripotent stem cells.
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Influence of CYP2A6*4 Genotypes on Maternal Serum Cotinine Among Chinese Nonsmoking Pregnant Women.
Nicotine Tob. Res.
PUBLISHED: 10-25-2013
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Serum cotinine is a common biomarker for smoking and secondhand smoke (SHS) exposure, but it can be affected by the activity of nicotine-metabolizing enzymes. This study investigated the influence of CYP2A6*4 genotypes on serum cotinine among nonsmoking pregnant women.
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Oxidative dehydrogenation on nanocarbon: identification and quantification of active sites by chemical titration.
Angew. Chem. Int. Ed. Engl.
PUBLISHED: 08-04-2013
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Ketonic carbonyl groups are catalytic active sites for oxidative dehydrogenation (ODH) reactions on carbon nanotubes. The quantity of these groups could be calculated from chemical titration with hydrazine compounds. ODH catalytic activity of nanocarbon is directly correlated with surface concentration of ketonic carbonyl groups, and the turnover frequency normalized by the number of active sites reflects the intrinsic activity of nanocarbon catalysts.
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PACAP induces the dimerization of PAC1 on the nucleus associated with the cAMP increase in the nucleus.
Neurosci. Lett.
PUBLISHED: 03-11-2013
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PAC1 is PACAP (pituitary adenylate cyclase-activating polypeptide) preferring receptor belonging to class B G protein couple receptor (GPCR) mediating the most effects of PACAP. The dimerization of PAC1 has been proven by our previous research. The bimolecular fluorescence complementation (BiFC) combined with fluorescence confocal microscope image was used in this research to explore the profiles of PAC1 dimers during the activation by PACAP. Fluorescence metry and cAMP assays were both used to detect the functions of the dimerization of PAC1 on the nucleus induced by PACAP. It was found that PACAP in concentration lower than 10nM induced the de-dimerization of PAC1 on the plasma membranes and the re-dimerization of PAC1 on the nucleus. While PACAP in concentration higher than 10nM, the nuclear localized PAC1 dimers were further translocated from outside/on the nucleus into the nucleus. In addition, it was also found that the more PAC1 dimers on the nucleus produced the higher cAMP level in the nucleus, and the levels of cAMP in the nucleus varied synchronously with functions of PACAP on the proliferation of PAC1-CHO cells. These results indicated the dimerization of PAC1 on the nucleus may be involved in the cell signals produced by PACAP. The finding and the research on the dimerization of PAC1 on the nucleus will help us to step forward to clarify the physiological and pharmacological role of PAC1.
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Down-regulation of programmed cell death 4 leads to epithelial to mesenchymal transition and promotes metastasis in mice.
Eur. J. Cancer
PUBLISHED: 01-10-2013
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In this study, we demonstrated that knockdown of programmed cell death 4 (Pdcd4), a novel tumour suppressor, decreased the expressions of epithelial-specific proteins and increased the expressions of mesenchymal-specific proteins in vitro and in vivo, suggesting that knockdown of Pdcd4 results in epithelial to mesenchymal transition (EMT). Knockdown of Pdcd4 increased the rate of wound closure and migration capacity in wound-healing assays and Boyden chamber migration assays, respectively, indicating that Pdcd4 knockdown promotes cell migration. Pdcd4 knockdown also altered the adhesion capacity of GEO cells to extracellular matrix including laminin, collagen IV and fibronectin. To test whether knockdown of Pdcd4 promotes metastasis in vivo, parental, control and Pdcd4 knockdown cells were injected into the caecal wall (orthotopic implantation) of nude mice. Tumours are formed on caecum in all injected mice. However, only mice injected with Pdcd4 knockdown cells developed hepatic and local lymph node metastases. Immunohistochemical staining analyses showed that c-Myc and Snail/Slug expressions were up-regulated in the tumours derived from injection of Pdcd4 knockdown cells. These results implicated that promotion of metastasis by Pdcd4 knockdown was contributed by up-regulation of c-Myc and Snail/Slug in nude mice. Taken together, our data demonstrated that knockdown of Pdcd4 leads to EMT, alternation of adhesion and promotion of migration and metastasis.
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Airway tissue factor-dependent coagulation activity in response to sulfur mustard analog 2-chloroethyl ethyl sulfide.
Am. J. Physiol. Lung Cell Mol. Physiol.
