Interaction between the cardiac rapidly (IKr) and slowly (IKs) activating delayed rectifier potassium channels revealed by low K+-induced hERG endocytic degradation.

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Guo, Jun; Wang, Tingzhong; Yang, Tonghua; Xu, Jianmin; Li, Wentao; Fridman, Michael D; Fisher, John T; Zhang, Shetuan

2011-10-07

Cardiac repolarization is controlled by the rapidly (I(Kr)) and slowly (I(Ks)) activating delayed rectifier potassium channels. The human ether-a-go-go-related gene (hERG) encodes I(Kr), whereas KCNQ1 and KCNE1 together encode I(Ks). Decreases in I(Kr) or I(Ks) cause long QT syndrome (LQTS), a cardiac disorder with a high risk of sudden death. A reduction in extracellular K(+) concentration ([K(+)](o)) induces LQTS and selectively causes endocytic degradation of mature hERG channels from the plasma membrane. In the present study, we investigated whether I(Ks) compensates for the reduced I(Kr) under low K(+) conditions. Our data show that when hERG and KCNQ1 were expressed separately in human embryonic kidney (HEK) cells, exposure to 0 mM K(+) for 6 h completely eliminated the mature hERG channel expression but had no effect on KCNQ1. When hERG and KCNQ1 were co-expressed, KCNQ1 significantly delayed 0 mM K(+)-induced hERG reduction. Also, hERG degradation led to a significant reduction in KCNQ1 in 0 mM K(+) conditions. An interaction between hERG and KCNQ1 was identified in hERG+KCNQ1-expressing HEK cells. Furthermore, KCNQ1 preferentially co-immunoprecipitated with mature hERG channels that are localized in the plasma membrane. Biophysical and pharmacological analyses indicate that although hERG and KCNQ1 closely interact with each other, they form distinct hERG and KCNQ1 channels. These data extend our understanding of delayed rectifier potassium channel trafficking and regulation, as well as the pathology of LQTS.

Endocytosis of HERG is clathrin-independent and involves arf6.

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Rucha Karnik

Full Text Available The hERG potassium channel is critical for repolarisation of the cardiac action potential. Reduced expression of hERG at the plasma membrane, whether caused by hereditary mutations or drugs, results in long QT syndrome and increases the risk of ventricular arrhythmias. Thus, it is of fundamental importance to understand how the density of this channel at the plasma membrane is regulated. We used antibodies to an extracellular native or engineered epitope, in conjunction with immunofluorescence and ELISA, to investigate the mechanism of hERG endocytosis in recombinant cells and validated the findings in rat neonatal cardiac myocytes. The data reveal that this channel undergoes rapid internalisation, which is inhibited by neither dynasore, an inhibitor of dynamin, nor a dominant negative construct of Rab5a, into endosomes that are largely devoid of the transferrin receptor. These results support a clathrin-independent mechanism of endocytosis and exclude involvement of dynamin-dependent caveolin and RhoA mechanisms. In agreement, internalised hERG displayed marked overlap with glycosylphosphatidylinositol-anchored GFP, a clathrin-independent cargo. Endocytosis was significantly affected by cholesterol extraction with methyl-β-cyclodextrin and inhibition of Arf6 function with dominant negative Arf6-T27N-eGFP. Taken together, we conclude that hERG undergoes clathrin-independent endocytosis via a mechanism involving Arf6.

Inhibition of HERG potassium channels by celecoxib and its mechanism.

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Roman V Frolov

Full Text Available Celecoxib (Celebrex, a widely prescribed selective inhibitor of cyclooxygenase-2, can modulate ion channels independently of cyclooxygenase inhibition. Clinically relevant concentrations of celecoxib can affect ionic currents and alter functioning of neurons and myocytes. In particular, inhibition of Kv2.1 channels by celecoxib leads to arrhythmic beating of Drosophila heart and of rat heart cells in culture. However, the spectrum of ion channels involved in human cardiac excitability differs from that in animal models, including mammalian models, making it difficult to evaluate the relevance of these observations to humans. Our aim was to examine the effects of celecoxib on hERG and other human channels critically involved in regulating human cardiac rhythm, and to explore the mechanisms of any observed effect on the hERG channels.Celecoxib inhibited the hERG, SCN5A, KCNQ1 and KCNQ1/MinK channels expressed in HEK-293 cells with IC(50s of 6.0 µM, 7.5 µM, 3.5 µM and 3.7 µM respectively, and the KCND3/KChiP2 channels expressed in CHO cells with an IC(50 of 10.6 µM. Analysis of celecoxib's effects on hERG channels suggested gating modification as the mechanism of drug action.The above channels play a significant role in drug-induced long QT syndrome (LQTS and short QT syndrome (SQTS. Regulatory guidelines require that all new drugs under development be tested for effects on the hERG channel prior to first administration in humans. Our observations raise the question of celecoxib's potential to induce cardiac arrhythmias or other channel related adverse effects, and make a case for examining such possibilities.

Indexing molecules for their hERG liability.

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Rayan, Anwar; Falah, Mizied; Raiyn, Jamal; Da'adoosh, Beny; Kadan, Sleman; Zaid, Hilal; Goldblum, Amiram

2013-07-01

The human Ether-a-go-go-Related-Gene (hERG) potassium (K(+)) channel is liable to drug-inducing blockage that prolongs the QT interval of the cardiac action potential, triggers arrhythmia and possibly causes sudden cardiac death. Early prediction of drug liability to hERG K(+) channel is therefore highly important and preferably obligatory at earlier stages of any drug discovery process. In vitro assessment of drug binding affinity to hERG K(+) channel involves substantial expenses, time, and labor; and therefore computational models for predicting liabilities of drug candidates for hERG toxicity is of much importance. In the present study, we apply the Iterative Stochastic Elimination (ISE) algorithm to construct a large number of rule-based models (filters) and exploit their combination for developing the concept of hERG Toxicity Index (ETI). ETI estimates the molecular risk to be a blocker of hERG potassium channel. The area under the curve (AUC) of the attained model is 0.94. The averaged ETI of hERG binders, drugs from CMC, clinical-MDDR, endogenous molecules, ACD and ZINC, were found to be 9.17, 2.53, 3.3, -1.98, -2.49 and -3.86 respectively. Applying the proposed hERG Toxicity Index Model on external test set composed of more than 1300 hERG blockers picked from chEMBL shows excellent performance (Matthews Correlation Coefficient of 0.89). The proposed strategy could be implemented for the evaluation of chemicals in the hit/lead optimization stages of the drug discovery process, improve the selection of drug candidates as well as the development of safe pharmaceutical products. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Endocytosis of hERG Is Clathrin-Independent and Involves Arf6

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Abuarab, Nada; Smith, Andrew J.; Hardy, Matthew E. L.; Elliott, David J. S.; Sivaprasadarao, Asipu

2013-01-01

The hERG potassium channel is critical for repolarisation of the cardiac action potential. Reduced expression of hERG at the plasma membrane, whether caused by hereditary mutations or drugs, results in long QT syndrome and increases the risk of ventricular arrhythmias. Thus, it is of fundamental importance to understand how the density of this channel at the plasma membrane is regulated. We used antibodies to an extracellular native or engineered epitope, in conjunction with immunofluorescence and ELISA, to investigate the mechanism of hERG endocytosis in recombinant cells and validated the findings in rat neonatal cardiac myocytes. The data reveal that this channel undergoes rapid internalisation, which is inhibited by neither dynasore, an inhibitor of dynamin, nor a dominant negative construct of Rab5a, into endosomes that are largely devoid of the transferrin receptor. These results support a clathrin-independent mechanism of endocytosis and exclude involvement of dynamin-dependent caveolin and RhoA mechanisms. In agreement, internalised hERG displayed marked overlap with glycosylphosphatidylinositol-anchored GFP, a clathrin-independent cargo. Endocytosis was significantly affected by cholesterol extraction with methyl-β-cyclodextrin and inhibition of Arf6 function with dominant negative Arf6-T27N-eGFP. Taken together, we conclude that hERG undergoes clathrin-independent endocytosis via a mechanism involving Arf6. PMID:24392021

Effect of beta-adrenoceptor blockers on human ether-a-go-go-related gene (HERG) potassium channels

DEFF Research Database (Denmark)

Dupuis, Delphine S; Klaerke, Dan A; Olesen, Søren-Peter

2005-01-01

Patients with congenital long QT syndrome may develop arrhythmias under conditions of increased sympathetic tone. We have addressed whether some of the beta-adrenoceptor blockers commonly used to prevent the development of these arrhythmias could per se block the cardiac HERG (Human Ether....... These data showed that HERG blockade by beta-adrenoceptor blockers occurred only at high micromolar concentrations, which are significantly above the recently established safe margin of 100 (Redfern et al., 2003).......-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride) blocked the HERG channel with similar affinity, whereas the beta1-receptor antagonists metoprolol and atenolol showed weak effects. Further, the four compounds blocked HERG channels expressed in a mammalian HEK293 cell line...

MiR-17-5p impairs trafficking of H-ERG K+ channel protein by targeting multiple er stress-related chaperones during chronic oxidative stress.

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Qi Wang

Full Text Available BACKGROUND: To investigate if microRNAs (miRNAs play a role in regulating h-ERG trafficking in the setting of chronic oxidative stress as a common deleterious factor for many cardiac disorders. METHODS: We treated neonatal rat ventricular myocytes and HEK293 cells with stable expression of h-ERG with H2O2 for 12 h and 48 h. Expression of miR-17-5p seed miRNAs was quantified by real-time RT-PCR. Protein levels of chaperones and h-ERG trafficking were measured by Western blot analysis. Luciferase reporter gene assay was used to study miRNA and target interactions. Whole-cell patch-clamp techniques were employed to record h-ERG K(+ current. RESULTS: H-ERG trafficking was impaired by H2O2 after 48 h treatment, accompanied by reciprocal changes of expression between miR-17-5p seed miRNAs and several chaperones (Hsp70, Hsc70, CANX, and Golga2, with the former upregulated and the latter downregulated. We established these chaperones as targets for miR-17-5p. Application miR-17-5p inhibitor rescued H2O2-induced impairment of h-ERG trafficking. Upregulation of endogenous by H2O2 or forced miR-17-5p expression either reduced h-ERG current. Sequestration of AP1 by its decoy molecule eliminated the upregulation of miR-17-5p, and ameliorated impairment of h-ERG trafficking. CONCLUSIONS: Collectively, deregulation of the miR-17-5p seed family miRNAs can cause severe impairment of h-ERG trafficking through targeting multiple ER stress-related chaperones, and activation of AP1 likely accounts for the deleterious upregulation of these miRNAs, in the setting of prolonged duration of oxidative stress. These findings revealed the role of miRNAs in h-ERG trafficking, which may contribute to the cardiac electrical disturbances associated with oxidative stress.

MiR-17-5p impairs trafficking of H-ERG K+ channel protein by targeting multiple er stress-related chaperones during chronic oxidative stress.

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Wang, Qi; Hu, Weina; Lei, Mingming; Wang, Yong; Yan, Bing; Liu, Jun; Zhang, Ren; Jin, Yuanzhe

2013-01-01

To investigate if microRNAs (miRNAs) play a role in regulating h-ERG trafficking in the setting of chronic oxidative stress as a common deleterious factor for many cardiac disorders. We treated neonatal rat ventricular myocytes and HEK293 cells with stable expression of h-ERG with H2O2 for 12 h and 48 h. Expression of miR-17-5p seed miRNAs was quantified by real-time RT-PCR. Protein levels of chaperones and h-ERG trafficking were measured by Western blot analysis. Luciferase reporter gene assay was used to study miRNA and target interactions. Whole-cell patch-clamp techniques were employed to record h-ERG K(+) current. H-ERG trafficking was impaired by H2O2 after 48 h treatment, accompanied by reciprocal changes of expression between miR-17-5p seed miRNAs and several chaperones (Hsp70, Hsc70, CANX, and Golga2), with the former upregulated and the latter downregulated. We established these chaperones as targets for miR-17-5p. Application miR-17-5p inhibitor rescued H2O2-induced impairment of h-ERG trafficking. Upregulation of endogenous by H2O2 or forced miR-17-5p expression either reduced h-ERG current. Sequestration of AP1 by its decoy molecule eliminated the upregulation of miR-17-5p, and ameliorated impairment of h-ERG trafficking. Collectively, deregulation of the miR-17-5p seed family miRNAs can cause severe impairment of h-ERG trafficking through targeting multiple ER stress-related chaperones, and activation of AP1 likely accounts for the deleterious upregulation of these miRNAs, in the setting of prolonged duration of oxidative stress. These findings revealed the role of miRNAs in h-ERG trafficking, which may contribute to the cardiac electrical disturbances associated with oxidative stress.

hERG1 channels are overexpressed in glioblastoma multiforme and modulate VEGF secretion in glioblastoma cell lines

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Masi, A; Becchetti, A; Restano-Cassulini, R; Polvani, S; Hofmann, G; Buccoliero, A M; Paglierani, M; Pollo, B; Taddei, G L; Gallina, P; Di Lorenzo, N; Franceschetti, S; Wanke, E; Arcangeli, A

2005-01-01

Recent studies have led to considerable advancement in our understanding of the molecular mechanisms that underlie the relentless cell growth and invasiveness of human gliomas. Partial understanding of these mechanisms has (1) improved the classification for gliomas, by identifying prognostic subgroups, and (2) pointed to novel potential therapeutic targets. Some classes of ion channels have turned out to be involved in the pathogenesis and malignancy of gliomas. We studied the expression and properties of K+ channels in primary cultures obtained from surgical specimens: human ether a gò-gò related (hERG)1 voltage-dependent K+ channels, which have been found to be overexpressed in various human cancers, and human ether a gò-gò-like 2 channels, that share many of hERG1's biophysical features. The expression pattern of these two channels was compared to that of the classical inward rectifying K+ channels, IRK, that are widely expressed in astrocytic cells and classically considered a marker of astrocytic differentiation. In our study, hERG1 was found to be specifically overexpressed in high-grade astrocytomas, that is, glioblastoma multiforme (GBM). In addition, we present evidence that, in GBM cell lines, hERG1 channel activity actively contributes to malignancy by promoting vascular endothelial growth factor secretion, thus stimulating the neoangiogenesis typical of high-grade gliomas. Our data provide important confirmation for studies proposing the hERG1 channel as a molecular marker of tumour progression and a possible target for novel anticancer therapies. PMID:16175187

Characterization of a Madin-Darby canine kidney cell line stably expressing TRPV5.

NARCIS (Netherlands)

Dekker, E. den; Schoeber, J.P.H.; Topala, C.N.; Graaf, S.F.J. van de; Hoenderop, J.G.J.; Bindels, R.J.M.

2005-01-01

To provide a cell model for studying specifically the regulation of Ca2+ entry by the epithelial calcium channel transient receptor potential-vanilloid-5 (TRPV5), green fluorescent protein (GFP)-tagged TRPV5 was expressed stably in Madin-Darby canine kidney type I (MDCK) cells. The localization of

MiR-17-5p Impairs Trafficking of H-ERG K+ Channel Protein by Targeting Multiple ER Stress-Related Chaperones during Chronic Oxidative Stress

OpenAIRE

Wang, Qi; Hu, Weina; Lei, Mingming; Wang, Yong; Yan, Bing; Liu, Jun; Zhang, Ren; Jin, Yuanzhe

2013-01-01

BACKGROUND: To investigate if microRNAs (miRNAs) play a role in regulating h-ERG trafficking in the setting of chronic oxidative stress as a common deleterious factor for many cardiac disorders. METHODS: We treated neonatal rat ventricular myocytes and HEK293 cells with stable expression of h-ERG with H2O2 for 12 h and 48 h. Expression of miR-17-5p seed miRNAs was quantified by real-time RT-PCR. Protein levels of chaperones and h-ERG trafficking were measured by Western blot analysis. Lucifer...

A novel hypothesis for the binding mode of HERG channel blockers

International Nuclear Information System (INIS)

Choe, Han; Nah, Kwang Hoon; Lee, Soo Nam; Lee, Han Sam; Lee, Hui Sun; Jo, Su Hyun; Leem, Chae Hun; Jang, Yeon Jin

2006-01-01

We present a new docking model for HERG channel blockade. Our new model suggests three key interactions such that (1) a protonated nitrogen of the channel blocker forms a hydrogen bond with the carbonyl oxygen of HERG residue T623; (2) an aromatic moiety of the channel blocker makes a π-π interaction with the aromatic ring of HERG residue Y652; and (3) a hydrophobic group of the channel blocker forms a hydrophobic interaction with the benzene ring of HERG residue F656. The previous model assumes two interactions such that (1) a protonated nitrogen of the channel blocker forms a cation-π interaction with the aromatic ring of HERG residue Y652; and (2) a hydrophobic group of the channel blocker forms a hydrophobic interaction with the benzene ring of HERG residue F656. To test these models, we classified 69 known HERG channel blockers into eight binding types based on their plausible binding modes, and further categorized them into two groups based on the number of interactions our model would predict with the HERG channel (two or three). We then compared the pIC 5 value distributions between these two groups. If the old hypothesis is correct, the distributions should not differ between the two groups (i.e., both groups show only two binding interactions). If our novel hypothesis is correct, the distributions should differ between Groups 1 and 2. Consistent with our hypothesis, the two groups differed with regard to pIC 5 , and the group having more predicted interactions with the HERG channel had a higher mean pIC 5 value. Although additional work will be required to further validate our hypothesis, this improved understanding of the HERG channel blocker binding mode may help promote the development of in silico predictions methods for identifying potential HERG channel blockers

The role of hERG1 ion channels in epithelial-mesenchymal transition and the capacity of riluzole to reduce cisplatin resistance in colorectal cancer cells.

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Fortunato, Angelo

2017-08-01

The transition of cells from the epithelial to the mesenchymal state (EMT) plays an important role in tumor progression. EMT allows cells to acquire mobility, stem-like behavior and resistance to apoptosis and drug treatment. These features turn EMT into a central process in tumor biology. Ion channels are attractive targets for the treatment of cancer since they play critical roles in controlling a wide range of physiological processes that are frequently deregulated in cancer. Here, we investigated the role of ether-a-go-go-related 1 (hERG1) ion channels in the EMT of colorectal cancer cells. We studied the epithelial-mesenchymal profile of different colorectal cancer-derived cell lines and the expression of hERG1 potassium channels in these cell lines using real-time PCR. Next, we knocked down hERG1 expression in HCT116 cells using lentivirus mediated RNA interference and characterized the hERG1 silenced cells in vitro and in vivo. Finally, we investigated the capacity of riluzole, an ion channel-modulating drug used in humans to treat amyotrophic lateral sclerosis, to reduce the resistance of the respective colorectal cancer cells to the chemotherapeutic drug cisplatin. We found that of the colorectal cancer-derived cell lines tested, HCT116 showed the highest mesenchymal profile and a high hERG1 expression. Subsequent hERG1 expression knockdown induced a change in cell morphology, which was accompanied by a reduction in the proliferative and tumorigenic capacities of the cells. Notably, we found that hERG1expression knockdown elicited a reversion of the EMT profile in HCT116 cells with a reacquisition of the epithelial-like profile. We also found that riluzole increased the sensitivity of HCT116 cisplatin-resistant cells to cisplatin. Our data indicate that hERG1 plays a role in the EMT of colorectal cancer cells and that its knockdown reduces the proliferative and tumorigenic capacities of these cells. In addition, we conclude that riluzole may be used in

In vitro chronic effects on hERG channel caused by the marine biotoxin Yessotoxin.

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Sara Fernández Ferreiro

2014-06-01

Currently, published evidence indicates that hERG channel dysfunction can be due to more than one mechanism for many drugs (Guth, 2007. Alterations of hERG channel trafficking are considered an important factor in hERG-related cardiotoxicity. Actually, a screening study revealed that almost 40% of the drugs that block Ikr have also trafficking effects (Wible et al., 2005. Although YTX does not block hERG channels, it has been historically described as cardiotoxic due to in vivo damage to cardiomyocytes. Our results show that YTX induces a significant increase of hERG channel levels on the extracellular side of the plasma membrane in vitro. YTX causes cell death in many cell lines (Korsnes and Espenes, 2011 and the alterations of surface hERG levels might be related to the apoptotic process. However, annexin-V, a relatively early marker of apoptosis (Vermes et al., 1995, occurs later than the increase of surface hERG. Additionally, staurosporine triggered apoptosis without a simultaneous increase of surface hERG, so events are not necessarily related. Therefore YTX-induced elevated hERG in the plasma membrane seem to be independent of apoptosis. Functional implications of hERG currents have been described after alterations of cell surface hERG density (Guth, 2007. YTX did not cause significant alterations of hERG currents. Furthermore the hERG levels after YTX treatment were duplicated, so the effect on currents should be clearly evidenced if these channels were functional. The hERG channels on the cell surface are regulated by its production, translocation to the plasma membrane and degradation. The increase of extracellular channel could be a consequence of a higher production and externalization or a slower degradation. Higher synthesis in our cell model would not be physiologically relevant but our results demonstrated that the amount of immature hERG is reduced instead of increased. Fully glycosylated hERG seems slightly increased in these conditions but it is

Voltage-Dependent Gating of hERG Potassium Channels

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Cheng, Yen May; Claydon, Tom W.

2012-01-01

The mechanisms by which voltage-gated channels sense changes in membrane voltage and energetically couple this with opening of the ion conducting pore has been the source of significant interest. In voltage-gated potassium (Kv) channels, much of our knowledge in this area comes from Shaker-type channels, for which voltage-dependent gating is quite rapid. In these channels, activation and deactivation are associated with rapid reconfiguration of the voltage-sensing domain unit that is electromechanically coupled, via the S4–S5 linker helix, to the rate-limiting opening of an intracellular pore gate. However, fast voltage-dependent gating kinetics are not typical of all Kv channels, such as Kv11.1 (human ether-à-go-go related gene, hERG), which activates and deactivates very slowly. Compared to Shaker channels, our understanding of the mechanisms underlying slow hERG gating is much poorer. Here, we present a comparative review of the structure–function relationships underlying activation and deactivation gating in Shaker and hERG channels, with a focus on the roles of the voltage-sensing domain and the S4–S5 linker that couples voltage sensor movements to the pore. Measurements of gating current kinetics and fluorimetric analysis of voltage sensor movement are consistent with models suggesting that the hERG activation pathway contains a voltage independent step, which limits voltage sensor transitions. Constraints upon hERG voltage sensor movement may result from loose packing of the S4 helices and additional intra-voltage sensor counter-charge interactions. More recent data suggest that key amino acid differences in the hERG voltage-sensing unit and S4–S5 linker, relative to fast activating Shaker-type Kv channels, may also contribute to the increased stability of the resting state of the voltage sensor. PMID:22586397

Voltage-dependent gating of hERG potassium channels

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Yen May eCheng

2012-05-01

Full Text Available The mechanisms by which voltage-gated channels sense changes in membrane voltage and energetically couple this with opening of the ion conducting pore has been the source of significant interest. In voltage-gated potassium (Kv channels, much of our knowledge in this area comes from Shaker-type channels, for which voltage-dependent gating is quite rapid. In these channels, activation and deactivation are associated with rapid reconfiguration of the voltage-sensing domain unit that is electromechanically coupled, via the S4-S5 linker helix, to the rate-limiting opening of an intracellular pore gate. However, fast voltage-dependent gating kinetics are not typical of all Kv channels, such as Kv11.1 (human ether-a-go-go related gene, hERG, which activates and deactivates very slowly. Compared to Shaker channels, our understanding of the mechanisms underlying slow hERG gating is much poorer. Here, we present a comparative review of the structure-function relationships underlying voltage-dependent gating in Shaker and hERG channels, with a focus on the roles of the voltage sensing domain and the S4-S5 linker that couples voltage sensor movements to the pore. Measurements of gating current kinetics and fluorimetric analysis of voltage sensor movement are consistent with models suggesting that the hERG activation pathway contains a voltage independent step, which limits voltage sensor transitions. Constraints upon hERG voltage sensor movement may result from loose packing of the S4 helices and additional intra-voltage sensor counter charge interactions. More recent data suggest that key amino acid differences in the hERG voltage sensing unit and S4-S5 linker, relative to fast activating Shaker-type Kv channels, may also contribute to the increased stability of the resting state of the voltage sensor.

Homozygous premature truncation of the HERG protein : the human HERG knockout

NARCIS (Netherlands)

Hoorntje, T.; Alders, M.; van Tintelen, P.; van der Lip, K.; Sreeram, N.; van der Wal, A.; Mannens, M.; Wilde, A.

1999-01-01

Background-In long-QT syndrome (LQTS), heterozygosity for a mutation in 1 of the K(+) channel genes leads to prolongation of the cardiac action potential, because the aberrant protein exhibits "loss of function." HERG, which is involved in LQT2, is the gene encoding the rapid component of the

Anti-HERG activity and the risk of drug-induced arrhythmias and sudden death

DEFF Research Database (Denmark)

De Bruin, M L; Pettersson, M; Meyboom, R H B

2005-01-01

AIMS: Drug-induced QTc-prolongation, resulting from inhibition of HERG potassium channels may lead to serious ventricular arrhythmias and sudden death. We studied the quantitative anti-HERG activity of pro-arrhythmic drugs as a risk factor for this outcome in day-to-day practice. METHODS...... defined as reports of cardiac arrest, sudden death, torsade de pointes, ventricular fibrillation, and ventricular tachycardia (n = 5591), and compared with non-cases regarding the anti-HERG activity, defined as the effective therapeutic plasma concentration (ETCPunbound) divided by the HERG IC50 value......, of suspected drugs. We identified a significant association of 1.93 (95% CI: 1.89-1.98) between the anti-HERG activity of drugs, measured as log10 (ETCPunbound/IC50), and reporting of serious ventricular arrhythmias and sudden death to the WHO-UMC database. CONCLUSION: Anti-HERG activity is associated...

Oxycodone is associated with dose-dependent QTc prolongation in patients and low-affinity inhibiting of hERG activity in vitro

DEFF Research Database (Denmark)

Fanoe, Søren; Jensen, Gorm Boje; Sjøgren, Per

2008-01-01

with the use of these drugs. WHAT THIS PAPER ADDS: This study is the first to show that oxycodone dose is associated with QT prolongation and in vitro blockade of hERG channels expressed in HEK293. Neither morphine nor tramadol doses are associated with the QT interval length. AIMS: During recent years some...... and TdP could be a more general problem associated with the use of these drugs. The aims of this study were to evaluate the association between different opioids and the QTc among patients and measure hERG activity under influence by opioids in vitro. METHODS: One hundred chronic nonmalignant pain...... patients treated with methadone, oxycodone, morphine or tramadol were recruited in a cross-sectional study. The QTc was estimated from a 12-lead ECG. To examine hERG activity in the presence of oxycodone, electrophysiological testing was conducted using Xenopus laevis oocytes and HEK293 cells expressing h...

Bag1 Co-chaperone Promotes TRC8 E3 Ligase-dependent Degradation of Misfolded Human Ether a Go-Go-related Gene (hERG) Potassium Channels.

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Hantouche, Christine; Williamson, Brittany; Valinsky, William C; Solomon, Joshua; Shrier, Alvin; Young, Jason C

2017-02-10

Cardiac long QT syndrome type 2 is caused by mutations in the human ether a go-go-related gene (hERG) potassium channel, many of which cause misfolding and degradation at the endoplasmic reticulum instead of normal trafficking to the cell surface. The Hsc70/Hsp70 chaperones assist the folding of the hERG cytosolic domains. Here, we demonstrate that the Hsp70 nucleotide exchange factor Bag1 promotes hERG degradation by the ubiquitin-proteasome system at the endoplasmic reticulum to regulate hERG levels and channel activity. Dissociation of hERG complexes containing Hsp70 and the E3 ubiquitin ligase CHIP requires the interaction of Bag1 with Hsp70, but this does not involve the Bag1 ubiquitin-like domain. The interaction with Bag1 then shifts hERG degradation to the membrane-anchored E3 ligase TRC8 and its E2-conjugating enzyme Ube2g2, as determined by siRNA screening. TRC8 interacts through the transmembrane region with hERG and decreases hERG functional expression. TRC8 also mediates degradation of the misfolded hERG-G601S disease mutant, but pharmacological stabilization of the mutant structure prevents degradation. Our results identify TRC8 as a previously unknown Hsp70-independent quality control E3 ligase for hERG. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

High yield purification of full-length functional hERG K+ channels produced in Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Molbaek, Karen; Scharff-Poulsen, Peter; Hélix-Nielsen, Claus

2015-01-01

knowledge this is the first reported high-yield production and purification of full length, tetrameric and functional hERG. This significant breakthrough will be paramount in obtaining hERG crystal structures, and in establishment of new high-throughput hERG drug safety screening assays....

A molecular switch driving inactivation in the cardiac K+ channel HERG.

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David A Köpfer

Full Text Available K(+ channels control transmembrane action potentials by gating open or closed in response to external stimuli. Inactivation gating, involving a conformational change at the K(+ selectivity filter, has recently been recognized as a major K(+ channel regulatory mechanism. In the K(+ channel hERG, inactivation controls the length of the human cardiac action potential. Mutations impairing hERG inactivation cause life-threatening cardiac arrhythmia, which also occur as undesired side effects of drugs. In this paper, we report atomistic molecular dynamics simulations, complemented by mutational and electrophysiological studies, which suggest that the selectivity filter adopts a collapsed conformation in the inactivated state of hERG. The selectivity filter is gated by an intricate hydrogen bond network around residues S620 and N629. Mutations of this hydrogen bond network are shown to cause inactivation deficiency in electrophysiological measurements. In addition, drug-related conformational changes around the central cavity and pore helix provide a functional mechanism for newly discovered hERG activators.

New aspects of HERG K⁺ channel function depending upon cardiac spatial heterogeneity.

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Pen Zhang

Full Text Available HERG K(+ channel, the genetic counterpart of rapid delayed rectifier K(+ current in cardiac cells, is responsible for many cases of inherited and drug-induced long QT syndromes. HERG has unusual biophysical properties distinct from those of other K(+ channels. While the conventional pulse protocols in patch-clamp studies have helped us elucidate these properties, their limitations in assessing HERG function have also been progressively noticed. We employed AP-clamp techniques using physiological action potential waveforms recorded from various regions of canine heart to study HERG function in HEK293 cells and identified several novel aspects of HERG function. We showed that under AP-clamp IHERG increased gradually with membrane repolarization, peaked at potentials around 20-30 mV more negative than revealed by pulse protocols and at action potential duration (APD to 60%-70% full repolarization, and fell rapidly at the terminal phase of repolarization. We found that the rising phase of IHERG was conferred by removal of inactivation and the decaying phase resulted from a fall in driving force, which were all determined by the rate of membrane repolarization. We identified regional heterogeneity and transmural gradient of IHERG when quantified with the area covered by IHERG trace. In addition, we observed regional and transmural differences of IHERG in response to dofetilide blockade. Finally, we characterized the influence of HERG function by selective inhibition of other ion currents. Based on our results, we conclude that the distinct biophysical properties of HERG reported by AP-clamp confer its unique function in cardiac repolarization thereby in antiarrhythmia and arrhythmogenesis.

Towards a Structural View of Drug Binding to hERG K+ Channels.

Science.gov (United States)

Vandenberg, Jamie I; Perozo, Eduardo; Allen, Toby W

2017-10-01

The human ether-a-go-go-related gene (hERG) K + channel is of great medical and pharmaceutical relevance. Inherited mutations in hERG result in congenital long-QT syndrome which is associated with a markedly increased risk of cardiac arrhythmia and sudden death. hERG K + channels are also remarkably susceptible to block by a wide range of drugs, which in turn can cause drug-induced long-QT syndrome and an increased risk of sudden death. The recent determination of the near-atomic resolution structure of the hERG K + channel, using single-particle cryo-electron microscopy (cryo-EM), provides tremendous insights into how these channels work. It also suggests a way forward in our quest to understand why these channels are so promiscuous with respect to drug binding. Copyright © 2017 Elsevier Ltd. All rights reserved.