PUBLISHED: 09-30-2011
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Acute lung injury is a principal cause of morbidity and mortality in response to mustard gas (SM) inhalation. Obstructive, fibrin-containing airway casts have recently been reported in a rat inhalation model employing the SM analog 2-chloroethyl ethyl sulfide (CEES). The present study was designed to identify the mechanism(s) causing activation of the coagulation cascade after CEES-induced airway injury. Here we report that CEES inhalation elevates tissue factor (TF) activity and numbers of detached epithelial cells present in lavage fluid (BALF) from rats after exposure (18 h). In vitro studies using 16HBE cells, or with rat BALF, indicated that detached epithelial cells could convert factor X (FX) to the active form FXa when incubated with factor VII and could elicit rapid clotting of plasma. In addition, immunocytochemical analysis demonstrated elevated cell surface (TF) expression on CEES-exposed 16HBE cells as a function of time. However, total cell TF expression did not increase. Since membrane surfaces bearing TF are important determinants of clot initiation, anticoagulants directed against these entities were tested for ability to limit plasma clotting or FX activation capacity of BALF or culture media. Addition of tifacogin, a TF pathway inhibitor, effectively blocked either activity, demonstrating that the procoagulant actions of CEES were TF pathway dependent. Lactadherin, a protein capable of competing with clotting factors for phospholipid-binding sites, was partially effective in limiting these procoagulant actions. These findings indicate that TF pathway inhibition could be an effective strategy to prevent airway obstruction after SM or CEES inhalation.
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AKT Activation by Pdcd4 Knockdown Up-Regulates Cyclin D1 Expression and Promotes Cell Proliferation.
Genes Cancer
PUBLISHED: 09-13-2011
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Programmed cell death 4 (Pdcd4), a novel tumor suppressor, inhibits neoplastic transformation and tumor invasion. In this study, the authors found that knockdown of Pdcd4 promoted cell proliferation and up-regulated cyclin D1 expression. Previously, the authors demonstrated that Pdcd4 knockdown activated NF-?B-dependent transcription. Mutations of NF-?B binding sites on the cyclin D1 promoter attenuated the cyclin D1 promoter activity induced by Pdcd4 knockdown. In addition, knockdown of NF-?B/I?B kinase (IKK) ? or IKK?, the kinase regulating NF-?B activation, inhibited cyclin D1 promoter activity and cyclin D1 expression, indicating that up-regulation of cyclin D1 by Pdcd4 knockdown is contributed, at least in part, by NF-?B activation. To investigate the mechanism of how Pdcd4 knockdown activates NF-?B, the authors found that the levels of AKT phosphorylation and AKT kinase activity were increased in the Pdcd4 knockdown cells. Conversely, ectopic expression of Pdcd4 inhibited AKT phosphorylation and cyclin D1 expression, suggesting that Pdcd4 regulates AKT activity and cyclin D1 expression. Furthermore, knockdown of AKT in the Pdcd4 knockdown cells inhibited IKK phosphorylation, NF-?B activation, cyclin D1 promoter activity, and cyclin D1 expression as well as cell proliferation. Taken together, these findings suggest that activation of NF-?B by Pdcd4 knockdown through AKT contributes to the elevated expression of cyclin D1, thus providing new insights into how loss of Pdcd4 expression promotes tumor development.
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Association of PPAR?2 polymorphisms with carcass and meat quality traits in a Pietrain x Jinhua F2 population.
Genet. Mol. Biol.
PUBLISHED: 03-01-2011
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The PPAR?2 gene is a key regulator of both proliferation and preadipocyte differentiation in mammals. Herein its genotype and allele frequencies were analyzed using PCR-SSCP in eight pig breeds (N = 416). Two kinds of polymorphisms of the PPAR?2 gene were detected, including a previously reported shift SNP A177G (Met59Val) in exon 1 and a novel silent mutation G876A in exon 5. The results revealed that European pig breeds carry a higher allele A frequency at the A177G locus and a fixed GG genotype at the G876A locus. Allele A at the G876A locus was only found in Jinhua pigs. The association between haplotype (A177G/G876A) and carcass and meat quality traits was analyzed in a Pietrain x Jinhua F2 population (N = 248). The PPAR?2 gene was found to be significantly associated with backfat thickness at the shoulder (p < 0.05), 6-7(th) ribs (p < 0.01), last rib (p < 0.01), gluteus medius (p <0.05) and ham weight (p < 0.01). Significant effects of different haplotypes on ham weight and backfat thickness at the 6-7(th) ribs, last rib, and gluteus medius were also observed.