Determinants of Isoform-Specific Gating Kinetics of hERG1 Channel: Combined Experimental and Simulation Study

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Laura L. Perissinotti

2018-04-01

Full Text Available IKr is the rapidly activating component of the delayed rectifier potassium current, the ion current largely responsible for the repolarization of the cardiac action potential. Inherited forms of long QT syndrome (LQTS (Lees-Miller et al., 1997 in humans are linked to functional modifications in the Kv11.1 (hERG ion channel and potentially life threatening arrhythmias. There is little doubt now that hERG-related component of IKr in the heart depends on the tetrameric (homo- or hetero- channels formed by two alternatively processed isoforms of hERG, termed hERG1a and hERG1b. Isoform composition (hERG1a- vs. the b-isoform has recently been reported to alter pharmacologic responses to some hERG blockers and was proposed to be an essential factor pre-disposing patients for drug-induced QT prolongation. Very little is known about the gating and pharmacological properties of two isoforms in heart membranes. For example, how gating mechanisms of the hERG1a channels differ from that of hERG1b is still unknown. The mechanisms by which hERG 1a/1b hetero-tetramers contribute to function in the heart, or what role hERG1b might play in disease are all questions to be answered. Structurally, the two isoforms differ only in the N-terminal region located in the cytoplasm: hERG1b is 340 residues shorter than hERG1a and the initial 36 residues of hERG1b are unique to this isoform. In this study, we combined electrophysiological measurements for HEK cells, kinetics and structural modeling to tease out the individual contributions of each isoform to Action Potential formation and then make predictions about the effects of having various mixture ratios of the two isoforms. By coupling electrophysiological data with computational kinetic modeling, two proposed mechanisms of hERG gating in two homo-tetramers were examined. Sets of data from various experimental stimulation protocols (HEK cells were analyzed simultaneously and fitted to Markov-chain models (M

A novel assessment of nefazodone-induced hERG inhibition by electrophysiological and stereochemical method

Energy Technology Data Exchange (ETDEWEB)

Shin, Dae-Seop; Park, Myoung Joo [Drug Discovery Platform Technology Research Group, Korea Research Institute of Chemical Technology, Daejeon (Korea, Republic of); Lee, Hyang-Ae [Korea Institute of Toxicology, Korea Research Institute of Chemical Technology, Daejeon (Korea, Republic of); Lee, Joo Yun; Chung, Hee-Chung; Yoo, Dae Seok; Chae, Chong Hak [Drug Discovery Platform Technology Research Group, Korea Research Institute of Chemical Technology, Daejeon (Korea, Republic of); Park, Sang-Joon [College of Veterinary Medicine, Kyungpook National University, Daegu (Korea, Republic of); Kim, Ki-Suk [Korea Institute of Toxicology, Korea Research Institute of Chemical Technology, Daejeon (Korea, Republic of); Bae, Myung Ae, E-mail: mbae@krict.re.kr [Drug Discovery Platform Technology Research Group, Korea Research Institute of Chemical Technology, Daejeon (Korea, Republic of)

2014-02-01

Nefazodone was used widely as an antidepressant until it was withdrawn from the U.S. market in 2004 due to hepatotoxicity. We have investigated methods to predict various toxic effects of drug candidates to reduce the failure rate of drug discovery. An electrophysiological method was used to assess the cardiotoxicity of drug candidates. Small molecules, including withdrawn drugs, were evaluated using a patch-clamp method to establish a database of hERG inhibition. Nefazodone inhibited hERG channel activity in our system. However, nefazodone-induced hERG inhibition indicated only a theoretical risk of cardiotoxicity. Nefazodone inhibited the hERG channel in a concentration-dependent manner with an IC{sub 50} of 45.3 nM in HEK-293 cells. Nefazodone accelerated both the recovery from inactivation and its onset. Nefazodone also accelerated steady-state inactivation, although it did not modify the voltage-dependent character. Alanine mutants of hERG S6 and pore region residues were used to identify the nefazodone-binding site on hERG. The hERG S6 point mutants Y652A and F656A largely abolished the inhibition by nefazodone. The pore region mutant S624A mildly reduced the inhibition by nefazodone but T623A had little effect. A docking study showed that the aromatic rings of nefazodone interact with Y652 and F656 via π–π interactions, while an amine interacted with the S624 residue in the pore region. In conclusion, Y652 and F656 in the S6 domain play critical roles in nefazodone binding. - Highlights: • Nefazodone inhibits hERG channels with an IC{sub 50} of 45.3 nM in HEK-293 cells. • Nefazodone blocks hERG channels by binding to the open channels. • Y652 and F656 are important for binding of nefazodone. • The aromatic rings of nefazodone interact with Y652 and F656 via π–π interactions.

Characterization of new cell line stably expressing CHI3L1 oncogene

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Chekhonin V. P.

2011-06-01

Full Text Available Aim. To characterize the immortalized 293 cell line after stable transfection with human oncogene (CHI3L1. Methods. 293 cells, stably transfected with pcDNA3.1_CHI3L1, and 293 cells, stably transfected with pcDNA3.1 as a negative control, were used throughout all experiments. The clones of CHI3L1-expressing 293 cells and 293 cells, transfected with pcDNA3.1, were analyzed by immunofluorescence and confocal microscopy. Cell proliferation was measured using MTT assay; analyses of ERK1/2 and AKT activation and their cellular localization were performed with anti-phospho-ERK and anti-phospho-AKT antibodies. Specific activation of MAP and PI3 kinases was measured by densitometric analysis of Western-blot signals. Results. The obtained results show quite modest ability of CHI3L1 to stimulate cell growth and reflect rather an improved cellular plating efficiency of the 293 cells stably transfected with pcDNA3.1_CHI3L1 as compared to the 293 cells transfected with an «empty» vector. ERK1/2 and AKT are activated in the 293_CHI3L1 cells. In these cells phosphorylated ERK1/2 were localized in both cell cytoplasm and nuclei while AKT only in cytoplasm. The 293_CHI3L1 cells differed from the 293 cells, transfected with an «empty» vector, in their size and ability to adhere to the culture plates. Conclusions. The overexpression of CHI3L1 is likely to have an important role in tumorigenesis via a mechanism which involves activation of PI3K and ERK1/2 pathways. The tumors which can be induced by orthotopic implantation of the transformed human cells with overexpressed human oncogene CHI3L1 into the rat brain can be used as a target for anticancer drug development.

A stably expressed llama single-domain intrabody targeting Rev displays broad-spectrum anti-HIV activity.

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Boons, Eline; Li, Guangdi; Vanstreels, Els; Vercruysse, Thomas; Pannecouque, Christophe; Vandamme, Anne-Mieke; Daelemans, Dirk

2014-12-01

The HIV Rev protein mediates the transport of partially and unspliced HIV mRNA from the nucleus to the cytoplasm. Rev multimerizes on a secondary stem-loop structure present in the viral intron-containing mRNA species and recruits the cellular karyopherin CRM1 to export viral mRNAs from the nucleus to the cytoplasm. Previously we have identified a single-domain intrabody (Nb(190)), derived from a llama heavy-chain antibody, which efficiently inhibits Rev multimerization and suppresses the production of infectious virus. We recently mapped the epitope of this nanobody and demonstrated that Rev residues K20 and Y23 are crucial for interaction while residues V16, H53 and L60 are important to a lesser extent. Here, we generated cell lines stably expressing Nb(190) and assessed the capacity of these cell lines to suppress the replication of different HIV-1 subtypes. These cells stably expressing the single-domain antibody are protected from virus-induced cytopathogenic effect even in the context of high multiplicity of infection. In addition, the replication of different subtypes of group M and one strain of group O is significantly suppressed in these cell lines. Next, we analysed the natural variations of Rev amino acids in sequence samples from HIV-1 infected patients worldwide and assessed the effect of Nb(190) on the most prevalent polymorphisms occurring at the key epitope positions (K20 and Y23) in Rev. We found that Nb(190) was able to suppress the function of these Rev variants except for the K20N mutant, which was present in only 0.7% of HIV-1 sequence populations (n = 4632). Cells stably expressing the single-domain intrabody Nb(190) are protected against virus-induced cytopathogenic effect and display a selective survival advantage upon infection. In addition, Nb(190) suppresses the replication of a wide range of different HIV-1 subtypes. Large-scale sequence analysis reveals that the Nb(190) epitope positions in Rev are well conserved across major HIV-1

Local anesthetic interaction with human ether-a-go-go-related gene (HERG) channels: role of aromatic amino acids Y652 and F656

DEFF Research Database (Denmark)

Siebrands, Cornelia C; Schmitt, Nicole; Friederich, Patrick

2005-01-01

was to determine the effect of the mutations Y652A and F656A in the putative drug binding region of HERG on the inhibition by bupivacaine, ropivacaine, and mepivacaine. METHODS: The authors examined the inhibition of wild-type and mutant HERG channels, transiently expressed in Chinese hamster ovary cells...... by bupivacaine, ropivacaine, and mepivacaine. Whole cell patch clamp recordings were performed at room temperature. RESULTS: Inhibition of HERG wild-type and mutant channels by the different local anesthetics was concentration dependent, stereoselective, and reversible. The sensitivity decreased in the order...... bupivacaine > ropivacaine > mepivacaine for wild-type and mutant channels. The mutant channels were approximately 4-30 times less sensitive to the inhibitory action of the different local anesthetics than the wild-type channel. The concentration-response data were described by Hill functions (bupivacaine...

Role of the activation gate in determining the extracellular potassium dependency of block of HERG by trapped drugs.

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Pareja, Kristeen; Chu, Elaine; Dodyk, Katrina; Richter, Kristofer; Miller, Alan

2013-01-01

Drug induced long QT syndrome (diLQTS) results primarily from block of the cardiac potassium channel HERG (human-ether-a-go-go related gene). In some cases long QT syndrome can result in the lethal arrhythmia torsade de pointes, an arrhythmia characterized by a rapid heart rate and severely compromised cardiac output. Many patients requiring medication present with serum potassium abnormalities due to a variety of conditions including gastrointestinal dysfunction, renal and endocrine disorders, diuretic use, and aging. Extracellular potassium influences HERG channel inactivation and can alter block of HERG by some drugs. However, block of HERG by a number of drugs is not sensitive to extracellular potassium. In this study, we show that block of WT HERG by bepridil and terfenadine, two drugs previously shown to be trapped inside the HERG channel after the channel closes, is insensitive to extracellular potassium over the range of 0 mM to 20 mM. We also show that bepridil block of the HERG mutant D540K, a mutant channel that is unable to trap drugs, is dependent on extracellular potassium, correlates with the permeant ion, and is independent of HERG inactivation. These results suggest that the lack of extracellular potassium dependency of block of HERG by some drugs may in part be related to the ability of these drugs to be trapped inside the channel after the channel closes.

Overcoming HERG affinity in the discovery of the CCR5 antagonist maraviroc.

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Price, David A; Armour, Duncan; de Groot, Marcel; Leishman, Derek; Napier, Carolyn; Perros, Manos; Stammen, Blanda L; Wood, Anthony

2006-09-01

The discovery of maraviroc 17 is described with particular reference to the generation of high selectivity over affinity for the HERG potassium channel. This was achieved through the use of a high throughput binding assay for the HERG channel that is known to show an excellent correlation with functional effects.

Stereoselective inhibition of the hERG1 potassium channel

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Liliana eSintra Grilo

2010-11-01

Full Text Available A growing number of drugs have been shown to prolong cardiac repolarization, predisposing individuals to life-threatening ventricular arrhythmias known as Torsades de Pointes. Most of these drugs are known to interfere with the human ether à-gogo related gene 1 (hERG1 channel, whose current is one of the main determinants of action potential duration. Prolonged repolarization is reflected by lengthening of the QT interval of the electrocardiogram, as seen in the suitably named drug-induced long QT syndrome. Chirality (presence of an asymmetric atom is a common feature of marketed drugs, which can therefore exist in at least two enantiomers with distinct three-dimensional structures and possibly distinct biological fates. Both the pharmacokinetic and pharmacodynamic properties can differ between enantiomers, as well as also between individuals who take the drug due to metabolic polymorphisms. Despite the large number of reports about drugs reducing the hERG1 current, potential stereoselective contributions have only been scarcely investigated. In this review, we present a non-exhaustive list of clinically important molecules which display chiral toxicity that may be related to hERG1-blocking properties. We particularly focus on methadone cardiotoxicity, which illustrates the importance of the stereoselective effect of drug chirality as well as individual variations resulting from pharmacogenetics. Furthermore, it seems likely that, during drug development, consideration of chirality in lead optimization and systematic assessment of the hERG1 current block with all enantiomers could contribute to the reduction of the risk of drug-induced LQTS.

Early identification of hERG liability in drug discovery programs by automated patch clamp

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Timm eDanker

2014-09-01

Full Text Available Blockade of the cardiac ion channel coded by hERG can lead to cardiac arrhythmia, which has become a major concern in drug discovery and development. Automated electrophysiological patch clamp allows assessment of hERG channel effects early in drug development to aid medicinal chemistry programs and has become routine in pharmaceutical companies. However, a number of potential sources of errors in setting up hERG channel assays by automated patch clamp can lead to misinterpretation of data or false effects being reported. This article describes protocols for automated electrophysiology screening of compound effects on the hERG channel current. Protocol details and the translation of criteria known from manual patch clamp experiments to automated patch clamp experiments to achieve good quality data are emphasized. Typical pitfalls and artifacts that may lead to misinterpretation of data are discussed. While this article focuses on hERG channel recordings using the QPatch (Sophion A/S, Copenhagen, Denmark technology, many of the assay and protocol details given in this article can be transferred for setting up different ion channel assays by automated patch clamp and are similar on other planar patch clamp platforms.

Overcoming hERG affinity in the discovery of maraviroc; a CCR5 antagonist for the treatment of HIV.

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Price, David A; Armour, Duncan; de Groot, Marcel; Leishman, Derek; Napier, Carolyn; Perros, Manos; Stammen, Blanda L; Wood, Anthony

2008-01-01

Avoiding cardiac liability associated with blockade of hERG (human ether a go-go) is key for successful drug discovery and development. This paper describes the work undertaken in the discovery of a potent CCR5 antagonist, maraviroc 34, for the treatment of HIV. In particular the use of a pharmacophore model of the hERG channel and a high throughput binding assay for the hERG channel are described that were critical to elucidate SAR to overcome hERG liabilities. The key SAR involves the introduction of polar substituents into regions of the molecule where it is postulated to undergo hydrophobic interactions with the ion channel. Within the CCR5 project there appeared to be no strong correlation between hERG affinity and physiochemical parameters such as pKa or lipophilicity. It is believed that chemists could apply these same strategies early in drug discovery to remove hERG interactions associated with lead compounds while retaining potency at the primary target.

Structural implications of hERG K+ channel block by a high-affinity minimally structured blocker

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Helliwell, Matthew V.; Zhang, Yihong; El Harchi, Aziza; Du, Chunyun; Hancox, Jules C.; Dempsey, Christopher E.

2018-01-01

Cardiac potassium channels encoded by human ether-à-go-go–related gene (hERG) are major targets for structurally diverse drugs associated with acquired long QT syndrome. This study characterized hERG channel inhibition by a minimally structured high-affinity hERG inhibitor, Cavalli-2, composed of three phenyl groups linked by polymethylene spacers around a central amino group, chosen to probe the spatial arrangement of side chain groups in the high-affinity drug-binding site of the hERG pore. hERG current (IhERG) recorded at physiological temperature from HEK293 cells was inhibited with an IC50 of 35.6 nm with time and voltage dependence characteristic of blockade contingent upon channel gating. Potency of Cavalli-2 action was markedly reduced for attenuated inactivation mutants located near (S620T; 54-fold) and remote from (N588K; 15-fold) the channel pore. The S6 Y652A and F656A mutations decreased inhibitory potency 17- and 75-fold, respectively, whereas T623A and S624A at the base of the selectivity filter also decreased potency (16- and 7-fold, respectively). The S5 helix F557L mutation decreased potency 10-fold, and both F557L and Y652A mutations eliminated voltage dependence of inhibition. Computational docking using the recent cryo-EM structure of an open channel hERG construct could only partially recapitulate experimental data, and the high dependence of Cavalli-2 block on Phe-656 is not readily explainable in that structure. A small clockwise rotation of the inner (S6) helix of the hERG pore from its configuration in the cryo-EM structure may be required to optimize Phe-656 side chain orientations compatible with high-affinity block. PMID:29545312

Asporin stably expressed in the surface layer of mandibular condylar cartilage and augmented in the deeper layer with age.

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Miyamoto, Yutaka; Kanzaki, Hiroyuki; Wada, Satoshi; Tsuruoka, Sari; Itohiya, Kanako; Kumagai, Kenichi; Hamada, Yoshiki; Nakamura, Yoshiki

2017-12-01

Mandibular condylar cartilage (MCC) exhibits dual roles both articular cartilage and growth center. Of many growth factors, TGF-β has been implicated in the growth of articular cartilage including MCC. Recently, Asporin, decoy to TGF-β, was discovered and it blocks TGF-β signaling. Asporin is expressed in a variety of tissues including osteoarthritic articular cartilage, though there was no report of Asporin expression in MCC. In the present study, we investigated the temporal and spatial expression of Asporin in MCC. Gene expression profile of MCC and epiphyseal cartilage in tibia of 5 weeks old ICR mice were firstly compared with microarray analysis using the laser capture microdissected samples. Variance of gene expression was further confirmed by real-time RT-PCR and immunohistochemical staining at 1,3,10, and 20 weeks old. TGF-β and its signaling molecule, phosphorylated Smad-2/3 (p-Smad2/3), were also examined by immunohistochemical staining. Microarray analysis revealed that Asporin was highly expressed in MCC. Real-time RT-PCR analysis confirmed that the fibrous layer of MCC exhibited stable higher Asporin expression at any time points as compared to epiphyseal cartilage. This was also observed in immunohistochemical staining. Deeper layer in MCC augmented Asporin expression with age. Whereas, TGF-β was stably highly observed in the layer. The fibrous layer of MCC exhibited weak staining of p-Smad2/3, though the proliferating layer of MCC was strongly stained as compared to epiphyseal cartilage of tibia at early time point. Consistent with the increase of Asporin expression in the deeper layer of MCC, the intensity of p-Smad-2/3 staining was decreased with age. In conclusion, we discovered that Asporin was stably expressed at the fibrous layer of MCC, which makes it possible to manage both articular cartilage and growth center at the same time.

Characteristics of stably expressed human dopamine D1a and D1b receptors: atypical behavior of the dopamine D1b receptor

DEFF Research Database (Denmark)

Pedersen, U B; Norby, B; Jensen, Anders A.

1994-01-01

Human dopamine D1a and D1b receptors were stably expressed in Baby Hamster Kidney (BHK) or Chinese Hamster Ovary (CHO) cells. [3H]SCH23390 saturation experiments indicated the presence of only a single binding site in the D1a expressing cell line with a Kd of 0.5 nM. In D1b expressing cell lines...

Role of the pH in state-dependent blockade of hERG currents

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Wang, Yibo; Guo, Jiqing; Perissinotti, Laura L.; Lees-Miller, James; Teng, Guoqi; Durdagi, Serdar; Duff, Henry J.; Noskov, Sergei Yu.

2016-10-01

Mutations that reduce inactivation of the voltage-gated Kv11.1 potassium channel (hERG) reduce binding for a number of blockers. State specific block of the inactivated state of hERG block may increase risks of drug-induced Torsade de pointes. In this study, molecular simulations of dofetilide binding to the previously developed and experimentally validated models of the hERG channel in open and open-inactivated states were combined with voltage-clamp experiments to unravel the mechanism(s) of state-dependent blockade. The computations of the free energy profiles associated with the drug block to its binding pocket in the intra-cavitary site display startling differences in the open and open-inactivated states of the channel. It was also found that drug ionization may play a crucial role in preferential targeting to the open-inactivated state of the pore domain. pH-dependent hERG blockade by dofetilie was studied with patch-clamp recordings. The results show that low pH increases the extent and speed of drug-induced block. Both experimental and computational findings indicate that binding to the open-inactivated state is of key importance to our understanding of the dofetilide’s mode of action.

A radiolabeled peptide ligand of the hERG channel, [125I]-BeKm-1

DEFF Research Database (Denmark)

Angelo, Kamilla; Korolkova, Yuliya V; Grunnet, Morten

2003-01-01

The wild-type scorpion toxin BeKm-1, which selectively blocks human ether-a-go-go related (hERG) channels, was radiolabeled with iodine at tyrosine 11. Both the mono- and di-iodinated derivatives were found to be biologically active. In electrophysiological patch-clamp recordings mono-[127I]-BeKm-1...... had a concentration of half-maximal inhibition (IC50 value) of 27 nM, while wild-type BeKm-1 inhibited hERG channels with an IC50 value of 7 nM. Mono-[125I]-BeKm-1 was found to bind in a concentration-dependent manner and with picomolar affinity to hERG channel protein in purified membrane vesicles...... of [125I]-BeKm-1 to the hERG channel to an IC50 of 7 nM. In autoradiographic studies on rat hearts, binding of [125I]-BeKm-1 was dose-dependent and could partially be displaced by the addition of excess amounts of non-radioactive BeKm-1. The density of the radioactive signal was equally distributed...

Modeling of the hERG K+ Channel Blockage Using Online Chemical Database and Modeling Environment (OCHEM).

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Li, Xiao; Zhang, Yuan; Li, Huanhuan; Zhao, Yong

2017-12-01

Human ether-a-go-go related gene (hERG) K+ channel plays an important role in cardiac action potential. Blockage of hERG channel may result in long QT syndrome (LQTS), even cause sudden cardiac death. Many drugs have been withdrawn from the market because of the serious hERG-related cardiotoxicity. Therefore, it is quite essential to estimate the chemical blockage of hERG in the early stage of drug discovery. In this study, a diverse set of 3721 compounds with hERG inhibition data was assembled from literature. Then, we make full use of the Online Chemical Modeling Environment (OCHEM), which supplies rich machine learning methods and descriptor sets, to build a series of classification models for hERG blockage. We also generated two consensus models based on the top-performing individual models. The consensus models performed much better than the individual models both on 5-fold cross validation and external validation. Especially, consensus model II yielded the prediction accuracy of 89.5 % and MCC of 0.670 on external validation. This result indicated that the predictive power of consensus model II should be stronger than most of the previously reported models. The 17 top-performing individual models and the consensus models and the data sets used for model development are available at https://ochem.eu/article/103592. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Irresponsiveness of two retinoblastoma cases to conservative therapy correlates with up- regulation of hERG1 channels and of the VEGF-A pathway

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La Torre Agostino

2010-09-01

Full Text Available Abstract Background Treatment strategies for Retinoblastoma (RB, the most common primary intraocular tumor in children, have evolved over the past few decades and chemoreduction is currently the most popular treatment strategy. Despite success, systemic chemotherapeutic treatment has relevant toxicity, especially in the pediatric population. Antiangiogenic therapy has thus been proposed as a valuable alternative for pediatric malignancies, in particolar RB. Indeed, it has been shown that vessel density correlates with both local invasive growth and presence of metastases in RB, suggesting that angiogenesis could play a pivotal role for both local and systemic invasive growth in RB. We present here two cases of sporadic, bilateral RB that did not benefit from the conservative treatment and we provide evidence that the VEGF-A pathway is significantly up-regulated in both RB cases along with an over expression of hERG1 K+ channels. Case presentation Two patients showed a sporadic, bilateral RB, classified at Stage II of the Reese-Elsworth Classification. Neither of them got benefits from conservative treatment, and the two eyes were enucleated. In samples from both RB cases we studied the VEGF-A pathway: VEGF-A showed high levels in the vitreous, the vegf-a, flt-1, kdr, and hif1-α transcripts were over-expressed. Moreover, both the transcripts and proteins of the hERG1 K+ channels turned out to be up-regulated in the two RB cases compared to the non cancerous retinal tissue. Conclusions We provide evidence that the VEGF-A pathway is up-regulated in two particular aggressive cases of bilateral RB, which did not experience any benefit from conservative treatment, showing the overexpression of the vegf-a, flt-1, kdr and hif1-α transcripts and the high secretion of VEGF-A. Moreover we also show for the first time that the herg1 gene transcripts and protein are over expressed in RB, as occurs in several aggressive tumors. These results further stress

[Establishment and application of a Vero cell line stably expressing raccoon dog SLAM, the cellular receptor of canine distemper virus].

Science.gov (United States)

Zhao, Jianjun; Yan, Ruxun; Zhang, Hailing; Zhang, Lei; Hu, Bo; Bai, Xue; Shao, Xiqun; Chai, Xiuli; Yan, Xijun; Wu, Wei

2012-12-04

The signaling lymphocyte activation molecule (SLAM, also known as CD150), is used as a cellular receptor by canine distemper virus (CDV). Wild-type strains of CDVs can be isolated and propagated efficiently in non-lymphoid cells expressing this protein. Our aim is to establish a Vero cells expressing raccoon dog SLAM (rSLAM) to efficiently isolate CDV from pathological samples. A eukaryotic expression plasmid, pIRES2-EGFP-rSLAMhis, containing rSLAM gene fused with six histidine-coding sequence, EGFP gene, and neomycin resistance gene was constructed. After transfection with the plasmid, a stable cell line, Vero-rSLAM, was screened from Vero cells with the identification of EGFP reporter and G418 resistance. Three CD positive specimens from infected foxes and raccoon dogs were inoculated to Vero-rSLAM cells for CDV isolation. Foxes and raccoon dogs were inoculated subcutaneously LN (10)fl strain with 4 x 10(2.39)TCID50 dose to evaluate pathogenicity of CDV isolations. The rSLAMh fused gene was shown to transcript and express stably in Vero-rSLAM cells by RT-PCR and Immunohistochemistry assay. Three CDV strains were isolated successfully in Vero-rSLAM cells 36 -48 hours after inoculation with spleen or lung specimens from foxes and raccoon dogs with distemper. By contrast, no CDV was recovered from those CD positive specimens when Vero cells were used for virus isolation. Infected foxes and raccoon dogs with LN(10)f1 strain all showed typical CD symptoms and high mortality (2/3 for foxes and 3/3 for raccoon dogs) in 22 days post challenge. Our results indicate that Vero-rSLAM cells stably expressing raccoon dog SLAM are highly sensitive to CDV in clinical specimens and the CDV isolation can maintain high virulence to its host animals.

Microtubule reorganization in tobacco BY-2 cells stably expressing GFP-MBD

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Granger, C. L.; Cyr, R. J.

2000-01-01

Microtubule organization plays an important role in plant morphogenesis; however, little is known about how microtubule arrays transit from one organized state to another. The use of a genetically incorporated fluorescent marker would allow long-term observation of microtubule behavior in living cells. Here, we have characterized a Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cell line that had been stably transformed with a gfp-mbd construct previously demonstrated to label microtubules (J. Marc et al., 1998, Plant Cell 10: 1927-1939). Fluorescence levels were low, but interphase and mitotic microtubule arrays, as well as the transitions between these arrays, could be observed in individual gfp-mbd-transformed cells. By comparing several attributes of transformed and untransformed cells it was concluded that the transgenic cells are not adversely affected by low-level expression of the transgene and that these cells will serve as a useful and accurate model system for observing microtubule reorganization in vivo. Indeed, some initial observations were made that are consistent with the involvement of motor proteins in the transition between the spindle and phragmoplast arrays. Our observations also support the role of the perinuclear region in nucleating microtubules at the end of cell division with a progressive shift of these microtubules and/or nucleating activity to the cortex to form the interphase cortical array.

Establishment of a novel immortalized human prostatic epithelial cell line stably expressing androgen receptor and its application for the functional screening of androgen receptor modulators

International Nuclear Information System (INIS)

Yu, Shan; Wang, Ming-Wei; Yao, Xiaoqiang; Chan, F.L.

2009-01-01

In this study, we developed a human prostatic epithelial cell line BPH-1-AR stably expressing AR by lentiviral transduction. Characterization by immunoblot and RT-PCR showed that AR was stably expressed in all representative BPH-1-AR clones. Androgen treatment induced a secretory differentiation phenotype in BPH-1-AR cells but suppressed their cell proliferation. Treatments with AR agonists induced transactivation of a transfected PSA-gene promoter reporter in BPH-1-AR cells, whereas this transactivation was suppressed by an AR antagonist flutamide, indicating that the transduced AR in BPH-1-AR cells was functional. Finally, we utilized BPH-1-AR cells to evaluate the androgenic activities and growth effects of five newly developed non-steroidal compounds. Results showed that these compounds showed androgenic activities and growth-inhibitory effects on BPH-1-AR cells. Our results showed that BPH-1-AR cell line would be a valuable in vitro model for the study of androgen-regulated processes in prostatic epithelial cells and identification of compounds with AR-modulating activities.

High potency inhibition of hERG potassium channels by the sodium–calcium exchange inhibitor KB-R7943

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Cheng, Hongwei; Zhang, Yihong; Du, Chunyun; Dempsey, Christopher E; Hancox, Jules C

2012-01-01

BACKGROUND AND PURPOSE KB-R7943 is an isothiourea derivative that is used widely as a pharmacological inhibitor of sodium–calcium exchange (NCX) in experiments on cardiac and other tissue types. This study investigated KB-R7943 inhibition of hERG (human ether-à-go-go-related gene) K+ channels that underpin the cardiac rapid delayed rectifier potassium current, IKr. EXPERIMENTAL APPROACH Whole-cell patch-clamp measurements were made of hERG current (IhERG) carried by wild-type or mutant hERG channels and of native rabbit ventricular IKr. Docking simulations utilized a hERG homology model built on a MthK-based template. KEY RESULTS KB-R7943 inhibited both IhERG and native IKr rapidly on membrane depolarization with IC50 values of ∼89 and ∼120 nM, respectively, for current tails at −40 mV following depolarizing voltage commands to +20 mV. Marked IhERG inhibition also occurred under ventricular action potential voltage clamp. IhERG inhibition by KB-R7943 exhibited both time- and voltage-dependence but showed no preference for inactivated over activated channels. Results of alanine mutagenesis and docking simulations indicate that KB-R7943 can bind to a pocket formed of the side chains of aromatic residues Y652 and F656, with the compound's nitrobenzyl group orientated towards the cytoplasmic side of the channel pore. The structurally related NCX inhibitor SN-6 also inhibited IhERG, but with a markedly reduced potency. CONCLUSIONS AND IMPLICATIONS KB-R7943 inhibits IhERG/IKr with a potency that exceeds that reported previously for acute cardiac NCX inhibition. Our results also support the feasibility of benzyloxyphenyl-containing NCX inhibitors with reduced potential, in comparison with KB-R7943, to inhibit hERG. PMID:21950687

Common variants in the hERG (KCNH2) voltage-gated potassium channel are associated with altered fasting and glucose-stimulated plasma incretin and glucagon responses

DEFF Research Database (Denmark)

Engelbrechtsen, Line; Mahendran, Yuvaraj; Jonsson, Anna

2018-01-01

BACKGROUND: Patients with long QT syndrome due to rare loss-of-function mutations in the human ether-á-go-go-related gene (hERG) have prolonged QT interval, risk of arrhythmias, increased secretion of insulin and incretins and impaired glucagon response to hypoglycemia. This is caused by a dysfun......BACKGROUND: Patients with long QT syndrome due to rare loss-of-function mutations in the human ether-á-go-go-related gene (hERG) have prolonged QT interval, risk of arrhythmias, increased secretion of insulin and incretins and impaired glucagon response to hypoglycemia. This is caused...... by a dysfunctional Kv11.1 voltage-gated potassium channel. Based on these findings in patients with rare variants in hERG, we hypothesized that common variants in hERG may also lead to alterations in glucose homeostasis. Subsequently, we aimed to evaluate the effect of two common gain-of-function variants in hERG...... in hERG on QT-interval and circulation levels of incretins, insulin and glucagon. The Danish population-based Inter99 cohort (n = 5895) was used to assess the effect of common variants on QT-interval. The Danish ADDITION-PRO cohort was used (n = 1329) to study genetic associations with levels of GLP-1...