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Mapping, cDNA cloning and tissue expression of the porcine thyrotropin-releasing hormone receptor gene.
Anim. Biotechnol.
PUBLISHED: 02-18-2011
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Thyrotropin-releasing hormone receptor (TRHR) is a G-protein-coupled receptor that plays a crucial role in regulating the hypothalamic-pituitary-thyroid axis by conveying the action of the hypothalamic tripeptide TRH, which is the primary central activator of this hormonal cascade. In the present study, the porcine TRHR (pTRHR) gene was localized to chromosome 4 by Radiation hybrid mapping. Quantitative trait loci affecting average backfat thickness, daily gain, and carcass and meat quality traits have been mapped to the region containing this gene. Further, the full-length cDNA of pTRHR was cloned and sequenced. pTRHR contains an open reading frame encoding 398 amino acids and shares 96.2% amino acid identity to human TRHR. Real-time quantitative RT-PCR showed that the mRNA of pTRHR is expressed in a variety of tissues, with high expression in the brain, hypothalamus, pituitary, testis, and fat tissue. The considerable expression level of TRHR mRNA found in fat tissue indicates potential direct action of TRH on lipocyte might exist. Additionally, two alternative spliced transcript variants of pTRHR were also isolated in this study. Our data provided basic molecular information which will be useful for further investigation on pTRHR gene.
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Activation and up-regulation of translation initiation factor 4B contribute to arsenic-induced transformation.
Mol. Carcinog.
PUBLISHED: 01-25-2011
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Arsenic is a known human carcinogen. However, the mechanism of how arsenic induces cell transformation remains unclear. In this study, we demonstrated that long-term exposure to sodium arsenite at low-dose (2?µM) increases cell proliferation and neoplastic transformation in a mouse epidermal cell model, JB6 promotion-susceptible cells. The phosphorylation of AKT and its downstream targets, 70-kDa ribosomal protein S6 kinase (p70S6K) and translation initiation factor 4B (eIF4B), are increased in the arsenite treated cells, indicating that long-term arsenite treatment activates AKT-p70S6K signaling pathway. In addition, long-term exposure to arsenite up-regulates eIF4B expression and increases the rate of translation. Knockdown of eIF4B expression resulted in inhibition of arsenic-induced cell proliferation, transformation, and translation. Moreover, the expression of c-Myc which is up-regulated by long-term arsenite treatment is inhibited by eIF4B knockdown. Taken together, these results indicate that activation and up-regulation of eIF4B contributes to arsenic-induced transformation in JB6 cells.
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Artesunate inhibiting angiogenesis induced by human myeloma RPMI8226 cells.
Int. J. Hematol.
PUBLISHED: 02-17-2010
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Multiple myeloma (MM) remains an incurable plasma cell disorder to date; therefore, new biologically target-based therapies are in urgent demand. Our previous studies showed that the antimalarial artesunate (ART) possessed anti-myeloma effect by inhibiting proliferation and inducing apoptosis of myeloma cells. The present study evaluated the effect of ART on human myeloma cell-induced angiogenesis and elucidated its mechanism. The human umbilical vein endothelial cells (HUVECs) migration test, aortic sprouting in fibrin gel in vitro and chicken chorioallantoic membrane (CAM) neovascularization in vivo model were used to examine the effect of ART on angiogenesis induced by human myeloma cells. The results showed that ART could inhibit HUVECs migration, even at a lower concentration (3 ?mol/l, P < 0.01, compared with the result of control group), and suppress efficiently the angiogenic ability of myeloma RPMI8226 cells in a dose-dependent pattern (3-12 ?mol/l, P < 0.05). The levels of VEGF and Ang-1 in the conditioned medium (CM) were quantified by enzyme-linked immunosorbent assay (ELISA). The results confirmed that 3 ?mol/l ART could significantly decrease VEGF and Ang-1 secretion by RPMI8226 cells (P < 0.05), which correlated well with the reduction of angiogenesis induced by myeloma RPMI8226 cells. The present study also showed that ART downregulated the expression of VEGF and Ang-1 in RPMI8226 cells and reduced the activation of extracellular signal-regulated kinase 1 (ERK1) as well. Therefore, ART can block ERK1/2 activation, downregulate VEGF and Ang-1 expression and inhibit angiogenesis induced by human multiple myeloma RPMI8226 cells. Combined with our previous published data, results from the present study indicate that ART possesses potential anti-myeloma effect.