In Silico Predictions of hERG Channel Blockers in Drug Discovery

DEFF Research Database (Denmark)

Taboureau, Olivier; Sørensen, Flemming Steen

2011-01-01

The risk for cardiotoxic side effects represents a major problem in clinical studies of drug candidates and regulatory agencies have explicitly recommended that all new drug candidates should be tested for blockage of the human Ether-a-go-go Related-Gene (hERG) potassium channel. Indeed, several ...

Voltage-sensing domain mode shift is coupled to the activation gate by the N-terminal tail of hERG channels.

Science.gov (United States)

Tan, Peter S; Perry, Matthew D; Ng, Chai Ann; Vandenberg, Jamie I; Hill, Adam P

2012-09-01

Human ether-a-go-go-related gene (hERG) potassium channels exhibit unique gating kinetics characterized by unusually slow activation and deactivation. The N terminus of the channel, which contains an amphipathic helix and an unstructured tail, has been shown to be involved in regulation of this slow deactivation. However, the mechanism of how this occurs and the connection between voltage-sensing domain (VSD) return and closing of the gate are unclear. To examine this relationship, we have used voltage-clamp fluorometry to simultaneously measure VSD motion and gate closure in N-terminally truncated constructs. We report that mode shifting of the hERG VSD results in a corresponding shift in the voltage-dependent equilibrium of channel closing and that at negative potentials, coupling of the mode-shifted VSD to the gate defines the rate of channel closure. Deletion of the first 25 aa from the N terminus of hERG does not alter mode shifting of the VSD but uncouples the shift from closure of the cytoplasmic gate. Based on these observations, we propose the N-terminal tail as an adaptor that couples voltage sensor return to gate closure to define slow deactivation gating in hERG channels. Furthermore, because the mode shift occurs on a time scale relevant to the cardiac action potential, we suggest a physiological role for this phenomenon in maximizing current flow through hERG channels during repolarization.

NF-kappa B activity in T cells stably expressing the Tax protein of human T cell lymphotropic virus type I

International Nuclear Information System (INIS)

Lacoste, J.; Cohen, L.; Hiscott, J.

1991-01-01

The effect of constitutive Tax expression on the interaction of NF-κ B with its recognition sequence and on NF-κ B-dependent gene expression was examined in T lymphoid Jurkat cell lines (19D and 9J) stably transformed with a Tax expression vector. Tax expressing T cell lines contained a constitutive level of NF-κ B binding activity, detectable by mobility shift assay and uv cross-linking using a palindromic NF-κ B probe homologous to the interferon beta PRDII site. In Jurkat and NC2.10 induction with phorbol esters resulted in the appearance of new DNA binding proteins of 85, 75, and 54 kDa, whereas in Tax expressing cells the 85-kDa protein and a 92-kDa DNA binding protein were constitutively induced. Expression of Tax protein in 19D and 9J resulted in transcription of the endogenous NF-kappa B-dependent granulocyte-macrophage colony stimulating factor gene and increased basal level expression of transfected NF-kappa B-regulated promoters. Nonetheless transcription of both the endogenous and the transfected gene was inducible by PMA treatment. Tax expression in Jurkat T cells may alter the stoichiometry of NF-kappa B DNA binding proteins and thus change the expression of NF-kappa B-regulated promoters

Generation of a Homozygous Transgenic Rat Strain Stably Expressing a Calcium Sensor Protein for Direct Examination of Calcium Signaling.

Science.gov (United States)

Szebényi, Kornélia; Füredi, András; Kolacsek, Orsolya; Pergel, Enikő; Bősze, Zsuzsanna; Bender, Balázs; Vajdovich, Péter; Tóvári, József; Homolya, László; Szakács, Gergely; Héja, László; Enyedi, Ágnes; Sarkadi, Balázs; Apáti, Ágota; Orbán, Tamás I

2015-08-03

In drug discovery, prediction of selectivity and toxicity require the evaluation of cellular calcium homeostasis. The rat is a preferred laboratory animal for pharmacology and toxicology studies, while currently no calcium indicator protein expressing rat model is available. We established a transgenic rat strain stably expressing the GCaMP2 fluorescent calcium sensor by a transposon-based methodology. Zygotes were co-injected with mRNA of transposase and a CAG-GCaMP2 expressing construct, and animals with one transgene copy were pre-selected by measuring fluorescence in blood cells. A homozygous rat strain was generated with high sensor protein expression in the heart, kidney, liver, and blood cells. No pathological alterations were found in these animals, and fluorescence measurements in cardiac tissue slices and primary cultures demonstrated the applicability of this system for studying calcium signaling. We show here that the GCaMP2 expressing rat cardiomyocytes allow the prediction of cardiotoxic drug side-effects, and provide evidence for the role of Na(+)/Ca(2+) exchanger and its beneficial pharmacological modulation in cardiac reperfusion. Our data indicate that drug-induced alterations and pathological processes can be followed by using this rat model, suggesting that transgenic rats expressing a calcium-sensitive protein provide a valuable system for pharmacological and toxicological studies.

Inducible and reversible suppression of Npm1 gene expression using stably integrated small interfering RNA vector in mouse embryonic stem cells

International Nuclear Information System (INIS)

Wang Beibei; Lu Rui; Wang Weicheng; Jin Ying

2006-01-01

The tetracycline (Tc)-inducible small interference RNA (siRNA) is a powerful tool for studying gene function in mammalian cells. However, the system is infrequently utilized in embryonic stem (ES) cells. Here, we present First application of the Tc-inducible, stably integrated plasmid-based siRNA system in mouse ES cells to down-regulate expression of Npm1, an essential gene for embryonic development. The physiological role of Npm1 in ES cells has not been defined. Our data show that the knock-down of Npm1 expression by this siRNA system was not only highly efficient, but also Tc- dose- and induction time-dependent. Particularly, the down-regulation of Npm1 expression was reversible. Importantly, suppression of Npm1 expression in ES cells resulted in reduced cell proliferation. Taken together, this system allows for studying gene function in a highly controlled manner, otherwise difficult to achieve in ES cells. Moreover, our results demonstrate that Npm1 is essential for ES cell proliferation

Magnetic cell sorting purification of differentiated embryonic stem cells stably expressing truncated human CD4 as surface marker.

Science.gov (United States)

David, Robert; Groebner, Michael; Franz, Wolfgang-Michael

2005-04-01

Embryonic stem (ES) cells offer great potential in regenerative medicine and tissue engineering. Clinical applications are still hampered by the lack of protocols for gentle, high-yield isolation of specific cell types for transplantation expressing no immunogenic markers. We describe labeling of stably transfected ES cells expressing a human CD4 molecule lacking its intracellular domain (DeltaCD4) under control of the phosphoglycerate kinase promoter for magnetic cell sorting (MACS). To track the labeled ES cells, we fused DeltaCD4 to an intracellular enhanced green fluorescent protein domain (DeltaCD4EGFP). We showed functionality of the membrane-bound fluorescent fusion protein and its suitability for MACS leading to purities greater than 97%. Likewise, expression of DeltaCD4 yielded up to 98.5% positive cells independently of their differentiation state. Purities were not limited by the initial percentage of DeltaCD4(+) cells, ranging from 0.6%-16%. The viability of MACS-selected cells was demonstrated by reaggregation and de novo formation of embryoid bodies developing all three germ layers. Thus, expression of DeltaCD4 in differentiated ES cells may enable rapid, high-yield purification of a desired cell type for tissue engineering and transplantation studies.

Enhanced functional recombinant factor VII production by HEK 293 cells stably transfected with VKORC1 where the gamma-carboxylase inhibitor calumenin is stably suppressed by shRNA transfection.

Science.gov (United States)

Wajih, Nadeem; Owen, John; Wallin, Reidar

2008-01-01

Recombinant members of the vitamin K-dependent protein family (factors IX and VII and protein C) have become important pharmaceuticals in treatment of bleeding disorders and sepsis. However, because the in vivo gamma-carboxylation system in stable cell lines used for transfection has a limited capacity of post translational gamma-carboxylation, the recovery of fully gamma-carboxylated and functional proteins is low. In this work we have engineered recombinant factor VII producing HEK 293 cells to stably overexpress VKORC1, the reduced vitamin K gamma-carboxylase cofactor and in addition stably silenced the gamma-carboxylase inhibitory protein calumenin. Stable cell lines transfected with only a factor VII cDNA had a 9% production of functional recombinant factor VII. On the other hand, these recombinant factor VII producing cells when engineered to overexpress VKORC1 and having calumenin stably suppressed more than 80% by shRNA expression, produced 68% functional factor VII. The technology presented should be applicable to all vertebrae members of the vitamin K-dependent protein family and should lower the production cost of the clinically used factors VII, IX and protein C.

Validation and Clinical Utility of the hERG IC50:Cmax Ratio to Determine the Risk of Drug-Induced Torsades de Pointes: A Meta-Analysis.

Science.gov (United States)

Lehmann, David F; Eggleston, William D; Wang, Dongliang

2018-03-01

Use of the QT interval corrected for heart rate (QTc) on the electrocardiogram (ECG) to predict torsades de pointes (TdP) risk from culprit drugs is neither sensitive nor specific. The ratio of the half-maximum inhibitory concentration of the hERG channel (hERG IC50) to the peak serum concentration of unbound drug (C max ) is used during drug development to screen out chemical entities likely to cause TdP. To validate the use of the hERG IC50:C max ratio to predict TdP risk from a culprit drug by its correlation with TdP incidence. Medline (between 1966 and March 2017) was accessed for hERG IC50 and C max values from the antihistamine, fluoroquinolone, and antipsychotic classes to identify cases of drug-induced TdP. Exposure to a culprit drug was estimated from annual revenues reported by the manufacturer. Inclusion criteria for TdP cases were provision of an ECG tracing that demonstrated QTc prolongation with TdP and normal serum values of potassium, calcium, and magnesium. Cases reported in patients with a prior rhythm disturbance and those involving a drug interaction were excluded. The Meta-Analysis of Observational Studies in Epidemiology checklist was used for epidemiological data extraction by two authors. Negligible risk drugs were defined by an hERG IC50:C max ratio that correlated with less than a 5% chance of one TdP event for every 100 million exposures (relative risk [RR] 1.0). The hERG IC50:C max ratio correlated with TdP risk (0.312; 95% confidence interval 0.205-0.476, pratio of 80 (RR 1.0). The RR from olanzapine is on par with loratadine; ziprasidone is comparable with ciprofloxacin. Drugs with an RR greater than 50 include astemizole, risperidone, haloperidol, and thioridazine. The hERG IC50:C max ratio was correlated with TdP incidence for culprit drugs. This validation provides support for the potential use of the hERG IC50:C max ratio for clinical decision making in instances of drug selection where TdP risk is a concern. © 2018

Delineation of the GPRC6A Receptor Signaling Pathways Using a Mammalian Cell Line Stably Expressing the Receptor

DEFF Research Database (Denmark)

Jacobsen, Stine Engesgaard; Nørskov-Lauritsen, Lenea; Thomsen, Alex Rojas Bie

2013-01-01

receptor has been suggested to couple to multiple G protein classes albeit via indirect methods. Thus, the exact ligand preferences and signaling pathways are yet to be elucidated. In the present study, we generated a Chinese hamster ovary (CHO) cell line that stably expresses mouse GPRC6A. In an effort...... and divalent cations, and for the first time, we conclusively show that these responses are mediated through the Gq pathway. We were not able to confirm previously published data demonstrating Gi- and Gs-mediated signaling; neither could we detect agonistic activity of testosterone and osteocalcin. Generation...... of the stable CHO cell line with robust receptor responsiveness and optimization of the highly sensitive homogeneous time resolved fluorescence technology allow fast assessment of Gq activation without previous manipulations like cotransfection of mutated G proteins. This cell-based assay system for GPRC6A...

Kuifjes katholieke jeugd. De katholieke achtergrond van Hergé. Deel 1 van 2

NARCIS (Netherlands)

de Groot, C.N.

2013-01-01

Following the launch of Steven Spielberg’s ‘Tintin and the Secret of the Unicorn’, l’Osservore Romano hailed Tintin as a ‘catholic hero’. This article demonstrates that the comic originates, more specifically, in conservative and reactionary milieus in Belgian Catholicism. Hergé (ps. for Georges

Large eddy simulation of stably stratified turbulence

International Nuclear Information System (INIS)

Shen Zhi; Zhang Zhaoshun; Cui Guixiang; Xu Chunxiao

2011-01-01

Stably stratified turbulence is a common phenomenon in atmosphere and ocean. In this paper the large eddy simulation is utilized for investigating homogeneous stably stratified turbulence numerically at Reynolds number Re = uL/v = 10 2 ∼10 3 and Froude number Fr = u/NL = 10 −2 ∼10 0 in which u is root mean square of velocity fluctuations, L is integral scale and N is Brunt-Vaïsälä frequency. Three sets of computation cases are designed with different initial conditions, namely isotropic turbulence, Taylor Green vortex and internal waves, to investigate the statistical properties from different origins. The computed horizontal and vertical energy spectra are consistent with observation in atmosphere and ocean when the composite parameter ReFr 2 is greater than O(1). It has also been found in this paper that the stratification turbulence can be developed under different initial velocity conditions and the internal wave energy is dominated in the developed stably stratified turbulence.

The S4-S5 linker acts as a signal integrator for HERG K+ channel activation and deactivation gating.

Directory of Open Access Journals (Sweden)

Chai Ann Ng

Full Text Available Human ether-à-go-go-related gene (hERG K(+ channels have unusual gating kinetics. Characterised by slow activation/deactivation but rapid inactivation/recovery from inactivation, the unique gating kinetics underlie the central role hERG channels play in cardiac repolarisation. The slow activation and deactivation kinetics are regulated in part by the S4-S5 linker, which couples movement of the voltage sensor domain to opening of the activation gate at the distal end of the inner helix of the pore domain. It has also been suggested that cytosolic domains may interact with the S4-S5 linker to regulate activation and deactivation kinetics. Here, we show that the solution structure of a peptide corresponding to the S4-S5 linker of hERG contains an amphipathic helix. The effects of mutations at the majority of residues in the S4-S5 linker of hERG were consistent with the previously identified role in coupling voltage sensor movement to the activation gate. However, mutations to Ser543, Tyr545, Gly546 and Ala548 had more complex phenotypes indicating that these residues are involved in additional interactions. We propose a model in which the S4-S5 linker, in addition to coupling VSD movement to the activation gate, also contributes to interactions that stabilise the closed state and a separate set of interactions that stabilise the open state. The S4-S5 linker therefore acts as a signal integrator and plays a crucial role in the slow deactivation kinetics of the channel.

Performance of Machine Learning Algorithms for Qualitative and Quantitative Prediction Drug Blockade of hERG1 channel.

Science.gov (United States)

Wacker, Soren; Noskov, Sergei Yu

2018-05-01

Drug-induced abnormal heart rhythm known as Torsades de Pointes (TdP) is a potential lethal ventricular tachycardia found in many patients. Even newly released anti-arrhythmic drugs, like ivabradine with HCN channel as a primary target, block the hERG potassium current in overlapping concentration interval. Promiscuous drug block to hERG channel may potentially lead to perturbation of the action potential duration (APD) and TdP, especially when with combined with polypharmacy and/or electrolyte disturbances. The example of novel anti-arrhythmic ivabradine illustrates clinically important and ongoing deficit in drug design and warrants for better screening methods. There is an urgent need to develop new approaches for rapid and accurate assessment of how drugs with complex interactions and multiple subcellular targets can predispose or protect from drug-induced TdP. One of the unexpected outcomes of compulsory hERG screening implemented in USA and European Union resulted in large datasets of IC 50 values for various molecules entering the market. The abundant data allows now to construct predictive machine-learning (ML) models. Novel ML algorithms and techniques promise better accuracy in determining IC 50 values of hERG blockade that is comparable or surpassing that of the earlier QSAR or molecular modeling technique. To test the performance of modern ML techniques, we have developed a computational platform integrating various workflows for quantitative structure activity relationship (QSAR) models using data from the ChEMBL database. To establish predictive powers of ML-based algorithms we computed IC 50 values for large dataset of molecules and compared it to automated patch clamp system for a large dataset of hERG blocking and non-blocking drugs, an industry gold standard in studies of cardiotoxicity. The optimal protocol with high sensitivity and predictive power is based on the novel eXtreme gradient boosting (XGBoost) algorithm. The ML-platform with XGBoost

Biophysical and Pharmacological Characterization of Nav1.9 Voltage Dependent Sodium Channels Stably Expressed in HEK-293 Cells.

Directory of Open Access Journals (Sweden)

Zhixin Lin

Full Text Available The voltage dependent sodium channel Nav1.9, is expressed preferentially in peripheral sensory neurons and has been linked to human genetic pain disorders, which makes it target of interest for the development of new pain therapeutics. However, characterization of Nav1.9 pharmacology has been limited due in part to the historical difficulty of functionally expressing recombinant channels. Here we report the successful generation and characterization of human, mouse and rat Nav1.9 stably expressed in human HEK-293 cells. These cells exhibit slowly activating and inactivating inward sodium channel currents that have characteristics of native Nav1.9. Optimal functional expression was achieved by coexpression of Nav1.9 with β1/β2 subunits. While recombinantly expressed Nav1.9 was found to be sensitive to sodium channel inhibitors TC-N 1752 and tetracaine, potency was up to 100-fold less than reported for other Nav channel subtypes despite evidence to support an interaction with the canonical local anesthetic (LA binding region on Domain 4 S6. Nav1.9 Domain 2 S6 pore domain contains a unique lysine residue (K799 which is predicted to be spatially near the local anesthetic interaction site. Mutation of this residue to the consensus asparagine (K799N resulted in an increase in potency for tetracaine, but a decrease for TC-N 1752, suggesting that this residue can influence interaction of inhibitors with the Nav1.9 pore. In summary, we have shown that stable functional expression of Nav1.9 in the widely used HEK-293 cells is possible, which opens up opportunities to better understand channel properties and may potentially aid identification of novel Nav1.9 based pharmacotherapies.

Functional characterization of a novel hERG variant in a family with recurrent sudden infant death syndrome: Retracting a genetic diagnosis.

Science.gov (United States)

Sergeev, Valentine; Perry, Frances; Roston, Thomas M; Sanatani, Shubhayan; Tibbits, Glen F; Claydon, Thomas W

2018-03-01

Long QT syndrome (LQTS) is the most common cardiac ion channelopathy and has been found to be responsible for approximately 10% of sudden infant death syndrome (SIDS) cases. Despite increasing use of broad panels and now whole exome sequencing (WES) in the investigation of SIDS, the probability of identifying a pathogenic mutation in a SIDS victim is low. We report a family-based study who are afflicted by recurrent SIDS in which several members harbor a variant, p.Pro963Thr, in the C-terminal region of the human-ether-a-go-go (hERG) gene, published to be responsible for cases of LQTS type 2. Functional characterization was undertaken due to the variable phenotype in carriers, the discrepancy with published cases, and the importance of identifying a cause for recurrent deaths in a single family. Studies of the mutated ion channel in in vitro heterologous expression systems revealed that the mutation has no detectable impact on membrane surface expression, biophysical gating properties such as activation, deactivation and inactivation, or the amplitude of the protective current conducted by hERG channels during early repolarization. These observations suggest that the p.Pro963Thr mutation is not a monogenic disease-causing LQTS mutation despite evidence of co-segregation in two siblings affected by SIDS. Our findings demonstrate some of the potential pitfalls in post-mortem molecular testing and the importance of functional testing of gene variants in determining disease-causation, especially where the impacts of cascade screening can affect multiple generations. Copyright © 2017 Elsevier B.V. All rights reserved.

Construction of a CD147 Lentiviral Expression Vector and Establishment of Its Stably Transfected A549 Cell Line

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Shaoxing YANG

2012-12-01

Full Text Available Background and objective CD147, a type of transmembrane glycoprotein embedded on the surface of tumor cells, can promote tumor invasion and metastasis. This aim of this study is to construct a CD147 lentiviral expression vector, establish its stably transfected A549 cell line, and observe the effect of CD147 on MMP-9 proliferation as well as on the invasive ability of human lung adenocarcinoma cells. Methods Full-length CD147 gene was amplified by real-time polymerase chain reaction (RT-PCR, inserted into a pEGFP vector to construct pEGFP-CD147 and pEGFP vectors, and then transfected into 293FT cells to precede the lentivirus equipment package. Subsequently, we collected the lentivirus venom to infect the A549 cells and establish a stable, overexpressed cell line named A549-CD147. The mRNA expression of MMP-9 was examined by RT-PCR. The proliferation and invasive ability of the human lung cancer cells before and after transfection were examined by the CCK-8 and Transwell methods. Results A CD147 lentiviral expression vector (pEGFP-CD147 was successfully constructed by restrictive enzyme digestion and plasmid sequencing. RT-PCR and Western blot analyses revealed increased mRNA and protein expression of CD147 gene in cells transfected with pEGFP-CD147 compared with the control groups. Therefore, the A549-CD147 cell line was successfully established through the experiment. The mRNA expression of MMP-9 also significantly increased after the upregulation of CD147 expression. Meanwhile, CCK-8 and Transwell assays indicated that the proliferation and invasive ability significantly increased in the A549-CD147 cells. Conclusion A lentiviral CD147 expression vector and its A549 cell line (A549-CD14 were successfully constructed. CD147 overexpression upregulated the protein expression of MMP-9, and strengthened the proliferation and invasive ability of human lung adenocarcinoma cells.

Quantitative Evaluation of Myostatin Gene in Stably Transfected Caprine Fibroblast Cells by Anti-Myostatin shRNA.

Science.gov (United States)

Jain, Sudhir Kumar; Jain, Hemlata; Kumar, Dharmendra; Bedekar, Megha Kadam; Pandey, Akhilesh Kumar; Sarkhel, Bikash Chandra

2015-09-01

Skeletal muscle is the major component of lean tissue that is used for consumption, and myostatin is a negative regulator of skeletal muscle growth. Downregulation of this gene therefore offers a strategy for developing superior animals with enhanced muscle growth. Knockdown of myostatin was achieved by RNA interference technology. The anti-myostatin shRNA were designed and stably transfected in caprine fibroblast cells. The reduced expression of target gene was achieved and measured in clonal fibroblast cells by real-time PCR. Two single-cell clones induced significant decrease of myostatin gene expression by 73.96 and 72.66 %, respectively (P < 0.05). To ensure the appropriate growth of transfected cell, seven media were tested. The best suited media was used for transfected fibroblast cell proliferation. The findings suggest that shRNA provides a novel potential tool for gene knockdown and these stably transfected cells can be used as the donor cells for animal cloning.

Antibody-dependent cellular cytotoxicity and cytokine/chemokine secretion by KHYG-1 cells stably expressing FcγRIIIA.

Science.gov (United States)

Kobayashi, Eiji; Motoi, Sotaro; Sugiura, Masahito; Kajikawa, Masunori; Kojima, Shuji; Kohroki, Junya; Masuho, Yasuhiko

2014-09-01

Antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer (NK) cells is a major mechanism of tumor therapy with antibodies. NK cells not only manifest cytotoxicity but also secrete a variety of cytokines/chemokines that regulate immune responses. Using a retroviral vector, in this study we established a KHYG-1 cell line that stably expresses FcγRIIIA (CD16A). The KHYG-1/FcγRIIIA cells exerted potent antibody concentration-dependent ADCC, whereas parental KHYG-1 cells did not. In contrast, without antibody, the natural killer activity of KHYG-1/FcγRIIIA cells was less potent than that of parental KHYG-1 cells. During the course of ADCC, KHYG-1/FcγRIIIA cells secreted IFN-γ and MIP-1α dependent upon antibody concentration, but parental KHYG-1 cells did not. These results suggest that KHYG-1/FcγRIIIA cells would be useful in studies to elucidate the function of NK cells and the mechanism of ADCC. Copyright © 2014 Elsevier B.V. All rights reserved.

Structural refinement of the hERG1 pore and voltage-sensing domains with ROSETTA-membrane and molecular dynamics simulations.

Science.gov (United States)

Subbotina, Julia; Yarov-Yarovoy, Vladimir; Lees-Miller, James; Durdagi, Serdar; Guo, Jiqing; Duff, Henry J; Noskov, Sergei Yu

2010-11-01

The hERG1 gene (Kv11.1) encodes a voltage-gated potassium channel. Mutations in this gene lead to one form of the Long QT Syndrome (LQTS) in humans. Promiscuous binding of drugs to hERG1 is known to alter the structure/function of the channel leading to an acquired form of the LQTS. Expectably, creation and validation of reliable 3D model of the channel have been a key target in molecular cardiology and pharmacology for the last decade. Although many models were built, they all were limited to pore domain. In this work, a full model of the hERG1 channel is developed which includes all transmembrane segments. We tested a template-driven de-novo design with ROSETTA-membrane modeling using side-chain placements optimized by subsequent molecular dynamics (MD) simulations. Although backbone templates for the homology modeled parts of the pore and voltage sensors were based on the available structures of KvAP, Kv1.2 and Kv1.2-Kv2.1 chimera channels, the missing parts are modeled de-novo. The impact of several alignments on the structure of the S4 helix in the voltage-sensing domain was also tested. Herein, final models are evaluated for consistency to the reported structural elements discovered mainly on the basis of mutagenesis and electrophysiology. These structural elements include salt bridges and close contacts in the voltage-sensor domain; and the topology of the extracellular S5-pore linker compared with that established by toxin foot-printing and nuclear magnetic resonance studies. Implications of the refined hERG1 model to binding of blockers and channels activators (potent new ligands for channel activations) are discussed. © 2010 Wiley-Liss, Inc.

In silico analysis of conformational changes induced by mutation of aromatic binding residues: consequences for drug binding in the hERG K+ channel.

Directory of Open Access Journals (Sweden)

Kirsten Knape

Full Text Available Pharmacological inhibition of cardiac hERG K(+ channels is associated with increased risk of lethal arrhythmias. Many drugs reduce hERG current by directly binding to the channel, thereby blocking ion conduction. Mutation of two aromatic residues (F656 and Y652 substantially decreases the potency of numerous structurally diverse compounds. Nevertheless, some drugs are only weakly affected by mutation Y652A. In this study we utilize molecular dynamics simulations and docking studies to analyze the different effects of mutation Y652A on a selected number of hERG blockers. MD simulations reveal conformational changes in the binding site induced by mutation Y652A. Loss of π-π-stacking between the two aromatic residues induces a conformational change of the F656 side chain from a cavity facing to cavity lining orientation. Docking studies and MD simulations qualitatively reproduce the diverse experimentally observed modulatory effects of mutation Y652A and provide a new structural interpretation for the sensitivity differences.

An NMR investigation of the structure, function and role of the hERG channel selectivity filter in the long QT syndrome.

Science.gov (United States)

Gravel, Andrée E; Arnold, Alexandre A; Dufourc, Erick J; Marcotte, Isabelle

2013-06-01

The human ether-a-go-go-related gene (hERG) voltage-gated K(+) channels are located in heart cell membranes and hold a unique selectivity filter (SF) amino acid sequence (SVGFG) as compared to other K(+) channels (TVGYG). The hERG provokes the acquired long QT syndrome (ALQTS) when blocked, as a side effect of drugs, leading to arrhythmia or heart failure. Its pore domain - including the SF - is believed to be a cardiotoxic drug target. In this study combining solution and solid-state NMR experiments we examine the structure and function of hERG's L(622)-K(638) segment which comprises the SF, as well as its role in the ALQTS using reported active drugs. We first show that the SF segment is unstructured in solution with and without K(+) ions in its surroundings, consistent with the expected flexibility required for the change between the different channel conductive states predicted by computational studies. We also show that the SF segment has the potential to perturb the membrane, but that the presence of K(+) ions cancels this interaction. The SF moiety appears to be a possible target for promethazine in the ALQTS mechanism, but not as much for bepridil, cetirizine, diphenhydramine and fluvoxamine. The membrane affinity of the SF is also affected by the presence of drugs which also perturb model DMPC-based membranes. These results thus suggest that the membrane could play a role in the ALQTS by promoting the access to transmembrane or intracellular targets on the hERG channel, or perturbing the lipid-protein synergy. Copyright © 2013 Elsevier B.V. All rights reserved.

Causal boundary for stably causal space-times

International Nuclear Information System (INIS)

Racz, I.

1987-12-01

The usual boundary constructions for space-times often yield an unsatisfactory boundary set. This problem is reviewed and a new solution is proposed. An explicit identification rule is given on the set of the ideal points of the space-time. This construction leads to a satisfactory boundary point set structure for stably causal space-times. The topological properties of the resulting causal boundary construction are examined. For the stably causal space-times each causal curve has a unique endpoint on the boundary set according to the extended Alexandrov topology. The extension of the space-time through the boundary is discussed. To describe the singularities the defined boundary sets have to be separated into two disjoint sets. (D.Gy.) 8 refs

hERG classification model based on a combination of support vector machine method and GRIND descriptors

DEFF Research Database (Denmark)

Li, Qiyuan; Jorgensen, Flemming Steen; Oprea, Tudor

2008-01-01

and diverse library of 495 compounds. The models combine pharmacophore-based GRIND descriptors with a support vector machine (SVM) classifier in order to discriminate between hERG blockers and nonblockers. Our models were applied at different thresholds from 1 to 40 mu m and achieved an overall accuracy up...

Visualization of mole fraction distribution of slow jet forming stably stratified field

International Nuclear Information System (INIS)

Fumizawa, Motoo; Hishida, Makoto

1990-01-01

An experimental study has been performed to investigate the behavior of flow and mass transfer in gaseous slow jet in which buoyancy force opposed the flow forming stably stratified field. The study has been performed to understand the basic features of air ingress phenomena at pipe rupture accident of the high temperature gas-cooled reactor. A displacement fringe technique was adopted in Mach-Zehnder interferometer to visualize the mole fraction distribution. As the result, the followings were obtained: (1) The stably stratified fields were formed in the vicinity of the outlet of the slow jet. The penetration distance of the stably stratified fields increased with Froude number. (2) Mass fraction distributions in the stably stratified fields were well correlated with the present model using the ramp mole velocity profile. (author)

Tiamulin inhibits human CYP3A4 activity in an NIH/3T3 cell line stably expressing CYP3A4 cDNA.

Science.gov (United States)

De Groene, E M; Nijmeijer, S M; Horbach, G J; Witkamp, R F

1995-09-07

Tiamulin is an antibiotic frequently used in veterinary medicine. The drug has been shown to produce clinically important interactions with other compounds that are administered simultaneously. An NIH/3T3 cell line, stably expressing human cytochrome P450 (EC 1.14.14.1) cDNA (CYP3A4), was used to study the effect of tiamulin on CYP3A4 activity. The 6 beta-hydroxylation activity of testosterone, which is increased in CYP3A4-expressing cells compared to vector-transfected cells, showed reduced activity after incubation with 1 microM tiamulin and was completely reduced to background level after incubation with 2, 5 and 10 microM tiamulin. The CYP3A4-expressing cell line was used in combination with a shuttle vector containing the bacterial lacZ' gene to study the effect of tiamulin on CYP3A4-mediated mutagenicity of aflatoxin B1. The mutation frequency of aflatoxin B1 could be completely inhibited by tiamulin in CYP3A4-expressing cells, but no effect was observed on the mutation frequency of the direct mutagen ethylmethanesulphonate. Western blotting of homogenates of the CYP3A4-expressing cell line showed stabilization of CYP3A4 protein after incubation with tiamulin, supporting the hypothesis that the mechanism of inhibition is by binding of tiamulin to the cytochrome.

Kuifjes katholieke jeugd. De katholieke achtergrond van Hergé. Deel 1 van 2

OpenAIRE

de Groot, C.N.

2013-01-01

Following the launch of Steven Spielberg’s ‘Tintin and the Secret of the Unicorn’, l’Osservore Romano hailed Tintin as a ‘catholic hero’. This article demonstrates that the comic originates, more specifically, in conservative and reactionary milieus in Belgian Catholicism. Hergé (ps. for Georges Rémi) designed Tintin for the children’s weekly of a newspaper that, in this period, shared its main themes with the Catholic fascist movement Rex: anti-communism, anti-capitalism, anti-semitism and t...

Characterization of calcium signals in human induced pluripotent stem cell-derived dentate gyrus neuronal progenitors and mature neurons, stably expressing an advanced calcium indicator protein.