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Adenosine A2A receptor is a unique angiogenic target of HIF-2alpha in pulmonary endothelial cells.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 06-17-2009
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Hypoxia, through the hypoxia-inducible transcription factors HIF-1alpha and HIF-2alpha (HIFs), induces angiogenesis by up-regulating a common set of angiogenic cytokines. Unlike HIF-1alpha, which regulates a unique set of genes, most genes regulated by HIF-2alpha overlap with those induced by HIF-1alpha. Thus, the unique contribution of HIF-2alpha remains largely obscure. By using adenoviral mutant HIF-1alpha and adenoviral mutant HIF-2alpha constructs, where the HIFs are transcriptionally active under normoxic conditions, we show that HIF-2alpha but not HIF-1alpha regulates adenosine A(2A) receptor in primary cultures of human lung endothelial cells. Further, siRNA knockdown of HIF-2alpha completely inhibits hypoxic induction of A(2A) receptor. Promoter studies show a 2.5-fold induction of luciferase activity with HIF-2alpha cotransfection. Analysis of the A(2A) receptor gene promoter revealed a hypoxia-responsive element in the region between -704 and -595 upstream of the transcription start site. By using a ChIP assay, we demonstrate that HIF-2alpha binding to this region is specific. In addition, we demonstrate that A(2A) receptor has angiogenic potential, as assessed by increases in cell proliferation, cell migration, and tube formation. Additional data show increased expression of A(2A) receptor in human lung tumor cancer samples relative to adjacent normal lung tissue. These data also demonstrate that A(2A) receptor is regulated by hypoxia and HIF-2alpha in human lung endothelial cells but not in mouse-derived endothelial cells.
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Bcl-2 suppresses sarcoplasmic/endoplasmic reticulum Ca2+-ATPase expression in cystic fibrosis airways: role in oxidant-mediated cell death.
Am. J. Respir. Crit. Care Med.
PUBLISHED: 02-06-2009
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Modulation of the activity of sarcoendoplasmic reticulum calcium ATPase (SERCA) can profoundly affect Ca(2+) homeostasis. Although altered calcium homeostasis is a characteristic of cystic fibrosis (CF), the role of SERCA is unknown.
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The N-terminal HSDCIF motif is required for cell surface trafficking and dimerization of family B G protein coupled receptor PAC1.
PLoS ONE
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PAC1 is PACAP (pituitary adenylate cyclase-activating polypeptide) preferring receptor belonging to class B G protein coupled receptor (GPCR) mediating the most effects of PACAP. The important role of G protein coupled receptor homo/heteromerization in receptor folding, maturation, trafficking, and cell surface expression has become increasingly evident. The bimolecular fluorescence complementation (BiFC) and bioluminescence resonance energy transfer (BRET) assay were used in this research to confirm the dimerization of PAC1 for the first time. The structure-activity relationship focused on the N-terminal HSDCIF motif, which locates behind the signal sequence and has high homology with PACAP (1-6), was assayed using a receptor mutant with the deletion of the HSDCIF motif. The fluorescence confocal microscope observation showed that the deletion of the HSDCIF motif impaired the cell delivery of PAC1. The results of BiFC, BRET and westernblot indicated that the deletion of HSDCIF motif and the replacement of the Cys residue with Ala in HSDCIF motif resulted in the disruption of receptor dimerization. And the exogenous chemically synthesized oligopeptide HSDCIF (100 nmol/L) not only down-regulated the dimerization of PAC1, induced the internalization of PAC1, but also inhibited the proliferation of CHO cells expressing PAC1 stably and decreased the activity of PACAP on the cell viability. All these data suggested that the N-terminal HSDCIF motif played key role in the trafficking and the dimerization of PAC1, and the exogenous oligopeptide HSDCIF had effects on the cell signaling, trafficking and the dimerization of PAC1.
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The expression of PAC1 increases in the degenerative thymus and low dose PACAP protects female mice from cyclophosphamide induced thymus atrophy.