Science.gov (United States)

Vőfély, Gergő; Berecz, Tünde; Szabó, Eszter; Szebényi, Kornélia; Hathy, Edit; Orbán, Tamás I; Sarkadi, Balázs; Homolya, László; Marchetto, Maria C; Réthelyi, János M; Apáti, Ágota

2018-04-01

Pluripotent stem cell derived human neuronal progenitor cells (hPSC-NPCs) and their mature neuronal cell culture derivatives may efficiently be used for central nervous system (CNS) drug screening, including the investigation of ligand-induced calcium signalization. We have established hippocampal NPC cultures derived from human induced PSCs, which were previously generated by non-integrating Sendai virus reprogramming. Using established protocols these NPCs were differentiated into hippocampal dentate gyrus neurons. In order to study calcium signaling without the need of dye loading, we have stably expressed an advanced calcium indicator protein (GCaMP6fast) in the NPCs using the Sleeping Beauty transposon system. We observed no significant effects of the long-term GCaMP6 expression on NPC morphology, gene expression pattern or neural differentiation capacity. In order to compare the functional properties of GCaMP6-expressing neural cells and the corresponding parental cells loaded with calcium indicator dye Fluo-4, a detailed characterization of calcium signals was performed. We found that the calcium signals induced by ATP, glutamate, LPA, or proteases - were similar in these two systems. Moreover, the presence of the calcium indicator protein allowed for a sensitive, repeatable detection of changes in calcium signaling during the process of neurogenesis and neuronal maturation. Copyright © 2018 Elsevier Inc. All rights reserved.

Computational analysis of the effects of the hERG channel opener NS1643 in a human ventricular cell model

DEFF Research Database (Denmark)

Peitersen, Torben; Grunnet, Morten; Benson, Alan P

2008-01-01

BACKGROUND: Dysfunction or pharmacologic inhibition of repolarizing cardiac ionic currents can lead to fatal arrhythmias. The hERG potassium channel underlies the repolarizing current I(Kr), and mutations therein can produce both long and short QT syndromes (LQT2 and SQT1). We previously reported...

RANS Modeling of Stably Stratified Turbulent Boundary Layer Flows in OpenFOAM®

Directory of Open Access Journals (Sweden)

Wilson Jordan M.

2015-01-01

Full Text Available Quantifying mixing processes relating to the transport of heat, momentum, and scalar quantities of stably stratified turbulent geophysical flows remains a substantial task. In a stably stratified flow, such as the stable atmospheric boundary layer (SABL, buoyancy forces have a significant impact on the flow characteristics. This study investigates constant and stability-dependent turbulent Prandtl number (Prt formulations linking the turbulent viscosity (νt and diffusivity (κt for modeling applications of boundary layer flows. Numerical simulations of plane Couette flow and pressure-driven channel flow are performed using the Reynolds-averaged Navier-Stokes (RANS framework with the standard k-ε turbulence model. Results are compared with DNS data to evaluate model efficacy for predicting mean velocity and density fields. In channel flow simulations, a Prandtl number formulation for wall-bounded flows is introduced to alleviate overmixing of the mean density field. This research reveals that appropriate specification of Prt can improve predictions of stably stratified turbulent boundary layer flows.

The N-terminal tail of hERG contains an amphipathic α-helix that regulates channel deactivation.

Directory of Open Access Journals (Sweden)

Chai Ann Ng

Full Text Available The cytoplasmic N-terminal domain of the human ether-a-go-go related gene (hERG K+ channel is critical for the slow deactivation kinetics of the channel. However, the mechanism(s by which the N-terminal domain regulates deactivation remains to be determined. Here we show that the solution NMR structure of the N-terminal 135 residues of hERG contains a previously described Per-Arnt-Sim (PAS domain (residues 26-135 as well as an amphipathic α-helix (residues 13-23 and an initial unstructured segment (residues 2-9. Deletion of residues 2-25, only the unstructured segment (residues 2-9 or replacement of the α-helix with a flexible linker all result in enhanced rates of deactivation. Thus, both the initial flexible segment and the α-helix are required but neither is sufficient to confer slow deactivation kinetics. Alanine scanning mutagenesis identified R5 and G6 in the initial flexible segment as critical for slow deactivation. Alanine mutants in the helical region had less dramatic phenotypes. We propose that the PAS domain is bound close to the central core of the channel and that the N-terminal α-helix ensures that the flexible tail is correctly orientated for interaction with the activation gating machinery to stabilize the open state of the channel.

Modeling the Conducting Stably-Stratified Layer of the Earth's Core

Science.gov (United States)

Petitdemange, L.; Philidet, J.; Gissinger, C.

2017-12-01

Observations of the Earth magnetic field as well as recent theoretical works tend to show that the Earth's outer liquid core is mostly comprised of a convective zone in which the Earth's magnetic field is generated - likely by dynamo action -, but also features a thin, stably stratified layer at the top of the core.We carry out direct numerical simulations by modeling this thin layer as an axisymmetric spherical Couette flow for a stably stratified fluid embedded in a dipolar magnetic field. The dynamo region is modeled by a conducting inner core rotating slightly faster than the insulating mantle due to magnetic torques acting on it, such that a weak differential rotation (low Rossby limit) can develop in the stably stratified layer.In the case of a non-stratified fluid, the combined action of the differential rotation and the magnetic field leads to the well known regime of `super-rotation', in which the fluid rotates faster than the inner core. Whereas in the classical case, this super-rotation is known to vanish in the magnetostrophic limit, we show here that the fluid stratification significantly extends the magnitude of the super-rotation, keeping this phenomenon relevant for the Earth core. Finally, we study how the shear layers generated by this new state might give birth to magnetohydrodynamic instabilities or waves impacting the secular variations or jerks of the Earth's magnetic field.

Calpain activation by ROS mediates human ether-a-go-go-related gene protein degradation by intermittent hypoxia.

Science.gov (United States)

Wang, N; Kang, H S; Ahmmed, G; Khan, S A; Makarenko, V V; Prabhakar, N R; Nanduri, J

2016-03-01

Human ether-a-go-go-related gene (hERG) channels conduct delayed rectifier K(+) current. However, little information is available on physiological situations affecting hERG channel protein and function. In the present study we examined the effects of intermittent hypoxia (IH), which is a hallmark manifestation of sleep apnea, on hERG channel protein and function. Experiments were performed on SH-SY5Y neuroblastoma cells, which express hERG protein. Cells were exposed to IH consisting of alternating cycles of 30 s of hypoxia (1.5% O2) and 5 min of 20% O2. IH decreased hERG protein expression in a stimulus-dependent manner. A similar reduction in hERG protein was also seen in adrenal medullary chromaffin cells from IH-exposed neonatal rats. The decreased hERG protein was associated with attenuated hERG K(+) current. IH-evoked hERG protein degradation was not due to reduced transcription or increased proteosome/lysomal degradation. Rather it was mediated by calcium-activated calpain proteases. Both COOH- and NH2-terminal sequences of the hERG protein were the targets of calpain-dependent degradation. IH increased reactive oxygen species (ROS) levels, intracellular Ca(2+) concentration ([Ca(2+)]i), calpain enzyme activity, and hERG protein degradation, and all these effects were prevented by manganese-(111)-tetrakis-(1-methyl-4-pyridyl)-porphyrin pentachloride, a membrane-permeable ROS scavenger. These results demonstrate that activation of calpains by ROS-dependent elevation of [Ca(2+)]i mediates hERG protein degradation by IH. Copyright © 2016 the American Physiological Society.

Turbulent circulation above the surface heat source in stably stratified atmosphere

Science.gov (United States)

Kurbatskii, A. F.; Kurbatskaya, L. I.

2016-10-01

The 3-level RANS approach for simulating a turbulent circulation over the heat island in a stably stratified environment under nearly calm conditions is formulated. The turbulent kinetic energy its spectral consumption (dissipation) and the dispersion of turbulent fluctuations of temperature are found from differential equations, thus the correct modeling of transport processes in the interface layer with the counter-gradient heat flux is assured. The three-parameter turbulence RANS approach minimizes difficulties in simulating the turbulent transport in a stably stratified environment and reduces efforts needed for the numerical implementation of the 3-level RANS approach. Numerical simulation of the turbulent structure of the penetrative convection over the heat island under conditions of stably stratified atmosphere demonstrates that the three-equation model is able to predict the thermal circulation induced by the heat island. The temperature distribution, root-mean-square fluctuations of the turbulent velocity and temperature fields and spectral turbulent kinetic energy flux are in good agreement with the experimental data. The model describes such thin physical effects, as a crossing of vertical profiles of temperature of a thermal plume with the formation of the negative buoyancy area testifying to development of the dome-shaped form at the top part of a plume in the form of "hat".

hERG blocking potential of acids and zwitterions characterized by three thresholds for acidity, size and reactivity

DEFF Research Database (Denmark)

Nikolov, Nikolai Georgiev; Dybdahl, Marianne; Jonsdottir, Svava Osk

2014-01-01

with a concordance of 91% by a decision tree based on the rule. Two external validations were performed with sets of 35 and 48 observations, respectively, both showing concordances of 91%. In addition, a global QSAR model of hERG blocking was constructed based on a large diverse training set of 1374 chemicals...... covering all ionization classes, externally validated showing high predictivity and compared to the decision tree. The decision tree was found to be superior for the acids and zwitterionic ampholytes classes....

Thermal response of rat fibroblasts stably transfected with the human 70-kDa heat shock protein-encoding gene

International Nuclear Information System (INIS)

Li, G.C.; Li, Ligeng; Liu, Yunkang; Mak, J.Y.; Chen, Lili; Lee, W.M.F.

1991-01-01

The major heat shock protein hsp70 is synthesized by cells of a wide variety of organisms in response to heat shock or other environmental stresses and is assumed to play an important role in protecting cells from thermal stress. The authors have tested this hypothesis directly by transfecting a constitutively expressed recombinant human hsp70-encoding gene into rat fibroblasts and examining the relationship between the levels of human hsp70 expressed and thermal resistance of the stably transfected rat cells. Successful transfection and expression of the gene for human hsp70 were characterized by RNA hybridization analysis, low-dimensional gel electrophoresis, and immunoblot analysis. When individual cloned cell lines were exposed to 45C and their thermal survivals were determined by colony-formation assay, they found that the expression of human hsp70 conferred heat resistance to the rat cells. These results reinforce the hypothesis that hsp70 has a protective function against thermal stress

Acute alteration of cardiac ECG, action potential, I{sub Kr} and the human ether-a-go-go-related gene (hERG) K{sup +} channel by PCB 126 and PCB 77

Energy Technology Data Exchange (ETDEWEB)

Park, Mi-Hyeong; Park, Won Sun; Jo, Su-Hyun, E-mail: suhyunjo@kangwon.ac.kr

2012-07-01

Polychlorinated biphenyls (PCBs) have been known as serious persistent organic pollutants (POPs), causing developmental delays and motor dysfunction. We have investigated the effects of two PCB congeners, 3,3′,4,4′-tetrachlorobiphenyl (PCB 77) and 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126) on ECG, action potential, and the rapidly activating delayed rectifier K{sup +} current (I{sub Kr}) of guinea pigs' hearts, and hERG K{sup +} current expressed in Xenopus oocytes. PCB 126 shortened the corrected QT interval (QTc) of ECG and decreased the action potential duration at 90% (APD{sub 90}), and 50% of repolarization (APD{sub 50}) (P < 0.05) without changing the action potential duration at 20% (APD{sub 20}). PCB 77 decreased APD{sub 20} (P < 0.05) without affecting QTc, APD{sub 90}, and APD{sub 50}. The PCB 126 increased the I{sub Kr} in guinea-pig ventricular myocytes held at 36 °C and hERG K{sup +} current amplitude at the end of the voltage steps in voltage-dependent mode (P < 0.05); however, PCB 77 did not change the hERG K{sup +} current amplitude. The PCB 77 increased the diastolic Ca{sup 2+} and decreased Ca{sup 2+} transient amplitude (P < 0.05), however PCB 126 did not change. The results suggest that PCB 126 shortened the QTc and decreased the APD{sub 90} possibly by increasing I{sub Kr}, while PCB 77 decreased the APD{sub 20} possibly by other modulation related with intracellular Ca{sup 2+}. The present data indicate that the environmental toxicants, PCBs, can acutely affect cardiac electrophysiology including ECG, action potential, intracellular Ca{sup 2+}, and channel activity, resulting in toxic effects on the cardiac function in view of the possible accumulation of the PCBs in human body. -- Highlights: ► PCBs are known as serious environmental pollutants and developmental disruptors. ► PCB 126 shortened QT interval of ECG and action potential duration. ► PCB 126 increased human ether-a-go-go-related K{sup +} current and I{sub Kr}. �

Identification and validation of suitable endogenous reference genes for gene expression studies in human peripheral blood

Directory of Open Access Journals (Sweden)

Turner Renee J

2009-08-01

Full Text Available Abstract Background Gene expression studies require appropriate normalization methods. One such method uses stably expressed reference genes. Since suitable reference genes appear to be unique for each tissue, we have identified an optimal set of the most stably expressed genes in human blood that can be used for normalization. Methods Whole-genome Affymetrix Human 2.0 Plus arrays were examined from 526 samples of males and females ages 2 to 78, including control subjects and patients with Tourette syndrome, stroke, migraine, muscular dystrophy, and autism. The top 100 most stably expressed genes with a broad range of expression levels were identified. To validate the best candidate genes, we performed quantitative RT-PCR on a subset of 10 genes (TRAP1, DECR1, FPGS, FARP1, MAPRE2, PEX16, GINS2, CRY2, CSNK1G2 and A4GALT, 4 commonly employed reference genes (GAPDH, ACTB, B2M and HMBS and PPIB, previously reported to be stably expressed in blood. Expression stability and ranking analysis were performed using GeNorm and NormFinder algorithms. Results Reference genes were ranked based on their expression stability and the minimum number of genes needed for nomalization as calculated using GeNorm showed that the fewest, most stably expressed genes needed for acurate normalization in RNA expression studies of human whole blood is a combination of TRAP1, FPGS, DECR1 and PPIB. We confirmed the ranking of the best candidate control genes by using an alternative algorithm (NormFinder. Conclusion The reference genes identified in this study are stably expressed in whole blood of humans of both genders with multiple disease conditions and ages 2 to 78. Importantly, they also have different functions within cells and thus should be expressed independently of each other. These genes should be useful as normalization genes for microarray and RT-PCR whole blood studies of human physiology, metabolism and disease.

Mitragynine and its potential blocking effects on specific cardiac potassium channels

Energy Technology Data Exchange (ETDEWEB)

Tay, Yea Lu; Teah, Yi Fan; Chong, Yoong Min [Malaysian Institute of Pharmaceuticals & Nutraceuticals, NIBM, Ministry of Science, Technology & Innovation (MOSTI), Pulau Pinang (Malaysia); Jamil, Mohd Fadzly Amar [Clinical Research Center, Hospital Seberang Jaya, Kementerian Kesihatan Malaysia, Pulau Pinang (Malaysia); Kollert, Sina [Institute of Physiology, University of Wurzburg, Wurzburg (Germany); Adenan, Mohd Ilham [Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM), Selangor Darul Ehsan (Malaysia); Wahab, Habibah Abdul [Pharmaceutical Design & Simulation (PhDS) Laboratory, School of Pharmaceutical Sciences, Universiti Sains Malaysia, Pulau Pinang (Malaysia); Döring, Frank; Wischmeyer, Erhard [Institute of Physiology, University of Wurzburg, Wurzburg (Germany); Tan, Mei Lan, E-mail: tanml@usm.my [Malaysian Institute of Pharmaceuticals & Nutraceuticals, NIBM, Ministry of Science, Technology & Innovation (MOSTI), Pulau Pinang (Malaysia); Advanced Medical and Dental Institute, Universiti Sains Malaysia, Pulau Pinang (Malaysia)

2016-08-15

Mitragyna speciosa Korth is known for its euphoric properties and is frequently used for recreational purposes. Several poisoning and fatal cases involving mitragynine have been reported but the underlying causes remain unclear. Human ether-a-go-go-related gene (hERG) encodes the cardiac I{sub Kr} current which is a determinant of the duration of ventricular action potentials and QT interval. On the other hand, I{sub K1}, a Kir current mediated by Kir2.1 channel and I{sub KACh}, a receptor-activated Kir current mediated by GIRK channel are also known to be important in maintaining the cardiac function. This study investigated the effects of mitragynine on the current, mRNA and protein expression of hERG channel in hERG-transfected HEK293 cells and Xenopus oocytes. The effects on Kir2.1 and GIRK channels currents were also determined in the oocytes. The hERG tail currents following depolarization pulses were inhibited by mitragynine with an IC{sub 50} value of 1.62 μM and 1.15 μM in the transfected cell line and Xenopus oocytes, respectively. The S6 point mutations of Y652A and F656A attenuated the inhibitor effects of mitragynine, indicating that mitragynine interacts with these high affinity drug-binding sites in the hERG channel pore cavity which was consistent with the molecular docking simulation. Interestingly, mitragynine does not affect the hERG expression at the transcriptional level but inhibits the protein expression. Mitragynine is also found to inhibit I{sub KACh} current with an IC{sub 50} value of 3.32 μM but has no significant effects on I{sub K1}. Blocking of both hERG and GIRK channels may cause additive cardiotoxicity risks. - Highlights: • The potential cardiac potassium channel blocking properties of mitragynine were investigated. • Mitragynine blocks hERG channel and I{sub Kr} in hERG-transfected HEK293 cells and hERG cRNA-injected Xenopus oocytes. • Mitragynine inhibits the hERG protein but not the mRNA expression. • Mitragynine

Mitragynine and its potential blocking effects on specific cardiac potassium channels

International Nuclear Information System (INIS)

Tay, Yea Lu; Teah, Yi Fan; Chong, Yoong Min; Jamil, Mohd Fadzly Amar; Kollert, Sina; Adenan, Mohd Ilham; Wahab, Habibah Abdul; Döring, Frank; Wischmeyer, Erhard; Tan, Mei Lan

2016-01-01

Mitragyna speciosa Korth is known for its euphoric properties and is frequently used for recreational purposes. Several poisoning and fatal cases involving mitragynine have been reported but the underlying causes remain unclear. Human ether-a-go-go-related gene (hERG) encodes the cardiac I Kr current which is a determinant of the duration of ventricular action potentials and QT interval. On the other hand, I K1 , a Kir current mediated by Kir2.1 channel and I KACh , a receptor-activated Kir current mediated by GIRK channel are also known to be important in maintaining the cardiac function. This study investigated the effects of mitragynine on the current, mRNA and protein expression of hERG channel in hERG-transfected HEK293 cells and Xenopus oocytes. The effects on Kir2.1 and GIRK channels currents were also determined in the oocytes. The hERG tail currents following depolarization pulses were inhibited by mitragynine with an IC 50 value of 1.62 μM and 1.15 μM in the transfected cell line and Xenopus oocytes, respectively. The S6 point mutations of Y652A and F656A attenuated the inhibitor effects of mitragynine, indicating that mitragynine interacts with these high affinity drug-binding sites in the hERG channel pore cavity which was consistent with the molecular docking simulation. Interestingly, mitragynine does not affect the hERG expression at the transcriptional level but inhibits the protein expression. Mitragynine is also found to inhibit I KACh current with an IC 50 value of 3.32 μM but has no significant effects on I K1 . Blocking of both hERG and GIRK channels may cause additive cardiotoxicity risks. - Highlights: • The potential cardiac potassium channel blocking properties of mitragynine were investigated. • Mitragynine blocks hERG channel and I Kr in hERG-transfected HEK293 cells and hERG cRNA-injected Xenopus oocytes. • Mitragynine inhibits the hERG protein but not the mRNA expression. • Mitragynine inhibits GIRK channel. • Simultaneous

Interactions between charged residues in the transmembrane segments of the voltage-sensing domain in the hERG channel.

Science.gov (United States)

Zhang, M; Liu, J; Jiang, M; Wu, D-M; Sonawane, K; Guy, H R; Tseng, G-N

2005-10-01

Studies on voltage-gated K channels such as Shaker have shown that positive charges in the voltage-sensor (S4) can form salt bridges with negative charges in the surrounding transmembrane segments in a state-dependent manner, and different charge pairings can stabilize the channels in closed or open states. The goal of this study is to identify such charge interactions in the hERG channel. This knowledge can provide constraints on the spatial relationship among transmembrane segments in the channel's voltage-sensing domain, which are necessary for modeling its structure. We first study the effects of reversing S4's positive charges on channel activation. Reversing positive charges at the outer (K525D) and inner (K538D) ends of S4 markedly accelerates hERG activation, whereas reversing the 4 positive charges in between either has no effect or slows activation. We then use the 'mutant cycle analysis' to test whether D456 (outer end of S2) and D411 (inner end of S1) can pair with K525 and K538, respectively. Other positive charges predicted to be able, or unable, to interact with D456 or D411 are also included in the analysis. The results are consistent with predictions based on the distribution of these charged residues, and confirm that there is functional coupling between D456 and K525 and between D411 and K538.

Sensitivity of the Geomagnetic Octupole to a Stably Stratified Layer in the Earth's Core

Science.gov (United States)

Yan, C.; Stanley, S.

2017-12-01

The presence of a stably stratified layer at the top of the core has long been proposed for Earth, based on evidence from seismology and geomagnetic secular variation. Geodynamo modeling offers a unique window to inspect the properties and dynamics in Earth's core. For example, numerical simulations have shown that magnetic field morphology is sensitive to the presence of stably stratified layers in a planet's core. Here we use the mMoSST numerical dynamo model to investigate the effects of a thin stably stratified layer at the top of the fluid outer core in Earth on the resulting large-scale geomagnetic field morphology. We find that the existence of a stable layer has significant influence on the octupolar component of the magnetic field in our models, whereas the quadrupole doesn't show an obvious trend. This suggests that observations of the geomagnetic field can be applied to provide information of the properties of this plausible stable layer, such as how thick and how stable this layer could be. Furthermore, we have examined whether the dominant thermal signature from mantle tomography at the core-mantle boundary (CMB) (a degree & order 2 spherical harmonic) can influence our results. We found that this heat flux pattern at the CMB has no outstanding effects on the quadrupole and octupole magnetic field components. Our studies suggest that if there is a stably stratified layer at the top of the Earth's core, it must be limited in terms of stability and thickness, in order to be compatible with the observed paleomagnetic record.

Large eddy simulation of turbulent and stably-stratified flows

International Nuclear Information System (INIS)

Fallon, Benoit

1994-01-01

The unsteady turbulent flow over a backward-facing step is studied by mean of Large Eddy Simulations with structure function sub grid model, both in isothermal and stably-stratified configurations. Without stratification, the flow develops highly-distorted Kelvin-Helmholtz billows, undergoing to helical pairing, with A-shaped vortices shed downstream. We show that forcing injected by recirculation fluctuations governs this oblique mode instabilities development. The statistical results show good agreements with the experimental measurements. For stably-stratified configurations, the flow remains more bi-dimensional. We show with increasing stratification, how the shear layer growth is frozen by inhibition of pairing process then of Kelvin-Helmholtz instabilities, and the development of gravity waves or stable density interfaces. Eddy structures of the flow present striking analogies with the stratified mixing layer. Additional computations show the development of secondary Kelvin-Helmholtz instabilities on the vorticity layers between two primary structures. This important mechanism based on baroclinic effects (horizontal density gradients) constitutes an additional part of the turbulent mixing process. Finally, the feasibility of Large Eddy Simulation is demonstrated for industrial flows, by studying a complex stratified cavity. Temperature fluctuations are compared to experimental measurements. We also develop three-dimensional un-stationary animations, in order to understand and visualize turbulent interactions. (author) [fr

Optimal energy growth in a stably stratified shear flow

Science.gov (United States)

Jose, Sharath; Roy, Anubhab; Bale, Rahul; Iyer, Krithika; Govindarajan, Rama

2018-02-01

Transient growth of perturbations by a linear non-modal evolution is studied here in a stably stratified bounded Couette flow. The density stratification is linear. Classical inviscid stability theory states that a parallel shear flow is stable to exponentially growing disturbances if the Richardson number (Ri) is greater than 1/4 everywhere in the flow. Experiments and numerical simulations at higher Ri show however that algebraically growing disturbances can lead to transient amplification. The complexity of a stably stratified shear flow stems from its ability to combine this transient amplification with propagating internal gravity waves (IGWs). The optimal perturbations associated with maximum energy amplification are numerically obtained at intermediate Reynolds numbers. It is shown that in this wall-bounded flow, the three-dimensional optimal perturbations are oblique, unlike in unstratified flow. A partitioning of energy into kinetic and potential helps in understanding the exchange of energies and how it modifies the transient growth. We show that the apportionment between potential and kinetic energy depends, in an interesting manner, on the Richardson number, and on time, as the transient growth proceeds from an optimal perturbation. The oft-quoted stabilizing role of stratification is also probed in the non-diffusive limit in the context of disturbance energy amplification.

Cholesterol Down-Regulates BK Channels Stably Expressed in HEK 293 Cells

Science.gov (United States)

Deng, Xiu-Ling; Sun, Hai-Ying; Li, Gui-Rong

2013-01-01

Cholesterol is one of the major lipid components of the plasma membrane in mammalian cells and is involved in the regulation of a number of ion channels. The present study investigates how large conductance Ca2+-activated K+ (BK) channels are regulated by membrane cholesterol in BK-HEK 293 cells expressing both the α-subunit hKCa1.1 and the auxiliary β1-subunit or in hKCa1.1-HEK 293 cells expressing only the α-subunit hKCa1.1 using approaches of electrophysiology, molecular biology, and immunocytochemistry. Membrane cholesterol was depleted in these cells with methyl-β-cyclodextrin (MβCD), and enriched with cholesterol-saturated MβCD (MβCD-cholesterol) or low-density lipoprotein (LDL). We found that BK current density was decreased by cholesterol enrichment in BK-HEK 293 cells, with a reduced expression of KCa1.1 protein, but not the β1-subunit protein. This effect was fully countered by the proteasome inhibitor lactacystin or the lysosome function inhibitor bafilomycin A1. Interestingly, in hKCa1.1-HEK 293 cells, the current density was not affected by cholesterol enrichment, but directly decreased by MβCD, suggesting that the down-regulation of BK channels by cholesterol depends on the auxiliary β1-subunit. The reduced KCa1.1 channel protein expression was also observed in cultured human coronary artery smooth muscle cells with cholesterol enrichment using MβCD-cholesterol or LDL. These results demonstrate the novel information that cholesterol down-regulates BK channels by reducing KCa1.1 protein expression via increasing the channel protein degradation, and the effect is dependent on the auxiliary β1-subunit. PMID:24260325

Scaffold-free 3D bio-printed human liver tissue stably maintains metabolic functions useful for drug discovery.

Science.gov (United States)

Kizawa, Hideki; Nagao, Eri; Shimamura, Mitsuru; Zhang, Guangyuan; Torii, Hitoshi

2017-07-01

The liver plays a central role in metabolism. Although many studies have described in vitro liver models for drug discovery, to date, no model has been described that can stably maintain liver function. Here, we used a unique, scaffold-free 3D bio-printing technology to construct a small portion of liver tissue that could stably maintain drug, glucose, and lipid metabolism, in addition to bile acid secretion. This bio-printed normal human liver tissue maintained expression of several kinds of hepatic drug transporters and metabolic enzymes that functioned for several weeks. The bio-printed liver tissue displayed glucose production via cAMP/protein kinase A signaling, which could be suppressed with insulin. Bile acid secretion was also observed from the printed liver tissue, and it accumulated in the culture medium over time. We observed both bile duct and sinusoid-like structures in the bio-printed liver tissue, which suggested that bile acid secretion occurred via a sinusoid-hepatocyte-bile duct route. These results demonstrated that our bio-printed liver tissue was unique, because it exerted diverse liver metabolic functions for several weeks. In future, we expect our bio-printed liver tissue to be applied to developing new models that can be used to improve preclinical predictions of long-term toxicity in humans, generate novel targets for metabolic liver disease, and evaluate biliary excretion in drug development.

Effect of microculture on cell metabolism and biochemistry: do cells get stressed in microchannels?

Science.gov (United States)

Su, Xiaojing; Theberge, Ashleigh B; January, Craig T; Beebe, David J

2013-02-05

Microfluidics is emerging as a promising platform for cell culture, enabling increased microenvironment control and potential for integrated analysis compared to conventional macroculture systems such as well plates and Petri dishes. To advance the use of microfluidic devices for cell culture, it is necessary to better understand how miniaturization affects cell behavior. In particular, microfluidic devices have significantly higher surface-area-to-volume ratios than conventional platforms, resulting in lower volumes of media per cell, which can lead to cell stress. We investigated cell stress under a variety of culture conditions using three cell lines: parental HEK (human embryonic kidney) cells and transfected HEK cells that stably express wild-type (WT) and mutant (G601S) human ether-a-go-go related gene (hERG) potassium channel protein. These three cell lines provide a unique model system through which to study cell-type-specific responses in microculture because mutant hERG is known to be sensitive to environmental conditions, making its expression a particularly sensitive readout through which to compare macro- and microculture. While expression of WT-hERG was similar in microchannel and well culture, the expression of mutant G601S-hERG was reduced in microchannels. Expression of the endoplasmic reticulum (ER) stress marker immunoglobulin binding protein (BiP) was upregulated in all three cell lines in microculture. Using BiP expression, glucose consumption, and lactate accumulation as readouts we developed methods for reducing ER stress including properly increasing the frequency of media replacement, reducing cell seeding density, and adjusting the serum concentration and buffering capacity of culture medium. Indeed, increasing the buffering capacity of culture medium or frequency of media replacement partially restored the expression of the G601S-hERG in microculture. This work illuminates how biochemical properties of cells differ in macro- and

Construction of PVX virus-expression vector to express enterotoxin ...

African Journals Online (AJOL)

Potato X potyvirus (PVX)-based vector has been comprehensively applied in transient expression system. In order to produce the heterologous proteins more quickly and stably, the ClaI and NotI enzyme sites were introduced into the Enterotoxin fusion gene LTB-ST by polymerase chain reaction (PCR) and the LTB-ST ...

BmTx3, a scorpion toxin with two putative functional faces separately active on A-type K+ and HERG currents.

OpenAIRE

Huys, Isabelle; Xu, Chen-Qi; Wang, Cheng-Zhong; Vacher, Hélène; Martin-Eauclaire, Marie-France; Chi, Cheng-Wu; Tytgat, Jan

2004-01-01

A novel HERG channel blocker was isolated from the venom of the scorpion Buthus martensi Karsch, sequenced and characterized at the pharmacological level after chemical synthesis. According to the determined amino acid sequence, the cDNA and genomic genes were then cloned. The genomic gene consists of two exons interrupted by an intron of 65 bp at position -6 upstream from the mature toxin. The protein sequence of this toxin was completely identical with that of a known A-type K+ current bloc...

Transgenic Parasites Stably Expressing Full-Length Plasmodium falciparum Circumsporozoite Protein as a Model for Vaccine Down-Selection in Mice Using Sterile Protection as an Endpoint

Science.gov (United States)

Porter, Michael D.; Nicki, Jennifer; Pool, Christopher D.; DeBot, Margot; Illam, Ratish M.; Brando, Clara; Bozick, Brooke; De La Vega, Patricia; Angra, Divya; Spaccapelo, Roberta; Crisanti, Andrea; Murphy, Jittawadee R.; Bennett, Jason W.; Schwenk, Robert J.; Ockenhouse, Christian F.

2013-01-01

Circumsporozoite protein (CSP) of Plasmodium falciparum is a protective human malaria vaccine candidate. There is an urgent need for models that can rapidly down-select novel CSP-based vaccine candidates. In the present study, the mouse-mosquito transmission cycle of a transgenic Plasmodium berghei malaria parasite stably expressing a functional full-length P. falciparum CSP was optimized to consistently produce infective sporozoites for protection studies. A minimal sporozoite challenge dose was established, and protection was defined as the absence of blood-stage parasites 14 days after intravenous challenge. The specificity of protection was confirmed by vaccinating mice with multiple CSP constructs of differing lengths and compositions. Constructs that induced high NANP repeat-specific antibody titers in enzyme-linked immunosorbent assays were protective, and the degree of protection was dependent on the antigen dose. There was a positive correlation between antibody avidity and protection. The antibodies in the protected mice recognized the native CSP on the parasites and showed sporozoite invasion inhibitory activity. Passive transfer of anti-CSP antibodies into naive mice also induced protection. Thus, we have demonstrated the utility of a mouse efficacy model to down-select human CSP-based vaccine formulations. PMID:23536694

Effects of the small molecule HERG activator NS1643 on Kv11.3 channels.

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Arne Bilet

Full Text Available NS1643 is one of the small molecule HERG (Kv11.1 channel activators and has also been found to increase erg2 (Kv11.2 currents. We now investigated whether NS1643 is also able to act as an activator of Kv11.3 (erg3 channels expressed in CHO cells. Activation of rat Kv11.3 current occurred in a dose-dependent manner and maximal current increasing effects were obtained with 10 µM NS1643. At this concentration, steady-state outward current increased by about 80% and the current increase was associated with a significant shift in the voltage dependence of activation to more negative potentials by about 15 mV. In addition, activation kinetics were accelerated, whereas deactivation was slowed. There was no significant effect on the kinetics of inactivation and recovery from inactivation. The strong current-activating agonistic effect of NS1643 did not result from a shift in the voltage dependence of Kv11.3 channel inactivation and was independent from external Na(+ or Ca(2+. At the higher concentration of 20 µM, NS1643 induced clearly less current increase. The left shift in the voltage dependence of activation reversed and the voltage sensitivity of activation dramatically decreased along with a slowing of Kv11.3 channel activation. These data show that, in comparison to other Kv11 family members, NS1643 exerts distinct effects on Kv11.3 channels with especially pronounced partial antagonistic effects at higher concentration.

Estudio electrofisiológico de canales Herg en células de cáncer de colón

OpenAIRE

Granja del Río, Alejandra

2013-01-01

En el presente estudio, mostramos que en las células de colon normales (NCM460) y en las tumorales (HT29) las principales corrientes activadas por voltaje son corrientes de K+. Sin embargo, el hallazgo más interesante fue mostrar que, en contraste con las células normales de colon, las células tumorales, al parecer, expresan corrientes de K+ activadas por voltaje que podrían ser mediadas por canales herg. Departamento de Bioquímica y Biología Molecular y Fisiología Máster en Investigaci...

Alkaline polymer electrolyte fuel cells stably working at 80 °C

Science.gov (United States)

Peng, Hanqing; Li, Qihao; Hu, Meixue; Xiao, Li; Lu, Juntao; Zhuang, Lin

2018-06-01

Alkaline polymer electrolyte fuel cells are a new class of polymer electrolyte fuel cells that fundamentally enables the use of nonprecious metal catalysts. The cell performance mostly relies on the quality of alkaline polymer electrolytes, including the ionic conductivity and the chemical/mechanical stability. For a long time, alkaline polymer electrolytes are thought to be too weak in stability to allow the fuel cell to be operated at elevated temperatures, e.g., above 60 °C. In the present work, we report a progress in the state-of-the-art alkaline polymer electrolyte fuel cell technology. By using a newly developed alkaline polymer electrolyte, quaternary ammonia poly (N-methyl-piperidine-co-p-terphenyl), which simultaneously possesses high ionic conductivity and excellent chemical/mechanical stability, the fuel cell can now be stably operated at 80 °C with high power density. The peak power density reaches ca. 1.5 W/cm2 at 80 °C with Pt/C catalysts used in both the anode and the cathode. The cell works stably in a period of study over 100 h.

Pharmacological and functional characterisation of the wild-type and site-directed mutants of the human H1 histamine receptor stably expressed in CHO cells.

Science.gov (United States)

Moguilevsky, N; Varsalona, F; Guillaume, J P; Noyer, M; Gillard, M; Daliers, J; Henichart, J P; Bollen, A

1995-01-01

A cDNA clone for the human histamine H1 receptor was isolated from a lung cDNA library and stably expressed in CHO cells. The recombinant receptor protein present in the cell membranes, displayed the functional and binding characteristics of histamine H1 receptors. Mutation of Ser155 to Ala in the fourth transmembrane domain did not significantly change the affinity of the receptor for histamine and H1 antagonists. However, mutation of the fifth transmembrane Asn198 to Ala resulted in a dramatic decrease of the affinity for histamine binding, and for the histamine-induced polyphosphoinositides breakdown, whereas the affinity towards antagonists was not significantly modified. In addition, mutation of another fifth transmembrane amino acid, Thr194 to Ala also diminished, but to a lesser extent, the affinity for histamine. These data led us to propose a molecular model for histamine interaction with the human H1 receptor. In this model, the amide moiety of Asn198 and the hydroxyl group of Thr194 are involved in hydrogen bonding with the nitrogen atoms of the imidazole ring of histamine. Moreover, mutation of Thr194 to Ala demonstrated that this residue is responsible for the discrimination between enantiomers of cetirizine.

New binding site on common molecular scaffold provides HERG channel specificity of scorpion toxin BeKm-1

DEFF Research Database (Denmark)

Korolkova, Yuliya V; Bocharov, Eduard V; Angelo, Kamilla

2002-01-01

The scorpion toxin BeKm-1 is unique among a variety of known short scorpion toxins affecting potassium channels in its selective action on ether-a-go-go-related gene (ERG)-type channels. BeKm-1 shares the common molecular scaffold with other short scorpion toxins. The toxin spatial structure...... resolved by NMR consists of a short alpha-helix and a triple-stranded antiparallel beta-sheet. By toxin mutagenesis study we identified the residues that are important for the binding of BeKm-1 to the human ERG K+ (HERG) channel. The most critical residues (Tyr-11, Lys-18, Arg-20, Lys-23) are located...

"A Nightmare Land, a Place of Death": An Exploration of the Moon as a Motif in Herge's "Destination Moon" (1953) and "Explorers on the Moon" (1954)

Science.gov (United States)

Beauvais, Clementine

2010-01-01

This article analyses the symbolic meaning of the Moon in two "bande dessinee" books from the Tintin series, Herge's "Destination Moon" ("Objectif Lune," 1953) and its sequel "Explorers on the Moon" ("On a Marche sur la Lune," 1954). It argues that these two volumes stand out in the series for their graphic, narrative and philosophical emphasis on…

MicroRNA Expression Profiling to Identify and Validate Reference Genes for the Relative Quantification of microRNA in Rectal Cancer

DEFF Research Database (Denmark)

Eriksen, Anne Haahr Mellergaard; Andersen, Rikke Fredslund; Pallisgaard, Niels

2016-01-01

the miRNA profiling experiment, miR-645, miR-193a-5p, miR-27a and let-7g were identified as stably expressed, both in malignant and stromal tissue. In addition, NormFinder confirmed high expression stability for the four miRNAs. In the RT-qPCR based validation experiments, no significant difference...... management. Real-time quantitative polymerase chain reaction (RT-qPCR) is commonly used, when measuring miRNA expression. Appropriate normalisation of RT-qPCR data is important to ensure reliable results. The aim of the present study was to identify stably expressed miRNAs applicable as normaliser candidates...... in future studies of miRNA expression in rectal cancer.MATERIALS AND METHODS: We performed high-throughput miRNA profiling (OpenArray®) on ten pairs of laser micro-dissected rectal cancer tissue and adjacent stroma. A global mean expression normalisation strategy was applied to identify the most stably...

Mechanisms underlying probucol-induced hERG-channel deficiency

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Shi YQ

2015-07-01

Full Text Available Yuan-Qi Shi,1,* Cai-Chuan Yan,1,* Xiao Zhang,1 Meng Yan,1 Li-Rong Liu,1 Huai-Ze Geng,1 Lin Lv,1 Bao-Xin Li1,21Department of Pharmacology, Harbin Medical University, 2State-Province Key Laboratory of Biopharmaceutical Engineering, Harbin, Heilongjiang, People’s Republic of China*These authors contributed equally to this workAbstract: The hERG gene encodes the pore-forming α-subunit of the rapidly activating delayed rectifier potassium channel (IKr, which is important for cardiac repolarization. Reduction of IhERG due to genetic mutations or drug interferences causes long QT syndrome, leading to life-threatening cardiac arrhythmias (torsades de pointes or sudden death. Probucol is a cholesterol-lowering drug that could reduce hERG current by decreasing plasma membrane hERG protein expression and eventually cause long QT syndrome. Here, we investigated the mechanisms of probucol effects on IhERG and hERG-channel expression. Our data demonstrated that probucol reduces SGK1 expression, known as SGK isoform, in a concentration-dependent manner, resulting in downregulation of phosphorylated E3 ubiquitin ligase Nedd4-2 expression, but not the total level of Nedd4-2. As a result, the hERG protein reduces, due to the enhanced ubiquitination level. On the contrary, carbachol could enhance the phosphorylation level of Nedd4-2 as an alternative to SGK1, and thus rescue the ubiquitin-mediated degradation of hERG channels caused by probucol. These discoveries provide a novel mechanism of probucol-induced hERG-channel deficiency, and imply that carbachol or its analog may serve as potential therapeutic compounds for the handling of probucol cardiotoxicity.Keywords: long QT, hERG potassium channels, probucol, SGK1, Nedd4-2

Expression of hepatitis C virus envelope protein 2 induces apoptosis in cultured mammalian cells

Institute of Scientific and Technical Information of China (English)

Li-Xin Zhu; Jing Liu; You-Hua Xie; Yu-Ying Kong; Ye Ye; Chun-Lin Wang; Guang-Di Li; Yuan Wang

2004-01-01

AIM: To explore the role of hepatitis C virus (HCV) envelope protein 2 (E2) in the induction of apoptosis.METHODS: A carboxyterminal truncated E2 (E2-661) was transiently expressed in several cultured mammalian cell lines or stably expressed in Chinese hamster ovary (CHO)cell line. Cell proliferation was assessed by 3H thymidine uptake. Apoptosis was examined by Hoechst 33258staining, flow cytometry and DNA fragmentation analysis.RESULTS: Reduced proliferation was readily observed in the E2-661 expressing cells. These cells manifested the typical features of apoptosis, including cell shrinkage,chromatin condensation and hypodiploid genomic DNA content. Similar apoptotic cell death was observed in an E2-661 stably expressing cell line.CONCLUSION: HCV E2 can induce apoptosis in cultured mammalian cells.

Method of stably radiolabeling antibodies with technetium and rhenium

International Nuclear Information System (INIS)

Paik, C.H.; Reba, R.C.; Eckelman, W.C.

1987-01-01

A method is described for labeling antibodies or antibody fragments with radionuclides of technetium or rhenium to obtain stable labeling, comprising: reacting a reduced radioisotope of technetium or rhenium with an antibody or antibody fragment, or a diethylenetriaminepentaacetic acid conjugated antibody or antibody fragment, in the presence of free or carrier-bound diethylenetriaminepentaacetic acid (DTPA). The amount of DTPA is sufficient to substantially completely inhibit binding of the reduced technetium or rhenium to nonstable binding sites of the antibody or antibody fragment, or the DTPA-conjugated antibody or antibody fragment. The resultant stably labeled antibody or antibody fragment, or DTPA[conjugated antibody or antibody fragment is recovered

Nicotine Component of Cigarette Smoke Extract (CSE) Decreases the Cytotoxicity of CSE in BEAS-2B Cells Stably Expressing Human Cytochrome P450 2A13.

Science.gov (United States)

Ji, Minghui; Zhang, Yudong; Li, Na; Wang, Chao; Xia, Rong; Zhang, Zhan; Wang, Shou-Lin

2017-10-13

Cytochrome P450 2A13 (CYP2A13), an extrahepatic enzyme mainly expressed in the human respiratory system, has been reported to mediate the metabolism and toxicity of cigarette smoke. We previously found that nicotine inhibited 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) metabolism by CYP2A13, but its influence on other components of cigarette smoke remains unclear. The nicotine component of cigarette smoke extract (CSE) was separated, purified, and identified using high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), splitting CSE into a nicotine section (CSE-N) and nicotine-free section (CSE-O). Cell viability and apoptosis by Cell Counting Kit-8 (CCK-8) and flow cytometry assays were conducted on immortalized human bronchial epithelial (BEAS-2B) cells stably expressing CYP2A13 (B-2A13) or vector (B-V), respectively. Interestingly, CSE and CSE-O were toxic to BEAS-2B cells whereas CSE-N showed less cytotoxicity. CSE-O was more toxic to B-2A13 cells than to B-V cells (IC 50 of 2.49% vs. 7.06%), which was flatted by 8-methoxypsoralen (8-MOP), a CYP inhibitor. CSE-O rather than CSE or CSE-N increased apoptosis of B-2A13 cells rather than B-V cells. Accordingly, compared to CSE-N and CSE, CSE-O significantly changed the expression of three pairs of pro- and anti-apoptotic proteins, Bcl-2 Associated X Protein/B cell lymphoma-2 (Bax/Bcl-2), Cleaved Poly (Adenosine Diphosphate-Ribose) Polymerase/Poly (Adenosine Diphosphate-Ribose) Polymerase (C-PARP/PARP), and C-caspase-3/caspase-3, in B-2A13 cells. In addition, recombination of CSE-N and CSE-O (CSE-O/N) showed similar cytotoxicity and apoptosis to the original CSE. These results demonstrate that the nicotine component decreases the metabolic activation of CYP2A13 to CSE and aids in understanding the critical role of CYP2A13 in human respiratory diseases caused by cigarette smoking.

Cre recombinase expression can result in phenotypic aberrations in plants

NARCIS (Netherlands)

Coppoolse, E.; Vroomen, de M.J.; Roelofs, D.; Smit, J.; Gennip, van F.; Hersmus, B.J.M.; Nijkamp, H.J.J.; Haaren, van M.J.

2003-01-01

The cre recombinase gene was stably introduced and expressed in tomato, petunia and Nicotiana tabacum. Some plants expressing the cre gene driven by a CaMV 35S promoter displayed growth retardation and a distinct pattern of chlorosis in their leaves. Although no direct relation can be proven between

Cre recombinase expression can result in phenotypic aberrations in plants

NARCIS (Netherlands)

Coppoolse, Eric R; de Vroomen, Marianne J; Roelofs, Dick; Smit, Jaap; van Gennip, Femke; Hersmus, Bart J M; Nijkamp, H John J; van Haaren, Mark J J

The cre recombinase gene was stably introduced and expressed in tomato, petunia and Nicotiana tabacum. Some plants expressing the cre gene driven by a CaMV 35S promoter displayed growth retardation and a distinct pattern of chlorosis in their leaves. Although no direct relation can be proven between

Construction and characterization of a recombinant yellow fever virus stably expressing Gaussia luciferase

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TELISSA C. KASSAR

Full Text Available ABSTRACT Yellow fever is an arthropod-borne viral disease that still poses high public health concerns, despite the availability of an effective vaccine. The development of recombinant viruses is of utmost importance for several types of studies, such as those aimed to dissect virus-host interactions and to search for novel antiviral strategies. Moreover, recombinant viruses expressing reporter genes may greatly facilitate these studies. Here, we report the construction of a recombinant yellow fever virus (YFV expressing Gaussia luciferase (GLuc (YFV-GLuc. We show, through RT-PCR, sequencing and measurement of GLuc activity, that stability of the heterologous gene was maintained after six passages. Furthermore, a direct association between GLuc expression and viral replication was observed (r2=0.9967, indicating that measurement of GLuc activity may be used to assess viral replication in different applications. In addition, we evaluated the use of the recombinant virus in an antiviral assay with recombinant human alfa-2b interferon. A 60% inhibition of GLuc expression was observed in cells infected with YFV-GLuc and incubated with IFN alfa-2b. Previously tested on YFV inhibition by plaque assays indicated a similar fold-decrease in viral replication. These results are valuable as they show the stability of YFV-GLuc and one of several possible applications of this construct.

Reference gene validation for gene expression normalization in canine osteosarcoma : a geNorm algorithm approach

NARCIS (Netherlands)

Selvarajah, G.T.; Bonestroo, F.A.S.; Timmermans Sprang, E.P.M.; Kirpensteijn, J.|info:eu-repo/dai/nl/189846992; Mol, J.A.|info:eu-repo/dai/nl/070918775

2017-01-01

Background Quantitative PCR (qPCR) is a common method for quantifying mRNA expression. Given the heterogeneity present in tumor tissues, it is crucial to normalize target mRNA expression data using appropriate reference genes that are stably expressed under a variety of pathological and experimental

Impact of cell type and epitope tagging on heterologous expression of G protein-coupled receptor: a systematic study on angiotensin type II receptor.

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Lili Jiang

Full Text Available Despite heterologous expression of epitope-tagged GPCR is widely adopted for functional characterization, there is lacking of systematic analysis of the impact of expression host and epitope tag on GPCR expression. Angiotensin type II (AT2 receptor displays agonist-dependent and -independent activities, coupling to a spectrum of signaling molecules. However, consensus has not been reached on the subcellular distributions, signaling cascades and receptor-mediated actions. To examine the contributions of host cell and epitope tag on receptor expression and activity, epitope-tagged AT2 receptor variants were transiently or stably expressed in HEK293, CHO-K1 and PC12 cells. The epitope-tagged AT2 receptor variants were detected both on the cell membrane and in the perinuclear region. In transiently transfected HEK293 cells, Myc-AT2 existed predominantly as monomer. Additionally, a ladder of ubiquitinated AT2 receptor proteins was detected. By contrast, stably expressed epitope-tagged AT2 receptor variants existed as both monomer and high molecular weight complexes, and the latter was enriched in cell surface. Glycosylation promoted cell surface expression of Myc-AT2 but had no effect on AT2-GFP in HEK293 cells. In cells that stably expressed Myc-AT2, serum starvation induced apoptosis in CHO-K1 cells but not in HEK293 or PC12 cells. Instead, HEK293 and PC12 cells stably expressing Myc-AT2 exhibited partial cell cycle arrest with cells accumulating at G1 and S phases, respectively. Taken together, these results suggest that expression levels, subcellular distributions and ligand-independent constitutive activities of AT2 receptor were cell type-dependent while posttranslational processing of nascent AT2 receptor protein was modulated by epitope tag and mode of expression.

Construction of a high-EGFR expression cell line and its biological ...

African Journals Online (AJOL)

Targeted screening of EGFR compounds has become one of the medical research focuses for tumor therapy. A431, which naturally expresses high levels of EGFR, was compared with the stably high expressing EGFR cell line HEK293. Flow cytometry was used to analyze cell growth and Western blot was used to ...

Housekeeping genes for quantitative expression studies in the three-spined stickleback Gasterosteus aculeatus

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Becker Sven

2008-01-01

Full Text Available Abstract Background During the last years the quantification of immune response under immunological challenges, e.g. parasitation, has been a major focus of research. In this context, the expression of immune response genes in teleost fish has been surveyed for scientific and commercial purposes. Despite the fact that it was shown in teleostei and other taxa that the gene for beta-actin is not the most stably expressed housekeeping gene (HKG, depending on the tissue and experimental treatment, the gene has been used as a reference gene in such studies. In the three-spined stickleback, Gasterosteus aculeatus, other HKG than the one for beta-actin have not been established so far. Results To establish a reliable method for the measurement of immune gene expression in Gasterosteus aculeatus, sequences from the now available genome database and an EST library of the same species were used to select oligonucleotide primers for HKG, in order to perform quantitative reverse-transcription (RT PCR. The expression stability of ten candidate reference genes was evaluated in three different tissues, and in five parasite treatment groups, using the three algorithms BestKeeper, geNorm and NormFinder. Our results showed that in most of the tissues and treatments HKG that could not be used so far due to unknown sequences, proved to be more stably expressed than the one for beta-actin. Conclusion As they were the most stably expressed genes in all tissues examined, we suggest using the genes for the L13a ribosomal binding protein and ubiquitin as alternative or additional reference genes in expression analysis in Gasterosteus aculeatus.

Turbulent entrainment across turbulent-nonturbulent interfaces in stably stratified mixing layers

Science.gov (United States)

Watanabe, T.; Riley, J. J.; Nagata, K.

2017-10-01

The entrainment process in stably stratified mixing layers is studied in relation to the turbulent-nonturbulent interface (TNTI) using direct numerical simulations. The statistics are calculated with the interface coordinate in an Eulerian frame as well as with the Lagrangian fluid particles entrained from the nonturbulent to the turbulent regions. The characteristics of entrainment change as the buoyancy Reynolds number Reb decreases and the flow begins to layer. The baroclinic torque delays the enstrophy growth of the entrained fluids at small Reb, while this effect is less efficient for large Reb. The entrained particle movement within the TNTI layer is dominated by the small dissipative scales, and the rapid decay of the kinetic energy dissipation rate due to buoyancy causes the entrained particle movement relative to the interface location to become slower. Although the Eulerian statistics confirm that there exists turbulent fluid with strong vorticity or with large buoyancy frequency near the TNTI, the entrained fluid particles circumvent these regions by passing through the TNTI in strain-dominant regions or in regions with small buoyancy frequency. The multiparticle statistics show that once the nonturbulent fluid volumes are entrained, they are deformed into flattened shapes in the vertical direction and diffuse in the horizontal direction. When Reb is large enough for small-scale turbulence to exist, the entrained fluid is able to penetrate into the turbulent core region. Once the flow begins to layer with decreasing Reb, however, the entrained fluid volume remains near the outer edge of the turbulent region and forms a stably stratified layer without vertical overturning.

A germline chromothripsis event stably segregating in 11 individuals through three generations

DEFF Research Database (Denmark)

Bertelsen, Birgitte; Nazaryan-Petersen, Lusine; Sun, Wei

2016-01-01

PURPOSE: Parentally transmitted germ-line chromothripsis (G-CTH) has been identified in only a few cases. Most of these rearrangements were stably transmitted, in an unbalanced form, from a healthy mother to her child with congenital abnormalities probably caused by de novo copy-number changes...... of the DNA damage response, may be related to G-CTH formation. CONCLUSION: G-CTH rearrangements are not always associated with abnormal phenotypes and may be misinterpreted as balanced two-way translocations, suggesting that G-CTH is an underdiagnosed phenomenon.Genet Med 18 5, 494-500....

Turbulent fluxes in stably stratified boundary layers

International Nuclear Information System (INIS)

L'vov, Victor S; Procaccia, Itamar; Rudenko, Oleksii

2008-01-01

We present here an extended version of an invited talk we gave at the international conference 'Turbulent Mixing and Beyond'. The dynamical and statistical description of stably stratified turbulent boundary layers with the important example of the stable atmospheric boundary layer in mind is addressed. Traditional approaches to this problem, based on the profiles of mean quantities, velocity second-order correlations and dimensional estimates of the turbulent thermal flux, run into a well-known difficulty, predicting the suppression of turbulence at a small critical value of the Richardson number, in contradiction to observations. Phenomenological attempts to overcome this problem suffer from various theoretical inconsistencies. Here, we present an approach taking into full account all the second-order statistics, which allows us to respect the conservation of total mechanical energy. The analysis culminates in an analytic solution of the profiles of all mean quantities and all second-order correlations, removing the unphysical predictions of previous theories. We propose that the approach taken here is sufficient to describe the lower parts of the atmospheric boundary layer, as long as the Richardson number does not exceed an order of unity. For much higher Richardson numbers, the physics may change qualitatively, requiring careful consideration of the potential Kelvin-Helmoholtz waves and their interaction with the vortical turbulence.

Turbulent circulation above the surface heat source in a stably stratified environment

Science.gov (United States)

Kurbatskii, A. F.; Kurbatskaya, L. I.

2016-09-01

The results of the numerical modeling of turbulent structure of the penetrating convection above the urban heat island with a small aspect ratio in a stably stratified medium at rest are presented. The gradient diffusion representations for turbulent momentum and heat fluxes are used, which depend on three parameters — the turbulence kinetic energy, the velocity of its spectral expenditure, and the dispersion of temperature fluctuations. These parameters are found from the closed differential equations of balance in the RANS approach of turbulence description. The distributions of averaged velocity and temperature fields as well as turbulent characteristics agree well with measurement data.

Microstructure of Turbulence in the Stably Stratified Boundary Layer

Science.gov (United States)

Sorbjan, Zbigniew; Balsley, Ben B.

2008-11-01

The microstructure of a stably stratified boundary layer, with a significant low-level nocturnal jet, is investigated based on observations from the CASES-99 campaign in Kansas, U.S.A. The reported, high-resolution vertical profiles of the temperature, wind speed, wind direction, pressure, and the turbulent dissipation rate, were collected under nocturnal conditions on October 14, 1999, using the CIRES Tethered Lifting System. Two methods for evaluating instantaneous (1-sec) background profiles are applied to the raw data. The background potential temperature is calculated using the “bubble sort” algorithm to produce a monotonically increasing potential temperature with increasing height. Other scalar quantities are smoothed using a running vertical average. The behaviour of background flow, buoyant overturns, turbulent fluctuations, and their respective histograms are presented. Ratios of the considered length scales and the Ozmidov scale are nearly constant with height, a fact that can be applied in practice for estimating instantaneous profiles of the dissipation rate.

Generation of induced pluripotent stem cells (iPSCs) stably expressing CRISPR-based synergistic activation mediator (SAM)

DEFF Research Database (Denmark)

Xiong, Kai; Zhou, Yan; Hyttel, Poul

2016-01-01

Human fibroblasts were engineered to express the CRISPR-based synergistic activation mediator (SAM) complex: dCas9-VP64 and MS2-P65-HSF1. Two induced pluripotent stem cells (iPSCs) clones expressing SAM were established by transducing these fibroblasts with lentivirus expressing OCT4, SOX2, KLF4...... a novel, useful tool to investigate genetic regulation of stem cell proliferation and differentiation through CRISPR-mediated activation of endogenous genes....

Development of an Immunoperoxidase Monolayer Assay for the Detection of Antibodies against Peste des Petits Ruminants Virus Based on BHK-21 Cell Line Stably Expressing the Goat Signaling Lymphocyte Activation Molecule.

Directory of Open Access Journals (Sweden)

Jialin Zhang

Full Text Available From 2013 to 2015, peste des petits ruminants (PPR broke out in more than half of the provinces of China; thus, the application and development of diagnostic methods are very important for the control of PPR. Here, an immunoperoxidase monolayer assay (IPMA was developed to detect antibodies against PPR. However, during IPMA development, we found that Vero cells were not the appropriate choice because staining results were not easily observed. Therefore, we first established a baby hamster kidney-goat signaling lymphocyte activation molecule (BHK-SLAM cell line that could stably express goat SLAM for at least 20 generations. Compared with Vero cells, the PPR-mediated cytopathic effect occurred earlier in BHK-SLAM cells, and large syncytia appeared after virus infection. Based on this cell line and recombinant PPR virus expressing the green fluorescent protein (GFP (rPPRV-GFP, an IPMA for PPR diagnosis was developed. One hundred and ninety-eight PPR serum samples from goats or sheep were tested by the IPMA and virus neutralization test (VNT. Compared with the VNT, the sensitivity and specificity of the IPMA were 91% and 100%, respectively, and the coincidence rate of the two methods was 95.5%. The IPMA assay could be completed in 4 h, compared with more than 6 d for the VNT using rPPRV-GFP, and it is easily performed, as the staining results can be observed under a microscope. Additionally, unlike the VNT, the IPMA does not require antigen purification, which will reduce its cost. In conclusion, the established IPMA will be an alternative method that replaces the VNT for detecting antibodies against PPRV in the field.

High current relativistic beam propagates stably in gas surrounded by nonconducting walls

International Nuclear Information System (INIS)

Clark, J.C.

1977-01-01

LLL has been studying the propagation of high current electron beams for a number of years to understand their behavior for use in a variety of experimental uses. Our latest experiments have shown that a mildly relativistic electron beam of 10 to 15 kA and a pulse width of 30 to 40 ns can propagate stably and with no net current transfer in insulating tubes filled with neutral gases. These experiments have been performed in the Magnetic Fusion Energy program where Electronics Engineering has been operating an electron beam accelerator, designing some of the diagnostics, such as laser interferometers, and performing the experiments. This article briefly describes our experimental observations

Generation of induced pluripotent stem cells (iPSCs) stably expressing CRISPR-based synergistic activation mediator (SAM).

Science.gov (United States)

Xiong, Kai; Zhou, Yan; Hyttel, Poul; Bolund, Lars; Freude, Kristine Karla; Luo, Yonglun

2016-11-01

Human fibroblasts were engineered to express the CRISPR-based synergistic activation mediator (SAM) complex: dCas9-VP64 and MS2-P65-HSF1. Two induced pluripotent stem cells (iPSCs) clones expressing SAM were established by transducing these fibroblasts with lentivirus expressing OCT4, SOX2, KLF4 and C-MYC. We have validated that the reprogramming cassette is silenced in the SAM iPSC clones. Expression of pluripotency genes (OCT4, SOX2, LIN28A, NANOG, GDF3, SSEA4, and TRA-1-60), differentiation potential to all three germ layers, and normal karyotypes are validated. These SAM-iPSCs provide a novel, useful tool to investigate genetic regulation of stem cell proliferation and differentiation through CRISPR-mediated activation of endogenous genes. Copyright © 2016 Michael Boutros, German Cancer Research Center, Heidelberg, Germany. Published by Elsevier B.V. All rights reserved.

Gene expression analysis of interferon-beta treatment in multiple sclerosis

DEFF Research Database (Denmark)

Sellebjerg, F.; Datta, P.; Larsen, J.

2008-01-01

by treatment with IFN-beta. We use DNA microarrays to study gene expression in 10 multiple sclerosis (MS) patients who began de novo treatment with IFN-beta. After the first injection of IFN-beta, the expression of 74 out of 3428 genes changed at least two-fold and statistically significantly (after Bonferroni......Treatment with interferon-beta (IFN-beta) induces the expression of hundreds of genes in blood mononuclear cells, and the expression of several genes has been proposed as a marker of the effect of treatment with IFN-beta. However, to date no molecules have been identified that are stably induced...

A Lithium-Air Battery Stably Working at High Temperature with High Rate Performance.

Science.gov (United States)

Pan, Jian; Li, Houpu; Sun, Hao; Zhang, Ye; Wang, Lie; Liao, Meng; Sun, Xuemei; Peng, Huisheng

2018-02-01

Driven by the increasing requirements for energy supply in both modern life and the automobile industry, the lithium-air battery serves as a promising candidate due to its high energy density. However, organic solvents in electrolytes are likely to rapidly vaporize and form flammable gases under increasing temperatures. In this case, serious safety problems may occur and cause great harm to people. Therefore, a kind of lithium-air that can work stably under high temperature is desirable. Herein, through the use of an ionic liquid and aligned carbon nanotubes, and a fiber shaped design, a new type of lithium-air battery that can effectively work at high temperatures up to 140 °C is developed. Ionic liquids can offer wide electrochemical windows and low vapor pressures, as well as provide high thermal stability for lithium-air batteries. The aligned carbon nanotubes have good electric and heat conductivity. Meanwhile, the fiber format can offer both flexibility and weavability, and realize rapid heat conduction and uniform heat distribution of the battery. In addition, the high temperature has also largely improved the specific powers by increasing the ionic conductivity and catalytic activity of the cathode. Consequently, the lithium-air battery can work stably at 140 °C with a high specific current of 10 A g -1 for 380 cycles, indicating high stability and good rate performance at high temperatures. This work may provide an effective paradigm for the development of high-performance energy storage devices. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular determinants of interactions between the N-terminal domain and the transmembrane core that modulate hERG K+ channel gating.