Peptides
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Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with cytoprotective ability mediated by its specific receptor PAC1. In this research, firstly the thymus index and the expression of PAC1 in the normal and degenerative thymus with different gender were assayed; secondly PACAP in different dose was used to treat the female mice with cyclophosphamide (CPS) and the changes in thymus index, the expression of PAC1, histopathology, apoptosis, oxidative status and the caspase 3 activity in thymus were determined and compared. It was found that in the mice of age from 1 to 9 weeks in the stage of sex development, the thymus index was significantly higher in female mice than in male mice. And it was found for the first time that the PAC1 expression level in thymus of female mice was significantly higher than that of male mice and the expression of the PAC1 and PACAP increased significantly in the degenerative thymus induced by CPS. After PACAP was co-injected with CPS to the female mice, it was shown that only low dose (1 nmol/kg) of PACAP promoted the thymus index, inhibited the cell apoptosis, ameliorated the oxidative status and decreased the caspase activity significantly, while high dose (10 nmol/kg) of PACAP had no significant protective effects against CPS-induced thymus atrophy. It was concluded that the expression of PAC1 in the thymus changes in reverse ratio with thymus index and in direct ratio with cell apoptosis and only low dose of PACAP had positive effects against the CPS-induced thymus atrophy.
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The novel peptide PACAP-TAT with enhanced traversing ability attenuates the severe lung injury induced by repeated smoke inhalation.
Peptides
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Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potential therapeutic peptide with anti-inflammatory and anti-oxidative effects. In order to increase the efficiency of traversing biological barriers, a novel fusion peptide PACAP-TAT was produced by tagging PACAP at its C-terminus with 11-amino acid TAT protein transduction domain. The results of characteristic assays showed that PACAP-TAT activated PACAP specific receptor PAC1 with the same potency as PACAP and PACAP-TAT crossed blood-brain barrier (BBB), blood-air barrier (BAB) and blood-testis barrier (BTB) with the efficiency about 2.5-fold higher than that of PACAP. Both PACAP-TAT and PACAP were used treat the mice with lung injury induced by repeated smoke inhalation. It was shown that both PACAP-TAT and PACAP decreased the mortality, increased the body weight and inhibited the edema and vascular permeability in the lungs of the mice received repeated smoke inhalation, while PACAP-TAT displayed more marked effects than PACAP. PACAP-TAT decreased myeloperoxidase (MPO) activity, increased catalase (CAT) activity and down-regulated interleukin 6 (IL-6) and malondialdehyde (MDA) levels in the lungs with a significantly higher efficiency than PACAP. The histopathological analysis also showed that PACAP-TAT attenuated the cell filtration and bronchi epithelial hyperplasia more significantly than PACAP. Moreover the leukocyte count in blood and the serum superoxide dismutase (SOD) activity in the mice treated with PACAP-TAT were significantly different from that in mice treated with PACAP (p<0.05). All these data indicated that PACAP-TAT with increased traversing ability was more effective than PACAP in protecting the mice from the lung injury induced by repeated smoke inhalation.
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The TAT peptide endows PACAP with an enhanced ability to traverse bio-barriers.
Neurosci. Lett.
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Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potential therapeutic neuropeptide. The 11-amino acid human immunodeficiency virus TAT protein transduction domain is able to deliver protein cargoes across the cell membrane and the blood-brain barrier. A novel fusion protein PACAP-TAT, containing TAT at the C-terminus of PACAP was therefore produced and studied for the ability to cross blood barriers. The gene encoding PACAP-TAT was cloned into the expression vector pKYB, and the target peptide PACAP-TAT was purified using the Intein Mediated Purification with an Affinity Chitin-binding Tag (IMPACT) system. The results of cell assays showed that PACAP-TAT stimulated the cell viability of PAC1-CHO cells with the same potency as PACAP, which indicated that the fusion of TAT did not affect the ability of PACAP-TAT to activate the PACAP-specific receptor PAC1. The transfer efficiencies of PACAP-TAT and PACAP across the blood-brain barrier (BBB), blood-air barrier (BAB) and blood-testis barrier (BTB) were assayed using peptides labeled with fluorescein isothiocyanate (FITC). The results showed that PACAP-TAT traversed blood barriers with an efficiency approximately 2.5-fold greater than PACAP. Fluorescence microscopic examination showed that PACAP-TAT traversed the BBB significantly more efficiently than PACAP. Furthermore, intraperitoneal (i.p.) injection of PACAP-TAT induced a stronger inhibitory effect on food intake than PACAP (p<0.01, PACAP-TAT vs. PACAP), which indicated that TAT helped to increase the localization of PACAP-TAT in the brain. Preparation of PACAP-TAT with the enhanced ability to cross biological barriers will improve its route of administration and expand its scope of application.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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