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Jorge Fernández-Trillo

Full Text Available A conserved eag domain in the cytoplasmic amino terminus of the human ether-a-go-go-related gene (hERG potassium channel is critical for its slow deactivation gating. Introduction of gene fragments encoding the eag domain are able to restore normal deactivation properties of channels from which most of the amino terminus has been deleted, and also those lacking exclusively the eag domain or carrying a single point mutation in the initial residues of the N-terminus. Deactivation slowing in the presence of the recombinant domain is not observed with channels carrying a specific Y542C point mutation in the S4-S5 linker. On the other hand, mutations in some initial positions of the recombinant fragment also impair its ability to restore normal deactivation. Fluorescence resonance energy transfer (FRET analysis of fluorophore-tagged proteins under total internal reflection fluorescence (TIRF conditions revealed a substantial level of FRET between the introduced N-terminal eag fragments and the eag domain-deleted channels expressed at the membrane, but not between the recombinant eag domain and full-length channels with an intact amino terminus. The FRET signals were also minimized when the recombinant eag fragments carried single point mutations in the initial portion of their amino end, and when Y542C mutated channels were used. These data suggest that the restoration of normal deactivation gating by the N-terminal recombinant eag fragment is an intrinsic effect of this domain directed by the interaction of its N-terminal segment with the gating machinery, likely at the level of the S4-S5 linker.

Construction of an expression vector for Lactococcus lactis based on ...

African Journals Online (AJOL)

To construct an expression vector for Lactococcus lactis, the EmPMT fragment which contained the erythromycin resistance gene, P32 promoter, multiple cloning site (MCS) and terminator (T) was subcloned into the small cryptic plasmid pAR141. The resulting vector, designated as pAR1411, was found to be stably ...

Establishment of Sf9 transformants constitutively expressing PBAN receptor variants: application to functional evaluation

Science.gov (United States)

To facilitate further evaluation of pheromone biosynthesis activating neuropeptide receptor (PBANR) functionality and regulation, we generated cultured insect cell lines stably expressing a number of fluorescent Bombyx mori PBANR (BommoPBANR) and Pseudaletia separata PBANR (PsesePBANR) variants incl...

A vaccine grade of yeast Saccharomyces cerevisiae expressing mammalian myostatin

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Zhang Tingting

2012-12-01

Full Text Available Abstract Background Yeast Saccharomyces cerevisiae is a widely-used system for protein expression. We previously showed that heat-killed whole recombinant yeast vaccine expressing mammalian myostatin can modulate myostatin function in mice, resulting in increase of body weight and muscle composition in these animals. Foreign DNA introduced into yeast cells can be lost soon unless cells are continuously cultured in selection media, which usually contain antibiotics. For cost and safety concerns, it is essential to optimize conditions to produce quality food and pharmaceutical products. Results We developed a simple but effective method to engineer a yeast strain stably expressing mammalian myostatin. This method utilized high-copy-number integration of myostatin gene into the ribosomal DNA of Saccharomyces cerevisiae. In the final step, antibiotic selection marker was removed using the Cre-LoxP system to minimize any possible side-effects for animals. The resulting yeast strain can be maintained in rich culture media and stably express mammalian myostatin for two years. Oral administration of the recombinant yeast was able to induce immune response to myostatin and modulated the body weight of mice. Conclusions Establishment of such yeast strain is a step further toward transformation of yeast cells into edible vaccine to improve meat production in farm animals and treat human muscle-wasting diseases in the future.

The impact of intragenic CpG content on gene expression.

Science.gov (United States)

Bauer, Asli Petra; Leikam, Doris; Krinner, Simone; Notka, Frank; Ludwig, Christine; Längst, Gernot; Wagner, Ralf

2010-07-01

The development of vaccine components or recombinant therapeutics critically depends on sustained expression of the corresponding transgene. This study aimed to determine the contribution of intragenic CpG content to expression efficiency in transiently and stably transfected mammalian cells. Based upon a humanized version of green fluorescent protein (GFP) containing 60 CpGs within its coding sequence, a CpG-depleted variant of the GFP reporter was established by carefully modulating the codon usage. Interestingly, GFP reporter activity and detectable protein amounts in stably transfected CHO and 293 cells were significantly decreased upon CpG depletion and independent from promoter usage (CMV, EF1 alpha). The reduction in protein expression associated with CpG depletion was likewise observed for other unrelated reporter genes and was clearly reflected by a decline in mRNA copy numbers rather than translational efficiency. Moreover, decreased mRNA levels were neither due to nuclear export restrictions nor alternative splicing or mRNA instability. Rather, the intragenic CpG content influenced de novo transcriptional activity thus implying a common transcription-based mechanism of gene regulation via CpGs. Increased high CpG transcription correlated with changed nucleosomal positions in vitro albeit histone density at the two genes did not change in vivo as monitored by ChIP.

Characterization of a human cell line stably over-expressing the candidate oncogene, dual specificity phosphatase 12.

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Erica L Cain

2011-04-01

Full Text Available Analysis of chromosomal rearrangements within primary tumors has been influential in the identification of novel oncogenes. Identification of the "driver" gene(s within cancer-derived amplicons is, however, hampered by the fact that most amplicons contain many gene products. Amplification of 1q21-1q23 is strongly associated with liposarcomas and microarray-based comparative genomic hybridization narrowed down the likely candidate oncogenes to two: the activating transcription factor 6 (atf6 and the dual specificity phosphatase 12 (dusp12. While atf6 is an established transcriptional regulator of the unfolded protein response, the potential role of dusp12 in cancer remains uncharacterized.To evaluate the oncogenic potential of dusp12, we established stable cell lines that ectopically over-express dusp12 in isolation and determined whether this cell line acquired properties frequently associated with transformed cells. Here, we demonstrate that cells over-expressing dusp12 display increased cell motility and resistance to apoptosis. Additionally, over-expression of dusp12 promoted increased expression of the c-met proto-oncogene and the collagen and laminin receptor intergrin alpha 1 (itga1 which is implicated in metastasis.Collectively, these results suggest that dusp12 is oncologically relevant and exposes a potential association between dusp12 and established oncogenes that could be therapeutically targeted.

Whole-Transcriptome Selection and Evaluation of Internal Reference Genes for Expression Analysis in Protocorm Development of Dendrobium officinale Kimura et Migo.

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Hongqiang An

Full Text Available Dendrobium officinale Kimu et Migo has increased many researchers' interest for its high medical and horticultural values and the molecular mechanism of its protocorm development remains unclear. In this study, 19 genes from 26 most stably expressed genes in whole transcriptome of protocorms and 5 housekeeping genes were used as candidate reference genes and screened with 4 application softwares (geNorm, NormFinder, BestKeeper and RefFinder. The results showed that a few reference genes could effectively normalize expression level of specific genes in protocorm development and the optimal top 2 reference genes were ASS and APH1L. Meanwhile, validation of GNOM, AP2 and temperature induced gene (TIL for normalization demonstrates the usefulness of the validated candidate reference genes. The expression profiles of these genes varied under protocorms and temperature stress according to the stablest and unstablest reference genes, which proved the importance of the choice of appropriate reference genes. The first systematic evaluation of stably expressed genes will be very useful in the future analysis of specific genes expression in D. officinale.

HTLV-1 Tax mediated downregulation of miRNAs associated with chromatin remodeling factors in T cells with stably integrated viral promoter.

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Saifur Rahman

Full Text Available RNA interference (RNAi is a natural cellular mechanism to silence gene expression and is predominantly mediated by microRNAs (miRNAs that target messenger RNA. Viruses can manipulate the cellular processes necessary for their replication by targeting the host RNAi machinery. This study explores the effect of human T-cell leukemia virus type 1 (HTLV-1 transactivating protein Tax on the RNAi pathway in the context of a chromosomally integrated viral long terminal repeat (LTR using a CD4(+ T-cell line, Jurkat. Transcription factor profiling of the HTLV-1 LTR stably integrated T-cell clone transfected with Tax demonstrates increased activation of substrates and factors associated with chromatin remodeling complexes. Using a miRNA microarray and bioinformatics experimental approach, Tax was also shown to downregulate the expression of miRNAs associated with the translational regulation of factors required for chromatin remodeling. These observations were validated with selected miRNAs and an HTLV-1 infected T cells line, MT-2. miR-149 and miR-873 were found to be capable of directly targeting p300 and p/CAF, chromatin remodeling factors known to play critical role in HTLV-1 pathogenesis. Overall, these results are first in line establishing HTLV-1/Tax-miRNA-chromatin concept and open new avenues toward understanding retroviral latency and/or replication in a given cell type.

MicroRNA expression profiling to identify and validate reference genes for relative quantification in colorectal cancer.

LENUS (Irish Health Repository)

Chang, Kah Hoong

2010-01-01

BACKGROUND: Advances in high-throughput technologies and bioinformatics have transformed gene expression profiling methodologies. The results of microarray experiments are often validated using reverse transcription quantitative PCR (RT-qPCR), which is the most sensitive and reproducible method to quantify gene expression. Appropriate normalisation of RT-qPCR data using stably expressed reference genes is critical to ensure accurate and reliable results. Mi(cro)RNA expression profiles have been shown to be more accurate in disease classification than mRNA expression profiles. However, few reports detailed a robust identification and validation strategy for suitable reference genes for normalisation in miRNA RT-qPCR studies. METHODS: We adopt and report a systematic approach to identify the most stable reference genes for miRNA expression studies by RT-qPCR in colorectal cancer (CRC). High-throughput miRNA profiling was performed on ten pairs of CRC and normal tissues. By using the mean expression value of all expressed miRNAs, we identified the most stable candidate reference genes for subsequent validation. As such the stability of a panel of miRNAs was examined on 35 tumour and 39 normal tissues. The effects of normalisers on the relative quantity of established oncogenic (miR-21 and miR-31) and tumour suppressor (miR-143 and miR-145) target miRNAs were assessed. RESULTS: In the array experiment, miR-26a, miR-345, miR-425 and miR-454 were identified as having expression profiles closest to the global mean. From a panel of six miRNAs (let-7a, miR-16, miR-26a, miR-345, miR-425 and miR-454) and two small nucleolar RNA genes (RNU48 and Z30), miR-16 and miR-345 were identified as the most stably expressed reference genes. The combined use of miR-16 and miR-345 to normalise expression data enabled detection of a significant dysregulation of all four target miRNAs between tumour and normal colorectal tissue. CONCLUSIONS: Our study demonstrates that the top six most

MicroRNA expression profiling to identify and validate reference genes for relative quantification in colorectal cancer

LENUS (Irish Health Repository)

Chang, Kah Hoong

2010-04-29

Abstract Background Advances in high-throughput technologies and bioinformatics have transformed gene expression profiling methodologies. The results of microarray experiments are often validated using reverse transcription quantitative PCR (RT-qPCR), which is the most sensitive and reproducible method to quantify gene expression. Appropriate normalisation of RT-qPCR data using stably expressed reference genes is critical to ensure accurate and reliable results. Mi(cro)RNA expression profiles have been shown to be more accurate in disease classification than mRNA expression profiles. However, few reports detailed a robust identification and validation strategy for suitable reference genes for normalisation in miRNA RT-qPCR studies. Methods We adopt and report a systematic approach to identify the most stable reference genes for miRNA expression studies by RT-qPCR in colorectal cancer (CRC). High-throughput miRNA profiling was performed on ten pairs of CRC and normal tissues. By using the mean expression value of all expressed miRNAs, we identified the most stable candidate reference genes for subsequent validation. As such the stability of a panel of miRNAs was examined on 35 tumour and 39 normal tissues. The effects of normalisers on the relative quantity of established oncogenic (miR-21 and miR-31) and tumour suppressor (miR-143 and miR-145) target miRNAs were assessed. Results In the array experiment, miR-26a, miR-345, miR-425 and miR-454 were identified as having expression profiles closest to the global mean. From a panel of six miRNAs (let-7a, miR-16, miR-26a, miR-345, miR-425 and miR-454) and two small nucleolar RNA genes (RNU48 and Z30), miR-16 and miR-345 were identified as the most stably expressed reference genes. The combined use of miR-16 and miR-345 to normalise expression data enabled detection of a significant dysregulation of all four target miRNAs between tumour and normal colorectal tissue. Conclusions Our study demonstrates that the top six most

Evaluation of reference genes for gene expression analysis using quantitative RT-PCR in Azospirillum brasilense.

Science.gov (United States)

McMillan, Mary; Pereg, Lily

2014-01-01

Azospirillum brasilense is a nitrogen fixing bacterium that has been shown to have various beneficial effects on plant growth and yield. Under normal conditions A. brasilense exists in a motile flagellated form, which, under starvation or stress conditions, can undergo differentiation into an encapsulated, cyst-like form. Quantitative RT-PCR can be used to analyse changes in gene expression during this differentiation process. The accuracy of quantification of mRNA levels by qRT-PCR relies on the normalisation of data against stably expressed reference genes. No suitable set of reference genes has yet been described for A. brasilense. Here we evaluated the expression of ten candidate reference genes (16S rRNA, gapB, glyA, gyrA, proC, pykA, recA, recF, rpoD, and tpiA) in wild-type and mutant A. brasilense strains under different culture conditions, including conditions that induce differentiation. Analysis with the software programs BestKeeper, NormFinder and GeNorm indicated that gyrA, glyA and recA are the most stably expressed reference genes in A. brasilense. The results also suggested that the use of two reference genes (gyrA and glyA) is sufficient for effective normalisation of qRT-PCR data.

Evaluation of reference genes for gene expression analysis using quantitative RT-PCR in Azospirillum brasilense.

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Mary McMillan

Full Text Available Azospirillum brasilense is a nitrogen fixing bacterium that has been shown to have various beneficial effects on plant growth and yield. Under normal conditions A. brasilense exists in a motile flagellated form, which, under starvation or stress conditions, can undergo differentiation into an encapsulated, cyst-like form. Quantitative RT-PCR can be used to analyse changes in gene expression during this differentiation process. The accuracy of quantification of mRNA levels by qRT-PCR relies on the normalisation of data against stably expressed reference genes. No suitable set of reference genes has yet been described for A. brasilense. Here we evaluated the expression of ten candidate reference genes (16S rRNA, gapB, glyA, gyrA, proC, pykA, recA, recF, rpoD, and tpiA in wild-type and mutant A. brasilense strains under different culture conditions, including conditions that induce differentiation. Analysis with the software programs BestKeeper, NormFinder and GeNorm indicated that gyrA, glyA and recA are the most stably expressed reference genes in A. brasilense. The results also suggested that the use of two reference genes (gyrA and glyA is sufficient for effective normalisation of qRT-PCR data.

Genome-Wide Constitutively Expressed Gene Analysis and New Reference Gene Selection Based on Transcriptome Data: A Case Study from Poplar/Canker Disease Interaction

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Jiaping Zhao

2017-10-01

Full Text Available A number of transcriptome datasets for differential expression (DE genes have been widely used for understanding organismal biology, but these datasets also contain untapped information that can be used to develop more precise analytical tools. With the use of transcriptome data generated from poplar/canker disease interaction system, we describe a methodology to identify candidate reference genes from high-throughput sequencing data. This methodology will improve the accuracy of RT-qPCR and will lead to better standards for the normalization of expression data. Expression stability analysis from xylem and phloem of Populus bejingensis inoculated with the fungal canker pathogen Botryosphaeria dothidea revealed that 729 poplar transcripts (1.11% were stably expressed, at a threshold level of coefficient of variance (CV of FPKM < 20% and maximum fold change (MFC of FPKM < 2.0. Expression stability and bioinformatics analysis suggested that commonly used house-keeping (HK genes were not the most appropriate internal controls: 70 of the 72 commonly used HK genes were not stably expressed, 45 of the 72 produced multiple isoform transcripts, and some of their reported primers produced unspecific amplicons in PCR amplification. RT-qPCR analysis to compare and evaluate the expression stability of 10 commonly used poplar HK genes and 20 of the 729 newly-identified stably expressed transcripts showed that some of the newly-identified genes (such as SSU_S8e, LSU_L5e, and 20S_PSU had higher stability ranking than most of commonly used HK genes. Based on these results, we recommend a pipeline for deriving reference genes from transcriptome data. An appropriate candidate gene should have a unique transcript, constitutive expression, CV value of expression < 20% (or possibly 30% and MFC value of expression <2, and an expression level of 50–1,000 units. Lastly, when four of the newly identified HK genes were used in the normalization of expression data for 20

Development and characterization of enhanced green fluorescent protein and luciferase expressing cell line for non-destructive evaluation of tissue engineering constructs.

NARCIS (Netherlands)

Blum, J.S.; Temenoff, J.S.; Park, H.; Jansen, J.A.; Mikos, A.G.; Barry, M.A.

2004-01-01

This study investigates the utility of genetically modified cells developed for the qualitative and quantitative non-destructive evaluation of cells on biomaterials. The Fisher rat fibroblastic cell line has been genetically modified to stably express the reporter genes enhanced green fluorescence

New Trends in Cancer Therapy: Targeting Ion Channels and Transporters

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Annarosa Arcangeli

2010-04-01

Full Text Available The expression and activity of different channel types mark and regulate specific stages of cancer establishment and progression. Blocking channel activity impairs the growth of some tumors, both in vitro and in vivo, which opens a new field for pharmaceutical research. However, ion channel blockers may produce serious side effects, such as cardiac arrhythmias. For instance, Kv11.1 (hERG1 channels are aberrantly expressed in several human cancers, in which they control different aspects of the neoplastic cell behaviour. hERG1 blockers tend to inhibit cancer growth. However they also retard the cardiac repolarization, thus lengthening the electrocardiographic QT interval, which can lead to life-threatening ventricular arrhythmias. Several possibilities exist to produce less harmful compounds, such as developing specific drugs that bind hERG1 channels in the open state or disassemble the ion channel/integrin complex which appears to be crucial in certain stages of neoplastic progression. The potential approaches to improve the efficacy and safety of ion channel targeting in oncology include: (1 targeting specific conformational channel states; (2 finding ever more specific inhibitors, including peptide toxins, for channel subtypes mainly expressed in well-identified tumors; (3 using specific ligands to convey traceable or cytotoxic compounds; (4 developing channel blocking antibodies; (5 designing new molecular tools to decrease channel expression in selected cancer types. Similar concepts apply to ion transporters such as the Na+/K+ pump and the Na+/H+ exchanger. Pharmacological targeting of these transporters is also currently being considered in anti-neoplastic therapy.

Effects of recombinant plasmid pEgr-p53 transfected stably in combination with X-irradiation on cell cycle progression and proliferation in human SKOV-3 tumor cells in vitro

International Nuclear Information System (INIS)

Dong Lihua; Liu Feng; Li Yanbo; Fu Shibo; Gong Shouliang

2008-01-01

Objective: To investigate the effect of recombinant plasmid pEgr-hp53 transfected stably in combination with X-ray irradiation on the cell cycle progression and the proliferation in human SKOV-3 tumor cells. Methods: pEgr-hp53 and pcDNA3.1 packaged with liposome were stably transfected into SKOV-3 cells in vitro. SKOV-3-hp53 and SKOV-3-vect were irradiated with 0, 0.5, 2.0 and 5.0 Gy X-rays, respectively, i.e. 8 experimental groups. The SKOV-3 cell proliferation and the cell cycle progression were measured with flow cytometry and cell growth curve, respectively. Results: Compared with 0 Gy group, the cell counts in SKOV-3- hp53 plus different doses of irradiation groups 2 d after irradiation decreased significantly (P 0 /G 1 cells increased significantly (P 2 /M cells decreased in varying degrees. The cell counts in SKOV-3-hp53 plus irradiation group were significantly lower than those in corresponding SKOV-3-vect plus irradiation group, the cell counts 4-8 d after irradiation with 0.5 Gy, 2 d after 2.0 Gy irradiation and 6 d after 5.0 Gy irradiation decreased significantly (P 0 /G 1 cells increased significantly (P 2 /M cells decreased significantly (P 1 arrest in SKOV-3 cells and inhibits the cell proliferation. Ionizing radiation can activate early growth response-1 (Egr-1) gene promoter and increase the expression of p53 gene, and enhance the inhibition of tumor cell growth. (authors)

Differential Expression of Ccn4 and Other Genes Between Metastatic and Non-metastatic EL4 Mouse Lymphoma Cells

OpenAIRE

S. CHAHAL, MANPREET; TERESA KU, H.; ZHANG, ZHIHONG; M. LEGASPI, CHRISTIAN; LUO, ANGELA; M. HOPKINS, MANDI; E. MEIER, KATHRYN

2016-01-01

Background: Previous work characterized variants of the EL4 murine lymphoma cell line. Some are non-metastatic, and others metastatic, in syngenic mice. In addition, metastatic EL4 cells were stably transfected with phospholipase D2 (PLD2), which further enhanced metastasis. Materials and Methods: Microarray analyses of mRNA expression was performed for non-metastatic, metastatic, and PLD2-expressing metastatic EL4 cells. Results: Many differences were observed between non-metastatic and meta...

Generation of chickens expressing Cre recombinase.

Science.gov (United States)

Leighton, Philip A; Pedersen, Darlene; Ching, Kathryn; Collarini, Ellen J; Izquierdo, Shelley; Jacob, Roy; van de Lavoir, Marie-Cecile

2016-10-01

Cre recombinase has been extensively used for genome engineering in transgenic mice yet its use in other species has been more limited. Here we describe the generation of transgenic chickens expressing Cre recombinase. Green fluorescent protein (GFP)-positive chicken primordial germ cells were stably transfected with β-actin-Cre-recombinase using phiC31 integrase and transgenic chickens were generated. Cre recombinase activity was verified by mating Cre birds to birds carrying a floxed transgene. Floxed sequences were only excised in offspring from roosters that inherited the Cre recombinase but were excised in all offspring from hens carrying the Cre recombinase irrespective of the presence of the Cre transgene. The Cre recombinase transgenic birds were healthy and reproductively normal. The Cre and GFP genes in two of the lines were closely linked whereas the genes segregated independently in a third line. These founders allowed development of GFP-expressing and non-GFP-expressing Cre recombinase lines. These lines of birds create a myriad of opportunities to study developmentally-regulated and tissue-specific expression of transgenes in chickens.

Gene expression results in lipopolysaccharide-stimulated monocytes depend significantly on the choice of reference genes

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Øvstebø Reidun

2010-05-01

Full Text Available Abstract Background Gene expression in lipopolysaccharide (LPS-stimulated monocytes is mainly studied by quantitative real-time reverse transcription PCR (RT-qPCR using GAPDH (glyceraldehyde 3-phosphate dehydrogenase or ACTB (beta-actin as reference gene for normalization. Expression of traditional reference genes has been shown to vary substantially under certain conditions leading to invalid results. To investigate whether traditional reference genes are stably expressed in LPS-stimulated monocytes or if RT-qPCR results are dependent on the choice of reference genes, we have assessed and evaluated gene expression stability of twelve candidate reference genes in this model system. Results Twelve candidate reference genes were quantified by RT-qPCR in LPS-stimulated, human monocytes and evaluated using the programs geNorm, Normfinder and BestKeeper. geNorm ranked PPIB (cyclophilin B, B2M (beta-2-microglobulin and PPIA (cyclophilin A as the best combination for gene expression normalization in LPS-stimulated monocytes. Normfinder suggested TBP (TATA-box binding protein and B2M as the best combination. Compared to these combinations, normalization using GAPDH alone resulted in significantly higher changes of TNF-α (tumor necrosis factor-alpha and IL10 (interleukin 10 expression. Moreover, a significant difference in TNF-α expression between monocytes stimulated with equimolar concentrations of LPS from N. meningitides and E. coli, respectively, was identified when using the suggested combinations of reference genes for normalization, but stayed unrecognized when employing a single reference gene, ACTB or GAPDH. Conclusions Gene expression levels in LPS-stimulated monocytes based on RT-qPCR results differ significantly when normalized to a single gene or a combination of stably expressed reference genes. Proper evaluation of reference gene stabiliy is therefore mandatory before reporting RT-qPCR results in LPS-stimulated monocytes.

Tropoelastin regulates chemokine expression in fibroblasts in Costello syndrome

International Nuclear Information System (INIS)

Tatano, Yutaka; Fujinawa, Reiko; Kozutsumi, Yasunori; Takahashi, Tsutomu; Tsuji, Daisuke; Takeuchi, Naohiro; Tsuta, Kohji; Takada, Goro; Sakuraba, Hitoshi; Itoh, Kohji

2008-01-01

Costello syndrome is a multiple congenital anomaly associated with growth and mental retardation, cardiac and skeletal anomalies, and a predisposition to develop neoplasia. Comprehensive expression analysis revealed remarkable up-regulation of several cytokines and chemokines including Gro family proteins, interleukin-1β (IL-1β), IL-8 and MCP-1 but down-regulation of extracellular matrix components including collagens and proteoglycans of skin fibroblasts derived from a Japanese Costello syndrome patient characterized by significantly reduced tropoelastin mRNA, impaired elastogenesis and enhanced cell proliferation. In contrast, decreases in these chemokines and IL-1β expression were observed in Costello fibroblastic cell lines stably expressing the bovine tropoelastin (btEln) gene and in restored elastic fibers. These results strongly suggest that the human TE gene (ELN) transfer could be applicable for the gene therapy of a group of Costello syndrome patients with reduced ELN gene expression

[Biological characteristics of a chimeric rabies virus expressing canine parvovirus VP2 protein].

Science.gov (United States)

Niu, Xue-Feng; Liu, Xiao-Hui; Sun, Zhao-Jin; Shi, He-He; Chen, Jing; Jiang, Bido; Sun, Jing-Chen; Guo, Xiao-Feng

2009-09-01

To obtain a bivalence vaccine against canine rabies virus and canine parvovirus, a chimeric rabies virus expressing canine parvovirus VP2 protein was generated by the technique of reverse genetics. It was shown that the chimeric virus designated as HEP-Flury (VP2) grew well on BHK-21 cells and the VP2 gene could still be stably expressed after ten passages on BHK-21 cells. Experiments on the mice immunized with the chimeric virus HEP-Flury (VP2) demonstrated that specific antibodies against rabies virus and canine parvovirus were induced in immunized mice after vaccination with the live chimeric virus.

21-Methylpyrenyl-cholesterol stably and specifically associates with lipoprotein peripheral hemi-membrane: A new labelling tool

Energy Technology Data Exchange (ETDEWEB)

Gaibelet, Gérald [INSERM U563, CHU Purpan, Toulouse (France); CEA, SB2SM and UMR8221 CNRS, IBiTec-Saclay, Gif-sur-Yvette (France); Tercé, François [Université Toulouse III, UMR 1048, Toulouse (France); INSERM U1048, Toulouse (France); Bertrand-Michel, Justine [Université Toulouse III, UMR 1048, Toulouse (France); INSERM U1048, Lipidomic Platform Metatoul, Toulouse (France); Allart, Sophie [Plateau Technique d’Imagerie Cellulaire, INSERM U1043, Toulouse (France); Azalbert, Vincent [Université Toulouse III, UMR 1048, Toulouse (France); INSERM U1048, Toulouse (France); Lecompte, Marie-France [INSERM U563, Faculté de Médecine de Rangueil, Toulouse (France); Collet, Xavier [Université Toulouse III, UMR 1048, Toulouse (France); INSERM U1048, Toulouse (France); Orlowski, Stéphane, E-mail: stephane.orlowski@cea.fr [INSERM U563, CHU Purpan, Toulouse (France); CEA, SB2SM and UMR8221 CNRS, IBiTec-Saclay, Gif-sur-Yvette (France)

2013-11-01

Highlights: •21-Methylpyrenyl-cholesterol specifically and stably associates to lipoproteins. •It is not esterified by LCAT, and thus reliably labels their peripheral hemi-membrane. •HDL vs. LDL are well distinguishable by various fluorescent labelling characteristics. •LDL peripheral hemi-membrane harbors cholesterol-rich ordered lipid (micro)domains. •Cultured cells can be stained by such labelled lipoproteins-mediated delivery. -- Abstract: Lipoproteins are important biological components. However, they have few convenient fluorescent labelling probes currently reported, and their physiological reliability can be questioned. We compared the association of two fluorescent cholesterol derivatives, 22-nitrobenzoxadiazole-cholesterol (NBD-Chol) and 21-methylpyrenyl-cholesterol (Pyr-met-Chol), to serum lipoproteins and to purified HDL and LDL. Both lipoproteins could be stably labelled by Pyr-met-Chol, but virtually not by NBD-Chol. At variance with NBD-Chol, LCAT did not esterify Pyr-met-Chol. The labelling characteristics of lipoproteins by Pyr-met-Chol were well distinguishable between HDL and LDL, regarding dializability, associated probe amount and labelling kinetics. We took benefit of the pyrene labelling to approach the structural organization of LDL peripheral hemi-membrane, since Pyr-met-Chol-labelled LDL, but not HDL, presented a fluorescence emission of pyrene excimers, indicating that the probe was present in an ordered lipid micro-environment. Since the peripheral membrane of LDL contains more sphingomyelin (SM) than HDL, this excimer formation was consistent with the existence of cholesterol- and SM-enriched lipid microdomains in LDL, as already suggested in model membranes of similar composition and reminiscent to the well-described “lipid rafts” in bilayer membranes. Finally, we showed that Pyr-met-Chol could stain cultured PC-3 cells via lipoprotein-mediated delivery, with a staining pattern well different to that observed with NBD

21-Methylpyrenyl-cholesterol stably and specifically associates with lipoprotein peripheral hemi-membrane: A new labelling tool

International Nuclear Information System (INIS)

Gaibelet, Gérald; Tercé, François; Bertrand-Michel, Justine; Allart, Sophie; Azalbert, Vincent; Lecompte, Marie-France; Collet, Xavier; Orlowski, Stéphane

2013-01-01

Highlights: •21-Methylpyrenyl-cholesterol specifically and stably associates to lipoproteins. •It is not esterified by LCAT, and thus reliably labels their peripheral hemi-membrane. •HDL vs. LDL are well distinguishable by various fluorescent labelling characteristics. •LDL peripheral hemi-membrane harbors cholesterol-rich ordered lipid (micro)domains. •Cultured cells can be stained by such labelled lipoproteins-mediated delivery. -- Abstract: Lipoproteins are important biological components. However, they have few convenient fluorescent labelling probes currently reported, and their physiological reliability can be questioned. We compared the association of two fluorescent cholesterol derivatives, 22-nitrobenzoxadiazole-cholesterol (NBD-Chol) and 21-methylpyrenyl-cholesterol (Pyr-met-Chol), to serum lipoproteins and to purified HDL and LDL. Both lipoproteins could be stably labelled by Pyr-met-Chol, but virtually not by NBD-Chol. At variance with NBD-Chol, LCAT did not esterify Pyr-met-Chol. The labelling characteristics of lipoproteins by Pyr-met-Chol were well distinguishable between HDL and LDL, regarding dializability, associated probe amount and labelling kinetics. We took benefit of the pyrene labelling to approach the structural organization of LDL peripheral hemi-membrane, since Pyr-met-Chol-labelled LDL, but not HDL, presented a fluorescence emission of pyrene excimers, indicating that the probe was present in an ordered lipid micro-environment. Since the peripheral membrane of LDL contains more sphingomyelin (SM) than HDL, this excimer formation was consistent with the existence of cholesterol- and SM-enriched lipid microdomains in LDL, as already suggested in model membranes of similar composition and reminiscent to the well-described “lipid rafts” in bilayer membranes. Finally, we showed that Pyr-met-Chol could stain cultured PC-3 cells via lipoprotein-mediated delivery, with a staining pattern well different to that observed with NBD

Cholecystokinin-2 receptor mediated gene expression in neuronal PC12 cells

DEFF Research Database (Denmark)

Hansen, Thomas v O; Borup, Rehannah; Marstrand, Troels

2007-01-01

could be identified. Comparison with forskolin- and nerve growth factor (NGF)-treated PC12 cells showed that CCK induced a separate set of target genes. Taken together, we propose that neuronal CCK may have a role in the regulation of the circadian rhythm, the metabolism of cerebral cholesterol...... of neuronal CCK are incompletely understood. To identify genes regulated by neuronal CCK, we generated neuronal PC12 cells stably expressing the CCK-2 receptor (CCK-2R) and treated the cells with sulphated CCK-8 for 2-16 h, before the global expression profile was examined. The changes in gene expression...... peaked after 2 h, with 67 differentially expressed transcripts identified. A pathway analysis indicated that CCK was implicated in the regulation of the circadian clock system, the plasminogen system and cholesterol metabolism. But transcripts encoding proteins involved in dopamine signaling, ornithine...

Reference genes for gene expression analysis by real-time reverse transcription polymerase chain reaction of renal cell carcinoma.

Science.gov (United States)

Bjerregaard, Henriette; Pedersen, Shona; Kristensen, Søren Risom; Marcussen, Niels

2011-12-01

Differentiation between malignant renal cell carcinoma and benign oncocytoma is of great importance to choose the optimal treatment. Accurate preoperative diagnosis of renal tumor is therefore crucial; however, existing imaging techniques and histologic examinations are incapable of providing an optimal differentiation profile. Analysis of gene expression of molecular markers is a new possibility but relies on appropriate standardization to compare different samples. The aim of this study was to identify stably expressed reference genes suitable for the normalization of results extracted from gene expression analysis of renal tumors. Expression levels of 8 potential reference genes (ATP5J, HMBS, HPRT1, PPIA, TBP, 18S, GAPDH, and POLR2A) were examined by real-time reverse transcription polymerase chain reaction in tumor and normal tissue from removed kidneys from 13 patients with renal cell carcinoma and 5 patients with oncocytoma. The expression levels of genes were compared by gene stability value M, average gene stability M, pairwise variation V, and coefficient of variation CV. More candidates were not suitable for the purpose, but a combination of HMBS, PPIA, ATP5J, and TBP was found to be the best combination with an average gene stability value M of 0.9 and a CV of 0.4 in the 18 tumors and normal tissues. A combination of 4 genes, HMBS, PPIA, ATP5J, and TBP, is a possible reference in renal tumor gene expression analysis by reverse transcription polymerase chain reaction. A combination of four genes, HMBS, PPIA, ATP5J and TBP, being stably expressed in tissues from RCC is possible reference genes for gene expression analysis.

Efficient expression of SRK intracellular domain by a modeling-based protein engineering.

Science.gov (United States)

Murase, Kohji; Hirano, Yoshinori; Takayama, Seiji; Hakoshima, Toshio

2017-03-01

S-locus protein kinase (SRK) is a receptor kinase that plays a critical role in self-recognition in the Brassicaceae self-incompatibility (SI) response. SRK is activated by binding of its ligand S-locus protein 11 (SP11) and subsequently induced phosphorylation of the intracellular kinase domain. However, a detailed activation mechanism of SRK is still largely unknown because of the difficulty in stably expressing SRK recombinant proteins. Here, we performed modeling-based protein engineering of the SRK kinase domain for stable expression in Escherichia coli. The engineered SRK intracellular domain was expressed about 54-fold higher production than wild type SRK, without loss of the kinase activity, suggesting it could be useful for further biochemical and structural studies. Copyright © 2016 Elsevier Inc. All rights reserved.

Evaluation of Candidate Reference Genes for Quantitative Gene Expression Analysis in Spodoptera exigu a after Long-time Exposure to Cadmium

OpenAIRE

P?achetka-Bo?ek, Anna; Augustyniak, Maria

2017-01-01

Studies on the transcriptional control of gene expression play an important role in many areas of biology. Reference genes, which are often referred to as housekeeping genes, such as GAPDH, G3PDH, EF2, RpL7A, RpL10, TUB? and Actin, have traditionally been assumed to be stably expressed in all conditions, and they are frequently used to normalize mRNA levels between different samples in qPCR analysis. However, it is known that the expression of these genes is influenced by numerous factors, su...

Newly constructed stable reporter cell lines for mechanistic studies on electrophile-responsive element-mediated gene expression reveal a role for flavonoid planarity.

NARCIS (Netherlands)

Boerboom, A.M.A.; Vermeulen, M.; Woude, H. van der; Bremer, B.I.; Lee-Hilz, Y.Y.; Kampman, E.; Bladeren, P.J. van; Rietjens, I.M.C.M.; Aarts, J.

2006-01-01

The electrophile-responsive element (EpRE) is a transcriptional enhancer involved in cancer-chemoprotective gene expression modulation by certain food components. Two stably transfected luciferase reporter cell lines were developed, EpRE(hNQO1)-LUX and EpRE(mGST-Ya)-LUX, based on EpRE sequences from

Newly constructed stable reporter cell lines for mechanistic studies on electrophile-responsive element-mediated gene expression reveal a role for flavonoid planarity

NARCIS (Netherlands)

Boerboom, A.M.J.F.; Vermeulen, M.; Woude, H. van der; Bremer, B.I.; Lee-Hilz, Y.Y.; Kampman, E.; Bladeren, P.J. van; Rietjens, I.M.C.M.; Aarts, J.M.M.J.G.

2006-01-01

The electrophile-responsive element (EpRE) is a transcriptional enhancer involved in cancer-chemoprotective gene expression modulation by certain food components. Two stably transfected luciferase reporter cell lines were developed, EpRE(hNQO1)-LUX and EpRE(mGST-Ya)-LUX, based on EpRE sequences from

[Selection and construction of cell line stably expressing survivin gene in lower level through eukaryotic plasmid vector of shRNA].

Science.gov (United States)

Wang, Wen-Xia; Sun, Shan-Zhen; Song, Ying

2008-06-01

To construct a short hairpin RNA(shRNA) interference expression plasmid vector of survivin gene, transfect tongue squamous cell carcinoma line Tca8113 which expressed survivin gene in a high level, and choose the cells whose survivin gene were suppressed significantly. Two pairs of oligonucleotide sequences specific for survivin gene were designed and synthesized, and cloned into pSilencer-2.1U6-neo plasmid. The recombinant plasmids (named PS1 and PS2) were amplified in Ecoli. DH5alpha was identified by restriction digestion, PCR and sequencing. The vectors were transfected into Tca8113 cells with lipofectamine 2000. After selection with G418, the stable cell clones were attained. Survivn expression was assayed with real-time quantitative PCR and Western blotting. SAS8.0 software package was used for Student t test. Two vectors were constructed successfully and stable cell clones with PS1 or PS2 plasmid were obtained. As compared with those of control, survivin expression of transfected cell with PS1 or PS2 in mRNA level was significantly suppressed (P<0.05). In protein level, only those of transfected cell with PS2 was significantly suppressed (P<0.01). The shRNA interference expression plasmid vectors of survivin gene are successfully constructed, and Tca8113 cells which express survivin gene in a stable lower level are attained, which enable us to carry out further research on gene therapy of oral squamous cell carcinoma. Supported by National Natural Science Foundation of China (Grant No.30572056).

Identification of a novel herpes simplex virus type 1 transcript and protein (AL3) expressed during latency.

Science.gov (United States)

Jaber, Tareq; Henderson, Gail; Li, Sumin; Perng, Guey-Chuen; Carpenter, Dale; Wechsler, Steven L; Jones, Clinton

2009-10-01

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) is abundantly expressed in latently infected sensory neurons. In small animal models of infection, expression of the first 1.5 kb of LAT coding sequences is necessary and sufficient for wild-type reactivation from latency. The ability of LAT to inhibit apoptosis is important for reactivation from latency. Within the first 1.5 kb of LAT coding sequences and LAT promoter sequences, additional transcripts have been identified. For example, the anti-sense to LAT transcript (AL) is expressed in the opposite direction to LAT from the 5' end of LAT and LAT promoter sequences. In addition, the upstream of LAT (UOL) transcript is expressed in the LAT direction from sequences in the LAT promoter. Further examination of the first 1.5 kb of LAT coding sequences revealed two small ORFs that are anti-sense with respect to LAT (AL2 and AL3). A transcript spanning AL3 was detected in productively infected cells, mouse neuroblastoma cells stably expressing LAT and trigeminal ganglia (TG) of latently infected mice. Peptide-specific IgG directed against AL3 specifically recognized a protein migrating near 15 kDa in cells stably transfected with LAT, mouse neuroblastoma cells transfected with a plasmid containing the AL3 ORF and TG of latently infected mice. The inability to detect the AL3 protein during productive infection may have been because the 5' terminus of the AL3 transcript was downstream of the first in-frame methionine of the AL3 ORF during productive infection.

Transient and stable expression of marker genes in cotransformed Petunia protoplasts in relation to X-ray and UV-irradiation

International Nuclear Information System (INIS)

Benediktsson, I.; Köhler, F.; Schieder, O.

1991-01-01

Irradiation of protoplasts with X-rays or ultraviolet light does not seem to influence the level of transient expression of foreign DNA in Petunia protoplasts, whereas the number of stably transformed colonies is significantly raised. This may indicate that irradiation influences integration and/or the expression of marker genes and does not result in enhanced uptake rates of plasmids into protoplasts and cell nuclei. Co-transformation with plasmids carrying a gene for kanamycin resistance (neomycin phosphotransferase II) and a gene for hygromycin resistance (hygromycin phosphotransferase) revealed that the cotransformation rates were not stimulated by irradiation when measuring expression

Salicylic acid inhibits UV- and Cis-Pt-induced human immunodeficiency virus expression

International Nuclear Information System (INIS)

Woloschak, G.E.; Panozzo, J.; Libertin, C.R.; Schreck, S.; South Carolina Univ., Columbia, SC

1994-01-01

Previous studies have shown that exposure of HeLa cells stably transfected with a human immunodeficiency virus-long terminal repeat-chloramphenicol acetyl transferase (HIV-LTR-CAT) construct to UV light-induced expression from the HIV LTR. By culturing the cells with salicylic acid we demonstrated dose-dependent repression of this induced HIV expression. Repression was evident if salicylic acid was administered 2 h before, at the same time as, or up to 6 h after exposure to the DNA-damaging agent. The kinetics were similar for UV- and for cis-Pt-induced HIV expression, and induction was dependent on the UV dose or cis-Pt concentration added to the culture. These results suggest a role for the prostaglandins or the cyclooxygenase pathway or both in HIV induction mediated by DNA-damaging agents

Proapoptotic Role of Potassium Ions in Liver Cells

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Zhenglin Xia

2016-01-01

Full Text Available Potassium channels are transmembrane proteins that selectively promote the infiltration of potassium ions. The significance of these channels for tumor biology has become obvious. However, the effects of potassium ions on the tumor or normal cells have seldom been studied. To address this problem, we studied the biological effects of L02 and HepG2 cells with ectogenous potassium ions. Cell proliferation, cell cycle, and apoptosis rate were analyzed. Our results indicated that potassium ions inhibited proliferation of L02 and HepG2 cells and promoted their apoptosis. Potassium ions induced apoptosis through regulating Bcl-2 family members and depolarized the mitochondrial membrane, especially for HepG2 cell. These biological effects were associated with channel protein HERG. By facilitating expression of channel protein HERG, potassium ions may prevent it from being shunted to procancerous pathways by inducing apoptosis. These results demonstrated that potassium ions may be a key regulator of liver cell function. Thus, our findings suggest that potassium ions could inhibit tumorigenesis through inducing apoptosis of hepatoma cells by upregulating potassium ions transport channel proteins HERG and VDAC1.

Inhibition of tolbutamide 4-methylhydroxylation by a series of non-steroidal anti-inflammatory drugs in V79-NH cells expressing human cytochrome P4502C10

NARCIS (Netherlands)

Kappers, W.A.; Groene, E.M. de; Kleij, L.A.; Witkamp, R.F.; Zweers-Zeilmaker, W.M.; Feron, V.J.; Horbach, G.J.

1996-01-01

1. To study the role of cytochrome P4502C10 in the metabolism of the non-steroidal antiinflammatory drugs (NSAIDs) diclofenac, phenylbutazone, fenoprofen, ibuprofen, flurbiprofen, ketoprofen and naproxen, a cell line was developed stably expressing CYP2C10 cDNA. A retroviral vector construct,

Differential expression of hERG1 channel isoforms reproduces properties of native I(Kr and modulates cardiac action potential characteristics.

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Anders Peter Larsen

Full Text Available BACKGROUND: The repolarizing cardiac rapid delayed rectifier current, I(Kr, is composed of ERG1 channels. It has been suggested that two isoforms of the ERG1 protein, ERG1a and ERG1b, both contribute to I(Kr. Marked heterogeneity in the kinetic properties of native I(Kr has been described. We hypothesized that the heterogeneity of native I(Kr can be reproduced by differential expression of ERG1a and ERG1b isoforms. Furthermore, the functional consequences of differential expression of ERG1 isoforms were explored as a potential mechanism underlying native heterogeneity of action potential duration (APD and restitution. METHODOLOGY/PRINCIPAL FINDINGS: The results show that the heterogeneity of native I(Kr can be reproduced in heterologous expression systems by differential expression of ERG1a and ERG1b isoforms. Characterization of the macroscopic kinetics of ERG1 currents demonstrated that these were dependent on the relative abundance of ERG1a and ERG1b. Furthermore, we used a computational model of the ventricular cardiomyocyte to show that both APD and the slope of the restitution curve may be modulated by varying the relative abundance of ERG1a and ERG1b. As the relative abundance of ERG1b was increased, APD was gradually shortened and the slope of the restitution curve was decreased. CONCLUSIONS/SIGNIFICANCE: Our results show that differential expression of ERG1 isoforms may explain regional heterogeneity of I(Kr kinetics. The data demonstrate that subunit dependent changes in channel kinetics are important for the functional properties of ERG1 currents and hence I(Kr. Importantly, our results suggest that regional differences in the relative abundance of ERG1 isoforms may represent a potential mechanism underlying the heterogeneity of both APD and APD restitution observed in mammalian hearts.

Biologically active, magnICON®-expressed EPO-Fc from stably transformed Nicotiana benthamiana plants presenting tetra-antennary N-glycan structures.

Science.gov (United States)

Nagels, Bieke; Van Damme, Els J M; Callewaert, Nico; Zabeau, Lennart; Tavernier, Jan; Delanghe, Joris R; Boets, Annemie; Castilho, Alexandra; Weterings, Koen

2012-08-31

In the past two decades plants have emerged as a valuable alternative for the production of pharmaceutical proteins. Since N-glycosylation influences functionality and stability of therapeutic proteins, the plant N-glycosylation pathway should be humanized. Here, we report the transient magnICON(®) expression of the erythropoietin fusion protein (EPO-Fc) in Nicotiana benthamiana plants that produce multi-antennary N-glycans without the plant-specific β1,2-xylose and α1,3-fucose residues in a stable manner (Nagels et al., 2011). The EPO-Fc fusion protein consists of EPO with a C-terminal-linked IgG-Fc domain and is used for pulmonary delivery of recombinant EPO to patients (Bitonti et al., 2004). Plant expressed EPO-Fc was quantified using a paramagnetic-particle chemiluminescent immunoassay and shown to be active in vitro via receptor binding experiments in HEK293T cells. Mass spectrometry-based N-glycan analysis confirmed the presence of multi-antennary N-glycans on plant-expressed EPO-Fc. The described research is the next step towards the development of a production platform for pharmaceutical proteins in plants. Copyright © 2012 Elsevier B.V. All rights reserved.

Automated Electrophysiology Makes the Pace for Cardiac Ion Channel Safety Screening

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Clemens eMoeller

2011-11-01

Full Text Available The field of automated patch-clamp electrophysiology has emerged from the tension between the pharmaceutical industry’s need for high-throughput compound screening versus its need to be conservative due to regulatory requirements. On the one hand, hERG channel screening was increasingly requested for new chemical entities, as the correlation between blockade of the ion channel coded by hERG and Torsades de Pointes cardiac arrhythmia gained increasing attention. On the other hand, manual patch-clamping, typically quoted as the gold-standard for understanding ion channel function and modulation, was far too slow (and, consequently, too expensive for keeping pace with the numbers of compounds submitted for hERG channel investigations from pharmaceutical R&D departments. In consequence it became more common for some pharmaceutical companies to outsource safety pharmacological investigations, with a focus on hERG channel interactions. This outsourcing has allowed those pharmaceutical companies to build up operational flexibility and greater independence from internal resources, and allowed them to obtain access to the latest technological developments that emerged in automated patch-clamp electrophysiology – much of which arose in specialized biotech companies. Assays for nearly all major cardiac ion channels are now available by automated patch-clamping using heterologous expression systems, and recently, automated action potential recordings from stem-cell derived cardiomyocytes have been demonstrated. Today, most of the large pharmaceutical companies have acquired automated electrophysiology robots and have established various automated cardiac ion channel safety screening assays on these, in addition to outsourcing parts of their needs for safety screening.

Functional characterization of Kv11.1 (hERG) potassium channels split in the voltage-sensing domain.

Science.gov (United States)

de la Peña, Pilar; Domínguez, Pedro; Barros, Francisco

2018-03-23

Voltage-dependent KCNH family potassium channel functionality can be reconstructed using non-covalently linked voltage-sensing domain (VSD) and pore modules (split channels). However, the necessity of a covalent continuity for channel function has not been evaluated at other points within the two functionally independent channel modules. We find here that by cutting Kv11.1 (hERG, KCNH2) channels at the different loops linking the transmembrane spans of the channel core, not only channels split at the S4-S5 linker level, but also those split at the intracellular S2-S3 and the extracellular S3-S4 loops, yield fully functional channel proteins. Our data indicate that albeit less markedly, channels split after residue 482 in the S2-S3 linker resemble the uncoupled gating phenotype of those split at the C-terminal end of the VSD S4 transmembrane segment. Channels split after residues 514 and 518 in the S3-S4 linker show gating characteristics similar to those of the continuous wild-type channel. However, breaking the covalent link at this level strongly accelerates the voltage-dependent accessibility of a membrane impermeable methanethiosulfonate reagent to an engineered cysteine at the N-terminal region of the S4 transmembrane helix. Thus, besides that of the S4-S5 linker, structural integrity of the intracellular S2-S3 linker seems to constitute an important factor for proper transduction of VSD rearrangements to opening and closing the cytoplasmic gate. Furthermore, our data suggest that the short and probably rigid characteristics of the extracellular S3-S4 linker are not an essential component of the Kv11.1 voltage sensing machinery.

Profile of differentially expressed genes mediated by the type III epidermal growth factor receptor mutation expressed in a small-cell lung cancer cell line

DEFF Research Database (Denmark)

Pedersen, M.W.; Andersen, Thomas Thykjær; Ørntoft, Torben Falck

2001-01-01

Previous studies have shown a correlation between expression of the EGF receptor type III mutation (EGFRvIII) and a more malignant phenotype of various cancers including: non-small-cell lung cancer, glioblastoma multiforme, prostate cancer and breast cancer. Thus, a detailed molecular genetic...... understanding of how the EGFRvIII contributes to the malignant phenotype is of major importance for future therapy. The GeneChip Hu6800Set developed by Affymetrix was used to identify changes in gene expression caused by the expression of EGFRvIII. The cell line selected for the study was an EGF receptor...... negative small-cell-lung cancer cell line, GLC3, stably transfected with the EGFRvIII gene in a Tet-On system. By comparison of mRNA levels in EGFRvIII-GLC3 with those of Tet-On-GLC3, it was found that the levels of mRNAs encoding several transcription factors (ATF-3, JunD, and c-Myb), cell adhesion...

Establishment of a human cell line stably overexpressing mouse Nip45 and characterization of Nip45 subcellular localization

Energy Technology Data Exchange (ETDEWEB)

Hashiguchi, Kohtaro; Ozaki, Masumi [Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto (Japan); Kuraoka, Isao [Biological Chemistry Group, Division of Chemistry, Graduate School of Engineering Science, Osaka University, Osaka (Japan); Saitoh, Hisato, E-mail: hisa@kumamoto-u.ac.jp [Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto (Japan); Department of New Frontier Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto (Japan); Global COE (Centers of Excellence) Program, Global Initiative Center for Pulsed Power Engineering, Kumamoto University, Kumamoto (Japan)

2013-01-04

Highlights: Black-Right-Pointing-Pointer A human cell line expressing a mouse Nip45 has facilitated Nip45 analysis. Black-Right-Pointing-Pointer Nip45 does not effectively inhibit polySUMOylation in vivo. Black-Right-Pointing-Pointer Nip45 interacts directly with SUMO and SUMO chains. Black-Right-Pointing-Pointer Nip45 accumulates at PML bodies in response to proteasome inhibition. -- Abstract: The nuclear factor of activated T cells, cytoplasmic, calcineurin dependent 2 interacting protein, Nfatc2ip (Nip45), has been implicated as a crucial coordinator of the immune response and of cellular differentiation in humans and mice, and contains SUMO-like domains in its C-terminal region. However, the significance of its N-terminal region and its correlation to the SUMO modification pathway remain largely uncharacterized. In this study, a human cultured cell line was established, in which FLAG-tagged mouse Nip45 (FLAG-mNip45) was stably overexpressed. Under standard, non-stressful conditions, we detected FLAG-mNip45 diffusely distributed in the nucleus. Intriguingly, proteasome inhibition by MG132 caused FLAG-mNip45, together with SUMOylated proteins, to localize in nuclear domains associated with promyelocytic leukemia protein. Finally, using an in vitro binding assay, we showed interaction of the N-terminal region of mNip45 with both free SUMO-3 and SUMO-3 chains, indicating that Nip45 may, in part, exert its function via interaction with SUMO/SUMOylated proteins. Taken together, our study provides novel information on a poorly characterized mammalian protein and suggests that our newly established cell line will be useful for elucidating the physiological role of Nip45.

Validation of reference genes for quantitative RT-PCR studies of gene expression in perennial ryegrass (Lolium perenne L.

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Thrush Anthony

2010-01-01

Full Text Available Abstract Background Perennial ryegrass (Lolium perenne L. is an important pasture and turf crop. Biotechniques such as gene expression studies are being employed to improve traits in this temperate grass. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR is among the best methods available for determining changes in gene expression. Before analysis of target gene expression, it is essential to select an appropriate normalisation strategy to control for non-specific variation between samples. Reference genes that have stable expression at different biological and physiological states can be effectively used for normalisation; however, their expression stability must be validated before use. Results Existing Serial Analysis of Gene Expression data were queried to identify six moderately expressed genes that had relatively stable gene expression throughout the year. These six candidate reference genes (eukaryotic elongation factor 1 alpha, eEF1A; TAT-binding protein homolog 1, TBP-1; eukaryotic translation initiation factor 4 alpha, eIF4A; YT521-B-like protein family protein, YT521-B; histone 3, H3; ubiquitin-conjugating enzyme, E2 were validated for qRT-PCR normalisation in 442 diverse perennial ryegrass (Lolium perenne L. samples sourced from field- and laboratory-grown plants under a wide range of experimental conditions. Eukaryotic EF1A is encoded by members of a multigene family exhibiting differential expression and necessitated the expression analysis of different eEF1A encoding genes; a highly expressed eEF1A (h, a moderately, but stably expressed eEF1A (s, and combined expression of multigene eEF1A (m. NormFinder identified eEF1A (s and YT521-B as the best combination of two genes for normalisation of gene expression data in perennial ryegrass following different defoliation management in the field. Conclusions This study is unique in the magnitude of samples tested with the inclusion of numerous field-grown samples

Ectopic ERK Expression Induces Phenotypic Conversion of C10 Cells and Alters DNA Methyltransferase Expression

Energy Technology Data Exchange (ETDEWEB)

Sontag, Ryan L.; Weber, Thomas J.

2012-05-04

In some model systems constitutive extracellular signal regulated kinase (ERK) activation is sufficient to promote an oncogenic phenotype. Here we investigate whether constitutive ERK expression influences phenotypic conversion in murine C10 type II alveolar epithelial cells. C10 cells were stably transduced with an ERK1-green fluorescent protein (ERK1-GFP) chimera or empty vector and ectopic ERK expression was associated with the acquisition of soft agar focus-forming potential in late passage, but not early passage cells. Late passage ERK1-GFP cells exhibited a significant increase in the expression of DNA methyl transferases (DNMT1 and 3b) and a marked increase in sensitivity to 5-azacytidine (5-azaC)-mediated toxicity, relative to early passage ERK1-GFP cells and vector controls. The expression of xeroderma pigmentosum complementation group A (XPA) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) were significantly increased in late passage cells, suggesting enhanced DNA damage recognition and repair activity which we interpret as a reflection of genomic instability. Phospho-ERK levels were dramatically decreased in late passage ERK1-GFP cells, relative to early passage and vector controls, and phospho-ERK levels were restored by treatment with sodium orthovanadate, indicating a role for phosphatase activity in this response. Collectively these observations suggest that ectopic ERK expression promotes phenotypic conversion of C10 cells that is associated with latent effects on epigenetic programming and phosphatase activities.

Algevir: An Expression System for Microalgae Based on Viral Vectors

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Bernardo Bañuelos-Hernández

2017-06-01

Full Text Available The use of recombinant algae for the production of valuable compounds is opening promising biotechnological applications. However, the development of efficient expression approaches is still needed to expand the exploitation of microalgae in biotechnology. Herein, the concept of using viral expression vectors in microalgae was explored for the first time. An inducible geminiviral vector leading to Rep-mediated replication of the expression cassette allowed the production of antigenic proteins at high levels. This system, called Algevir, allows the production of complex viral proteins (GP1 from Zaire ebolavirus and bacterial toxin subunits (B subunit of the heat-labile Escherichia coli enterotoxin, which retained their antigenic activity. The highest achieved yield was 1.25 mg/g fresh biomass (6 mg/L of culture, which was attained 3 days after transformation. The Algevir system allows for a fast and efficient production of recombinant proteins, overcoming the difficulties imposed by the low yields and unstable expression patterns frequently observed in stably transformed microalgae at the nuclear level; as well as the toxicity of some target proteins.

Algevir: An Expression System for Microalgae Based on Viral Vectors

Science.gov (United States)

Bañuelos-Hernández, Bernardo; Monreal-Escalante, Elizabeth; González-Ortega, Omar; Angulo, Carlos; Rosales-Mendoza, Sergio

2017-01-01

The use of recombinant algae for the production of valuable compounds is opening promising biotechnological applications. However, the development of efficient expression approaches is still needed to expand the exploitation of microalgae in biotechnology. Herein, the concept of using viral expression vectors in microalgae was explored for the first time. An inducible geminiviral vector leading to Rep-mediated replication of the expression cassette allowed the production of antigenic proteins at high levels. This system, called Algevir, allows the production of complex viral proteins (GP1 from Zaire ebolavirus) and bacterial toxin subunits (B subunit of the heat-labile Escherichia coli enterotoxin), which retained their antigenic activity. The highest achieved yield was 1.25 mg/g fresh biomass (6 mg/L of culture), which was attained 3 days after transformation. The Algevir system allows for a fast and efficient production of recombinant proteins, overcoming the difficulties imposed by the low yields and unstable expression patterns frequently observed in stably transformed microalgae at the nuclear level; as well as the toxicity of some target proteins. PMID:28713333

Anti-addiction drug ibogaine inhibits voltage-gated ionic currents: A study to assess the drug's cardiac ion channel profile

Energy Technology Data Exchange (ETDEWEB)

Koenig, Xaver; Kovar, Michael; Rubi, Lena; Mike, Agnes K.; Lukacs, Peter; Gawali, Vaibhavkumar S.; Todt, Hannes [Center for Physiology and Pharmacology, Department of Neurophysiology and -pharmacology, Medical University of Vienna, 1090 Vienna (Austria); Hilber, Karlheinz, E-mail: karlheinz.hilber@meduniwien.ac.at [Center for Physiology and Pharmacology, Department of Neurophysiology and -pharmacology, Medical University of Vienna, 1090 Vienna (Austria); Sandtner, Walter [Center for Physiology and Pharmacology, Institute of Pharmacology, Medical University of Vienna, 1090 Vienna (Austria)

2013-12-01

The plant alkaloid ibogaine has promising anti-addictive properties. Albeit not licenced as a therapeutic drug, and despite hints that ibogaine may perturb the heart rhythm, this alkaloid is used to treat drug addicts. We have recently reported that ibogaine inhibits human ERG (hERG) potassium channels at concentrations similar to the drugs affinity for several of its known brain targets. Thereby the drug may disturb the heart's electrophysiology. Here, to assess the drug's cardiac ion channel profile in more detail, we studied the effects of ibogaine and its congener 18-Methoxycoronaridine (18-MC) on various cardiac voltage-gated ion channels. We confirmed that heterologously expressed hERG currents are reduced by ibogaine in low micromolar concentrations. Moreover, at higher concentrations, the drug also reduced human Na{sub v}1.5 sodium and Ca{sub v}1.2 calcium currents. Ion currents were as well reduced by 18-MC, yet with diminished potency. Unexpectedly, although blocking hERG channels, ibogaine did not prolong the action potential (AP) in guinea pig cardiomyocytes at low micromolar concentrations. Higher concentrations (≥ 10 μM) even shortened the AP. These findings can be explained by the drug's calcium channel inhibition, which counteracts the AP-prolonging effect generated by hERG blockade. Implementation of ibogaine's inhibitory effects on human ion channels in a computer model of a ventricular cardiomyocyte, on the other hand, suggested that ibogaine does prolong the AP in the human heart. We conclude that therapeutic concentrations of ibogaine have the propensity to prolong the QT interval of the electrocardiogram in humans. In some cases this may lead to cardiac arrhythmias. - Highlights: • We study effects of anti-addiction drug ibogaine on ionic currents in cardiomyocytes. • We assess the cardiac ion channel profile of ibogaine. • Ibogaine inhibits hERG potassium, sodium and calcium channels. • Ibogaine’s effects on

The Eag domain regulates the voltage-dependent inactivation of rat Eag1 K+ channels.

Directory of Open Access Journals (Sweden)

Ting-Feng Lin

Full Text Available Eag (Kv10 and Erg (Kv11 belong to two distinct subfamilies of the ether-à-go-go K+ channel family (KCNH. While Erg channels are characterized by an inward-rectifying current-voltage relationship that results from a C-type inactivation, mammalian Eag channels display little or no voltage-dependent inactivation. Although the amino (N-terminal region such as the eag domain is not required for the C-type inactivation of Erg channels, an N-terminal deletion in mouse Eag1 has been shown to produce a voltage-dependent inactivation. To further discern the role of the eag domain in the inactivation of Eag1 channels, we generated N-terminal chimeras between rat Eag (rEag1 and human Erg (hERG1 channels that involved swapping the eag domain alone or the complete cytoplasmic N-terminal region. Functional analyses indicated that introduction of the homologous hERG1 eag domain led to both a fast phase and a slow phase of channel inactivation in the rEag1 chimeras. By contrast, the inactivation features were retained in the reverse hERG1 chimeras. Furthermore, an eag domain-lacking rEag1 deletion mutant also showed the fast phase of inactivation that was notably attenuated upon co-expression with the rEag1 eag domain fragment, but not with the hERG1 eag domain fragment. Additionally, we have identified a point mutation in the S4-S5 linker region of rEag1 that resulted in a similar inactivation phenotype. Biophysical analyses of these mutant constructs suggested that the inactivation gating of rEag1 was distinctly different from that of hERG1. Overall, our findings are consistent with the notion that the eag domain plays a critical role in regulating the inactivation gating of rEag1. We propose that the eag domain may destabilize or mask an inherent voltage-dependent inactivation of rEag1 K+ channels.

Anti-addiction drug ibogaine inhibits voltage-gated ionic currents: A study to assess the drug's cardiac ion channel profile

International Nuclear Information System (INIS)

Koenig, Xaver; Kovar, Michael; Rubi, Lena; Mike, Agnes K.; Lukacs, Peter; Gawali, Vaibhavkumar S.; Todt, Hannes; Hilber, Karlheinz; Sandtner, Walter

2013-01-01

The plant alkaloid ibogaine has promising anti-addictive properties. Albeit not licenced as a therapeutic drug, and despite hints that ibogaine may perturb the heart rhythm, this alkaloid is used to treat drug addicts. We have recently reported that ibogaine inhibits human ERG (hERG) potassium channels at concentrations similar to the drugs affinity for several of its known brain targets. Thereby the drug may disturb the heart's electrophysiology. Here, to assess the drug's cardiac ion channel profile in more detail, we studied the effects of ibogaine and its congener 18-Methoxycoronaridine (18-MC) on various cardiac voltage-gated ion channels. We confirmed that heterologously expressed hERG currents are reduced by ibogaine in low micromolar concentrations. Moreover, at higher concentrations, the drug also reduced human Na v 1.5 sodium and Ca v 1.2 calcium currents. Ion currents were as well reduced by 18-MC, yet with diminished potency. Unexpectedly, although blocking hERG channels, ibogaine did not prolong the action potential (AP) in guinea pig cardiomyocytes at low micromolar concentrations. Higher concentrations (≥ 10 μM) even shortened the AP. These findings can be explained by the drug's calcium channel inhibition, which counteracts the AP-prolonging effect generated by hERG blockade. Implementation of ibogaine's inhibitory effects on human ion channels in a computer model of a ventricular cardiomyocyte, on the other hand, suggested that ibogaine does prolong the AP in the human heart. We conclude that therapeutic concentrations of ibogaine have the propensity to prolong the QT interval of the electrocardiogram in humans. In some cases this may lead to cardiac arrhythmias. - Highlights: • We study effects of anti-addiction drug ibogaine on ionic currents in cardiomyocytes. • We assess the cardiac ion channel profile of ibogaine. • Ibogaine inhibits hERG potassium, sodium and calcium channels. • Ibogaine’s effects on ion channels are a potential

Saponin determination, expression analysis and functional characterization of saponin biosynthetic genes in Chenopodium quinoa leaves.

Science.gov (United States)

Fiallos-Jurado, Jennifer; Pollier, Jacob; Moses, Tessa; Arendt, Philipp; Barriga-Medina, Noelia; Morillo, Eduardo; Arahana, Venancio; de Lourdes Torres, Maria; Goossens, Alain; Leon-Reyes, Antonio

2016-09-01

Quinoa (Chenopodium quinoa Willd.) is a highly nutritious pseudocereal with an outstanding protein, vitamin, mineral and nutraceutical content. The leaves, flowers and seed coat of quinoa contain triterpenoid saponins, which impart bitterness to the grain and make them unpalatable without postharvest removal of the saponins. In this study, we quantified saponin content in quinoa leaves from Ecuadorian sweet and bitter genotypes and assessed the expression of saponin biosynthetic genes in leaf samples elicited with methyl jasmonate. We found saponin accumulation in leaves after MeJA treatment in both ecotypes tested. As no reference genes were available to perform qPCR in quinoa, we mined publicly available RNA-Seq data for orthologs of 22 genes known to be stably expressed in Arabidopsis thaliana using geNorm, NormFinder and BestKeeper algorithms. The quinoa ortholog of At2g28390 (Monensin Sensitivity 1, MON1) was stably expressed and chosen as a suitable reference gene for qPCR analysis. Candidate saponin biosynthesis genes were screened in the quinoa RNA-Seq data and subsequent functional characterization in yeast led to the identification of CqbAS1, CqCYP716A78 and CqCYP716A79. These genes were found to be induced by MeJA, suggesting this phytohormone might also modulate saponin biosynthesis in quinoa leaves. Knowledge of the saponin biosynthesis and its regulation in quinoa may aid the further development of sweet cultivars that do not require postharvest processing. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Molekulare Klonierung, stabile Transfektion und funktionelle Expression der murinen Chemokinrezeptoren Ccr2 und Ccr5

OpenAIRE

Simonis, Christopher

2009-01-01

The two chemokine receptors CCR2 and CCR5 play important roles in the recruitment and activation of monocytes/macrophages and T-lymphocytes at sites of infection and inflammation. To further examine their function, I cloned the two murine chemokine receptors Ccr2 and Ccr5 from genomic mouse DNA by a PCR-based cloning strategy and functionally expressed them in stably transfected CHO-cells. These cells were used to generate the first monoclonal antibodies against Ccr2 and Ccr5.

Susceptibility to viral infection is enhanced by stable expression of 3A or 3AB proteins from foot-and-mouth disease virus

International Nuclear Information System (INIS)

Rosas, Maria F.; Vieira, Yuri A.; Postigo, Raul; Martin-Acebes, Miguel A.; Armas-Portela, Rosario; Martinez-Salas, Encarnacion; Sobrino, Francisco

2008-01-01

The foot-and-mouth disease virus (FMDV) 3A protein is involved in virulence and host range. A distinguishing feature of FMDV 3B among picornaviruses is that three non-identical copies are encoded in the viral RNA and required for optimal replication in cell culture. Here, we have studied the involvement of the 3AB region on viral infection using constitutive and transient expression systems. BHK-21 stably transformed clones expressed low levels of FMDV 3A or 3A(B) proteins in the cell cytoplasm. Transformed cells stably expressing these proteins did not exhibit inner cellular rearrangements detectable by electron microscope analysis. Upon FMDV infection, clones expressing either 3A alone or 3A(B) proteins showed a significant increase in the percentage of infected cells, the number of plaque forming units and the virus yield. The 3A-enhancing effect was specific for FMDV as no increase in viral multiplication was observed in transformed clones infected with another picornavirus, encephalomyocarditis virus, or the negative-strand RNA virus vesicular stomatitis virus. A potential role of 3A protein in viral RNA translation was discarded by the lack of effect on FMDV IRES-dependent translation. Increased viral susceptibility was not caused by a released factor; neither the supernatant of transformed clones nor the addition of purified 3A protein to the infection medium was responsible for this effect. Unlike stable expression, high levels of 3A or 3A(B) protein transient expression led to unspecific inhibition of viral infection. Therefore, the effect observed on viral yield, which inversely correlated with the intracellular levels of 3A protein, suggests a transacting role operating on the FMDV multiplication cycle

Effects of frequently used pharmaceutical excipients on the organic cation transporters 1-3 and peptide transporters 1/2 stably expressed in MDCKII cells.

Science.gov (United States)

Otter, Marcus; Oswald, Stefan; Siegmund, Werner; Keiser, Markus

2017-03-01

There is ample evidence that pharmaceutical excipients, which are supposed to be pharmacologically inactive, have an impact on drug metabolism and efflux transport. So far, little is known whether they also modulate uptake transporter proteins. We have recently shown that commonly used solubilizing agents exert significant effects on the function of organic anion uptake transporting polypeptides. Therefore, we investigated in this study the influence of frequently used pharmaceutical excipients on the transport activity of organic cation transporters OCT1, OCT2 and OCT3 and the peptide transporters PEPT1 and PEPT2. Inhibition of the OCTs and PEPTs by the excipients polyethylene glycol 400 (PEG), hydroxypropyl-β-cyclodextrin (HPCD), Solutol® HS15 (SOL), Cremophor® EL (CrEL), Tween® 20 (Tw20), Tween® 80 (Tw80), Kolliphor® P188 (P188) and Kolliphor® P407 (P407) was evaluated using stably transfected MDCKII cells with radio-labeled reference substrates and established inhibitors as controls. Intracellular accumulation of [3H]-1-methyl-4-phenylpyridinium (MPP + ) for the OCTs and [3H]-glycyl-sarcosine (Gly-Sar) for the PEPTs was measured by liquid scintillation counting after cell lysis. Our studies revealed that PEG, HPCD, SOL, CrEL, Tw20 and Tw80 were potent inhibitors of OCT1-3 (e.g., Tw20 IC 50 values<0.04%). Cellular uptake of Gly-Sar by PEPT1 and PEPT2 was strongly inhibited by both Tw20 and Tw80. SOL was also a strong inhibitor of PEPT1 and PEPT2 (e.g., SOL IC 50 values<0.02%), while CrEL showed significantly inhibition of only PEPT2. The substantial inhibitory effects of certain solubilizing agents on OCTs and PEPTs should be considered if they are to be used in dosage forms for new chemical entities and registered drugs to avoid misinterpretation of pharmacokinetic data and undesired drug interactions. Copyright © 2016 Elsevier B.V. All rights reserved.

Identification and validation of superior reference gene for gene expression normalization via RT-qPCR in staminate and pistillate flowers of Jatropha curcas - A biodiesel plant.

Science.gov (United States)

Karuppaiya, Palaniyandi; Yan, Xiao-Xue; Liao, Wang; Wu, Jun; Chen, Fang; Tang, Lin

2017-01-01

Physic nut (Jatropha curcas L) seed oil is a natural resource for the alternative production of fossil fuel. Seed oil production is mainly depended on seed yield, which was restricted by the low ratio of staminate flowers to pistillate flowers. Further, the mechanism of physic nut flower sex differentiation has not been fully understood yet. Quantitative Real Time-Polymerase Chain Reaction is a reliable and widely used technique to quantify the gene expression pattern in biological samples. However, for accuracy of qRT-PCR, appropriate reference gene is highly desirable to quantify the target gene level. Hence, the present study was aimed to identify the stable reference genes in staminate and pistillate flowers of J. curcas. In this study, 10 candidate reference genes were selected and evaluated for their expression stability in staminate and pistillate flowers, and their stability was validated by five different algorithms (ΔCt, BestKeeper, NormFinder, GeNorm and RefFinder). Resulting, TUB and EF found to be the two most stably expressed reference for staminate flower; while GAPDH1 and EF found to be the most stably expressed reference gene for pistillate flowers. Finally, RT-qPCR assays of target gene AGAMOUS using the identified most stable reference genes confirmed the reliability of selected reference genes in different stages of flower development. AGAMOUS gene expression levels at different stages were further proved by gene copy number analysis. Therefore, the present study provides guidance for selecting appropriate reference genes for analyzing the expression pattern of floral developmental genes in staminate and pistillate flowers of J. curcas.

Silicon homo-heterojunction solar cells: A promising candidate to realize high performance more stably

Directory of Open Access Journals (Sweden)

Miao Tan

2017-08-01

Full Text Available We have investigated the influences of diverse physical parameters on the performances of a silicon homo-heterojunction (H-H solar cell, which encompasses both homojunction and heterojunction, together with their underlying mechanisms by the aid of AFORS-HET simulation. It is found that the performances of H-H solar cell are less sensitive to (i the work function of the transparent conductive oxide layer, (ii the interfacial density of states at the front hydrogenated amorphous silicon/crystalline silicon (a-Si:H/c-Si interface, (iii the peak dangling bond defect densities within the p-type a-Si:H (p-a-Si:H layer, and (iv the doping concentration of the p-a-Si:H layer, when compared to that of the conventional heterojunction with intrinsic thin layer (HIT counterparts. These advantages are due to the fact that the interfacial recombination and the recombination within the a-Si:H region are less affected by all the above parameters, which fundamentally benefit from the field-effect passivation of the homojunction. Therefore, the design of H-H structure can provide an opportunity to produce high-efficiency solar cells more stably.

Silicon homo-heterojunction solar cells: A promising candidate to realize high performance more stably

Science.gov (United States)

Tan, Miao; Zhong, Sihua; Wang, Wenjie; Shen, Wenzhong

2017-08-01

We have investigated the influences of diverse physical parameters on the performances of a silicon homo-heterojunction (H-H) solar cell, which encompasses both homojunction and heterojunction, together with their underlying mechanisms by the aid of AFORS-HET simulation. It is found that the performances of H-H solar cell are less sensitive to (i) the work function of the transparent conductive oxide layer, (ii) the interfacial density of states at the front hydrogenated amorphous silicon/crystalline silicon (a-Si:H/c-Si) interface, (iii) the peak dangling bond defect densities within the p-type a-Si:H (p-a-Si:H) layer, and (iv) the doping concentration of the p-a-Si:H layer, when compared to that of the conventional heterojunction with intrinsic thin layer (HIT) counterparts. These advantages are due to the fact that the interfacial recombination and the recombination within the a-Si:H region are less affected by all the above parameters, which fundamentally benefit from the field-effect passivation of the homojunction. Therefore, the design of H-H structure can provide an opportunity to produce high-efficiency solar cells more stably.

Comparison of [{sup 18}F]FHBG and [{sup 14}C]FIAU for imaging of HSV1-tk reporter gene expression: adenoviral infection vs stable transfection

Energy Technology Data Exchange (ETDEWEB)

Min, Jung-Jun; Iyer, Meera [The Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, UCLA School of Medicine, B3-399A BRI 700 Westwood Plaza, CA 90095-1770, Los Angeles (United States); Gambhir, Sanjiv S. [The Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, UCLA School of Medicine, B3-399A BRI 700 Westwood Plaza, CA 90095-1770, Los Angeles (United States); Department of Radiology, Bio-X Program, Stanford University, Stanford, California (United States)

2003-11-01

Earlier studies involving comparison of different reporter probes have shown conflicting results between pyrimidine nucleosides [e.g., 2'-fluoro-2'-deoxy-1-{beta}-d-arabinofuranosyl-5-iodouracil (FIAU)] and acycloguanosine derivatives [e.g., penciclovir (PCV), 9-(4-fluoro-3-hydroxymethylbutyl)guanine (FHBG)]. We hypothesized that this reported discrepancy may be related to how the reporter gene is delivered to the cells - stably transfected vs adenoviral infection. We directly compared the uptake characteristics of [{sup 18}F]FHBG, [{sup 3}H]PCV, and [{sup 14}C]FIAU in cell culture and in vivo using an adenoviral mediated gene transfer model and stably transfected cells. We further compared the uptake of three reporter probes using both HSV1-tk and a mutant HSV1-sr39tk expressing cells to assess the optimal reporter probe/reporter gene combination. [{sup 14}C]FIAU accumulation was greater than that of [{sup 3}H]PCV and [{sup 18}F]FHBG in control cells and in HSV1-tk stably transfected cells (P<0.001). After infection of C6 cells with AdCMV-HSV1-tk (1.5 x 10{sup 8} pfu), [{sup 18}F]FHBG and [{sup 3}H]PCV accumulation was significantly greater than that of [{sup 14}C]FIAU (P<0.01). [{sup 18}F]FHBG and [{sup 3}H]PCV accumulated to a significantly greater extent than [{sup 14}C]FIAU in C6-stb-sr39tk+ and AdCMV-HSV1-sr39tk infected C6 cells (P<0.001). Results from the nude mice supported the results in cell culture. [{sup 14}C]FIAU led to significantly higher %ID/g in C6-stb-tk+ xenografts than [{sup 18}F]FHBG (P<0.05); however, compared with [{sup 14}C]FIAU, [{sup 18}F]FHBG led to as high %ID/g in HSV1-tk expressing hepatocytes and to significantly greater %ID/g in C6-stb-sr39tk+ xenografts and HSV1-sr39tk expressing hepatocytes. Dynamic sequential images showed that [{sup 18}F]FHBG was well retained in HSV1-sr39tk expressing cells (C6-stb-sr39tk+) for at least 4 h after injection, while it was rapidly cleared from HSV1-tk expressing cells (MH3924A

Plasmodium yoelii yoelii 17XNL constitutively expressing GFP throughout the life cycle.

Science.gov (United States)

Ono, Takeshi; Tadakuma, Takushi; Rodriguez, Ana

2007-03-01

Plasmodium yoelii is a rodent parasite commonly used as a model to study malaria infection. It is the preferred model parasite for liver-stage immunological studies and is also widely used to study hepatocyte, erythrocyte and mosquito infection. We have generated a P. yoelii yoelii 17XNL line that is stably transfected with the green fluorescent protein (gfp) gene. This parasite line constitutively expresses high levels of GFP during the complete parasite life cycle including liver, blood and mosquito stages. These fluorescent parasites can be used in combination with fluorescence activated cell sorting or live microscopy for a wide range of experimental applications.

Transcriptional expression of type I interferon response genes and stability of housekeeping genes in the human endometrium and endometriosis

DEFF Research Database (Denmark)

Vestergaard, Anna L; Knudsen, Ulla B; Munk, Torben

2011-01-01

Endometriosis is a painful chronic female disease defined by the presence of endometrial tissue implants in ectopic locations. The pathogenesis is much debated, and type I interferons could be involved. The expression of genes of the type I interferon response were profiled by a specific PCR Array...... of RNA obtained from ectopic and eutopic endometrium collected from 9 endometriosis patients and 9 healthy control women. Transcriptional expression levels of selected interferon-regulated and housekeeping genes were investigated by real-time quantitative reverse transcriptase PCR (qRT-PCR). Stably...... expressed housekeeping genes for valid normalization of transcriptional studies of endometrium and endometriosis have not yet been published. Here, seven housekeeping genes were evaluated for stability using the GeNorm and NormFinder software. A normalization factor based on HMBS, TBP, and YWHAZ expression...

GnRH receptor activation competes at a low level with growth signaling in stably transfected human breast cell lines

International Nuclear Information System (INIS)

Morgan, Kevin; Meyer, Colette; Miller, Nicola; Sims, Andrew H; Cagnan, Ilgin; Faratian, Dana; Harrison, David J; Millar, Robert P; Langdon, Simon P

2011-01-01

Gonadotrophin releasing hormone (GnRH) analogs lower estrogen levels in pre-menopausal breast cancer patients. GnRH receptor (GnRH-R) activation also directly inhibits the growth of certain cells. The applicability of GnRH anti-proliferation to breast cancer was therefore analyzed. GnRH-R expression in 298 primary breast cancer samples was measured by quantitative immunofluorescence. Levels of functional GnRH-R in breast-derived cell lines were assessed using 125 I-ligand binding and stimulation of 3 H-inositol phosphate production. Elevated levels of GnRH-R were stably expressed in cells by transfection. Effects of receptor activation on in vitro cell growth were investigated in comparison with IGF-I and EGF receptor inhibition, and correlated with intracellular signaling using western blotting. GnRH-R immunoscoring was highest in hormone receptor (triple) negative and grade 3 breast tumors. However prior to transfection, functional endogenous GnRH-R were undetectable in four commonly studied breast cancer cell lines (MCF-7, ZR-75-1, T47D and MDA-MB-231). After transfection with GnRH-R, high levels of cell surface GnRH-R were detected in SVCT and MDA-MB-231 clones while low-moderate levels of GnRH-R occurred in MCF-7 clones and ZR-75-1 clones. MCF-7 sub-clones with high levels of GnRH-R were isolated following hygromycin phosphotransferase transfection. High level cell surface GnRH-R enabled induction of high levels of 3 H-inositol phosphate and modest growth-inhibition in SVCT cells. In contrast, growth of MCF-7, ZR-75-1 or MDA-MB-231 clones was unaffected by GnRH-R activation. Cell growth was inhibited by IGF-I or EGF receptor inhibitors. IGF-I receptor inhibitor lowered levels of p-ERK1/2 in MCF-7 clones. Washout of IGF-I receptor inhibitor resulted in transient hyper-elevation of p-ERK1/2, but co-addition of GnRH-R agonist did not alter the dynamics of ERK1/2 re-phosphorylation. Breast cancers exhibit a range of GnRH-R immunostaining, with higher levels of

Phenomenology of two-dimensional stably stratified turbulence under large-scale forcing

KAUST Repository

Kumar, Abhishek; Verma, Mahendra K.; Sukhatme, Jai

2017-01-01

In this paper, we characterise the scaling of energy spectra, and the interscale transfer of energy and enstrophy, for strongly, moderately and weakly stably stratified two-dimensional (2D) turbulence, restricted in a vertical plane, under large-scale random forcing. In the strongly stratified case, a large-scale vertically sheared horizontal flow (VSHF) coexists with small scale turbulence. The VSHF consists of internal gravity waves and the turbulent flow has a kinetic energy (KE) spectrum that follows an approximate k−3 scaling with zero KE flux and a robust positive enstrophy flux. The spectrum of the turbulent potential energy (PE) also approximately follows a k−3 power-law and its flux is directed to small scales. For moderate stratification, there is no VSHF and the KE of the turbulent flow exhibits Bolgiano–Obukhov scaling that transitions from a shallow k−11/5 form at large scales, to a steeper approximate k−3 scaling at small scales. The entire range of scales shows a strong forward enstrophy flux, and interestingly, large (small) scales show an inverse (forward) KE flux. The PE flux in this regime is directed to small scales, and the PE spectrum is characterised by an approximate k−1.64 scaling. Finally, for weak stratification, KE is transferred upscale and its spectrum closely follows a k−2.5 scaling, while PE exhibits a forward transfer and its spectrum shows an approximate k−1.6 power-law. For all stratification strengths, the total energy always flows from large to small scales and almost all the spectral indicies are well explained by accounting for the scale-dependent nature of the corresponding flux.

Phenomenology of two-dimensional stably stratified turbulence under large-scale forcing

KAUST Repository

Kumar, Abhishek

2017-01-11

In this paper, we characterise the scaling of energy spectra, and the interscale transfer of energy and enstrophy, for strongly, moderately and weakly stably stratified two-dimensional (2D) turbulence, restricted in a vertical plane, under large-scale random forcing. In the strongly stratified case, a large-scale vertically sheared horizontal flow (VSHF) coexists with small scale turbulence. The VSHF consists of internal gravity waves and the turbulent flow has a kinetic energy (KE) spectrum that follows an approximate k−3 scaling with zero KE flux and a robust positive enstrophy flux. The spectrum of the turbulent potential energy (PE) also approximately follows a k−3 power-law and its flux is directed to small scales. For moderate stratification, there is no VSHF and the KE of the turbulent flow exhibits Bolgiano–Obukhov scaling that transitions from a shallow k−11/5 form at large scales, to a steeper approximate k−3 scaling at small scales. The entire range of scales shows a strong forward enstrophy flux, and interestingly, large (small) scales show an inverse (forward) KE flux. The PE flux in this regime is directed to small scales, and the PE spectrum is characterised by an approximate k−1.64 scaling. Finally, for weak stratification, KE is transferred upscale and its spectrum closely follows a k−2.5 scaling, while PE exhibits a forward transfer and its spectrum shows an approximate k−1.6 power-law. For all stratification strengths, the total energy always flows from large to small scales and almost all the spectral indicies are well explained by accounting for the scale-dependent nature of the corresponding flux.

Transient GFP expression in Nicotiana plumbaginifolia suspension cells: the role of gene silencing, cell death and T-DNA loss.

Science.gov (United States)

Weld, R; Heinemann, J; Eady, C

2001-03-01

The transient nature of T-DNA expression was studied with a gfp reporter gene transferred to Nicotiana plumbaginifolia suspension cells from Agrobacterium tumefaciens. Individual GFP-expressing protoplasts were isolated after 4 days' co-cultivation. The protoplasts were cultured without selection and 4 weeks later the surviving proto-calluses were again screened for GFP expression. Of the proto-calluses initially expressing GFP, 50% had lost detectable GFP activity during the first 4 weeks of culture. Multiple T-DNA copies of the gfp gene were detected in 10 of 17 proto-calluses lacking visible GFP activity. The remaining 7 cell lines contained no gfp sequences. Our results confirm that transiently expressed T-DNAs can be lost during growth of somatic cells and demonstrate that transiently expressing cells frequently integrate multiple T-DNAs that become silenced. In cells competent for DNA uptake, cell death and gene silencing were more important barriers to the recovery of stably expressing transformants than lack of T-DNA integration.

Gene expression analysis uncovers novel Hedgehog interacting protein (HHIP) effects in human bronchial epithelial cells

Science.gov (United States)

Zhou, Xiaobo; Qiu, Weiliang; Sathirapongsasuti, J. Fah.; Cho, Michael H.; Mancini, John D.; Lao, Taotao; Thibault, Derek M.; Litonjua, Gus; Bakke, Per S.; Gulsvik, Amund; Lomas, David A.; Beaty, Terri H.; Hersh, Craig P.; Anderson, Christopher; Geigenmuller, Ute; Raby, Benjamin A.; Rennard, Stephen I.; Perrella, Mark A.; Choi, Augustine M.K.; Quackenbush, John; Silverman, Edwin K.

2013-01-01

Hedgehog Interacting Protein (HHIP) was implicated in chronic obstructive pulmonary disease (COPD) by genome-wide association studies (GWAS). However, it remains unclear how HHIP contributes to COPD pathogenesis. To identify genes regulated by HHIP, we performed gene expression microarray analysis in a human bronchial epithelial cell line (Beas-2B) stably infected with HHIP shRNAs. HHIP silencing led to differential expression of 296 genes; enrichment for variants nominally associated with COPD was found. Eighteen of the differentially expressed genes were validated by real-time PCR in Beas-2B cells. Seven of 11 validated genes tested in human COPD and control lung tissues demonstrated significant gene expression differences. Functional annotation indicated enrichment for extracellular matrix and cell growth genes. Network modeling demonstrated that the extracellular matrix and cell proliferation genes influenced by HHIP tended to be interconnected. Thus, we identified potential HHIP targets in human bronchial epithelial cells that may contribute to COPD pathogenesis. PMID:23459001

Production and characterization of active recombinant interleukin-12/eGFP fusion protein in stably-transfected DF1 chicken cells.

Science.gov (United States)

Wu, Hsing Chieh; Chen, Yu San; Shen, Pin Chun; Shien, Jui Hung; Lee, Long Huw; Chiu, Hua Hsien

2015-01-01

The adjuvant activity of chicken interleukin-12 (chIL-12) protein has been described as similar to that of mammalian IL-12. Recombinant chIL-12 can be produced using several methods, but chIL-12 production in eukaryotic cells is lower than that in prokaryotic cells. Stimulating compounds, such as dimethyl sulfoxide (DMSO), can be added to animal cell cultures to overcome this drawback. In this study, we constructed a cell line, DF1/chIL-12 which stably expressed a fusion protein, chIL-12 and enhanced green fluorescent protein (eGFP) connected by a (G4 S)3 linker sequence. Fusion protein production was increased when cells were cultured in the presence of DMSO. When 1 × 10(6) DF1/chIL-12 cells were inoculated in a T-175 flask containing 30 mL of media, incubated for 15 h, and further cultivated in the presence of 4% DMSO for 48 h, the production of total fusion protein was mostly enhanced compared with the production of total fusion protein by using cell lysates induced with DMSO at other concentrations. The concentrations of the unpurified and purified total fusion proteins in cell lysates were 2,781 ± 2.72 ng mL(-1) and 2,207 ± 3.28 ng mL(-1) , respectively. The recovery rate was 79%. The fusion protein stimulated chicken splenocytes to produce IFN-γ, which was measured using an enzyme-linked immunosorbent assay, in the culture supernatant, indicating that treating DF1/chIL-12 cells with DMSO or producing chIL-12 in a fusion protein form does not have adverse effects on the bioactivity of chIL-12. © 2015 American Institute of Chemical Engineers.

The 3' region of Human Papillomavirus type 16 early mRNAs decrease expression

DEFF Research Database (Denmark)

Vinther, J.; Rosenstierne, M.W.; Kristiansen, Karen

2005-01-01

Background: High risk human papillomavirus (HR-HPV) infects mucosal surfaces and HR-HPV infection is required for development of cervical cancer. Accordingly, enforced expression of the early HR-HPV proteins can induce immortalisation of human cells. In most cervical cancers and cervical cancer...... cell lines the HR-HPV double stranded DNA genome has been integrated into the host cell genome. Methods: We have used a retroviral GUS reporter system to generate pools of stably transfected HaCaT and SiHa cells. The HPV-16 early sequences that are deleted upon integration of the HPV-16 genome...

Cellular expression of gH confers resistance to herpes simplex virus type-1 entry

International Nuclear Information System (INIS)

Scanlan, Perry M.; Tiwari, Vaibhav; Bommireddy, Susmita; Shukla, Deepak

2003-01-01

Entry of herpes simplex virus-1 (HSV-1) into cells requires a concerted action of four viral glycoproteins gB, gD, and gH-gL. Previously, cell surface expression of gD had been shown to confer resistance to HSV-1 entry. To investigate any similar effects caused by other entry glycoproteins, gB and gH-gL were coexpressed with Nectin-1 in Chinese hamster ovary (CHO) cells. Interestingly, cellular expression of gB had no effect on HSV-1(KOS) entry. In contrast, entry was significantly reduced in cells expressing gH-gL. This effect was further analyzed by expressing gH and gL separately. Cells expressing gL were normally susceptible, whereas gH-expressing cells were significantly resistant. Further experiments suggested that the gH-mediated interference phenomenon was not specific to any particular gD receptor and was also observed in gH-expressing HeLa cells. Moreover, contrary to a previous report, gL-independent cell surface expression of gH was detected in stably transfected CHO cells, possibly implicating cell surface gH in the interference phenomenon. Thus, taken together these findings indicate that cellular expression of gH interferes with HSV-1 entry

Acute and Chronic Toxicity, Cytochrome P450 Enzyme Inhibition, and hERG Channel Blockade Studies with a Polyherbal, Ayurvedic Formulation for Inflammation

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Debendranath Dey

2015-01-01

Full Text Available Ayurvedic plants are known for thousands of years to have anti-inflammatory and antiarthritic effect. We have recently shown that BV-9238, a proprietary formulation of Withania somnifera, Boswellia serrata, Zingiber officinale, and Curcuma longa, inhibits LPS-induced TNF-alpha and nitric oxide production from mouse macrophage and reduces inflammation in different animal models. To evaluate the safety parameters of BV-9238, we conducted a cytotoxicity study in RAW 264.7 cells (0.005–1 mg/mL by MTT/formazan method, an acute single dose (2–10 g/kg bodyweight toxicity study and a 180-day chronic study with 1 g and 2 g/kg bodyweight in Sprague Dawley rats. Some sedation, ptosis, and ataxia were observed for first 15–20 min in very high acute doses and hence not used for further chronic studies. At the end of 180 days, gross and histopathology, blood cell counts, liver and renal functions were all at normal levels. Further, a modest attempt was made to assess the effects of BV-9238 (0.5 µg/mL on six major human cytochrome P450 enzymes and 3H radioligand binding assay with human hERG receptors. BV-9238 did not show any significant inhibition of these enzymes at the tested dose. All these suggest that BV-9238 has potential as a safe and well tolerated anti-inflammatory formulation for future use.

Bean Yellow Dwarf Virus replicons for high-level transgene expression in transgenic plants and cell cultures.

Science.gov (United States)

Zhang, Xiuren; Mason, Hugh

2006-02-05

A novel stable transgenic plant expression system was developed using elements of the replication machinery of Bean Yellow Dwarf Virus (BeYDV). The system contains two transgenes: 1) The BeYDV replicon vector with an expression cassette flanked by cis-acting DNA elements of BeYDV, and 2) The viral replication initiator protein (Rep) controlled by an alcohol-inducible promoter. When Rep expression was triggered by treatment with ethanol, it induced release of the BeYDV replicon from stably integrated T-DNA and episomal replication to high copy number. Replicon amplification resulted in substantially increased transgene mRNA levels (up to 80-fold) and translation products (up to 10-fold) after induction of Rep expression by ethanol treatment in tobacco NT1 cells and leaves of whole potato plants. Thus, the BeYDV stable transformant replicon system is a powerful tool for plant-based production of recombinant proteins. (c) 2005 Wiley Periodicals, Inc.

Vector Development for the Expression of Foreign Proteins in the Vaccine Strain Brucella abortus S19

Science.gov (United States)

Comerci, Diego J.; Pollevick, Guido D.; Vigliocco, Ana M.; Frasch, Alberto C. C.; Ugalde, Rodolfo A.

1998-01-01

A vector for the expression of foreign antigens in the vaccine strain Brucella abortus S19 was developed by using a DNA fragment containing the regulatory sequences and the signal peptide of the Brucella bcsp31 gene. This fragment was cloned in broad-host-range plasmid pBBR4MCS, resulting in plasmid pBEV. As a reporter protein, a repetitive antigen of Trypanosoma cruzi was used. The recombinant fusion protein is stably expressed and secreted into the Brucella periplasmic space, inducing a good antibody response against the T. cruzi antigen. The expression of the repetitive antigen in Brucella neither altered its growth pattern nor generated a toxic or lethal effect during experimental infection. The application of this strategy for the generation of live recombinant vaccines and the tagging of B. abortus S19 vaccine is discussed. This is the first time that a recombinant protein has been expressed in the periplasm of brucellae. PMID:9673273

Experimental investigation on a diode-pumped cesium-vapor laser stably operated at continuous-wave and pulse regime.

Science.gov (United States)

Chen, Fei; Xu, Dongdong; Gao, Fei; Zheng, Changbin; Zhang, Kuo; He, Yang; Wang, Chunrui; Guo, Jin

2015-05-04

Employing a fiber-coupled diode-laser with a center wavelength of 852.25 nm and a line width of 0.17 nm, experimental investigation on diode-end-pumped cesium (Cs) vapor laser stably operated at continuous-wave (CW) and pulse regime is carried out. A 5 mm long cesium vapor cell filled with 60 kPa helium and 20 kPa ethane is used as laser medium. Using an output coupler with reflectivity of 48.79%, 1.26 W 894.57 nm CW laser is obtained at an incident pump power of 4.76 W, corresponding an optical-optical efficiency of 26.8% and a slope-efficiency of 28.8%, respectively. The threshold temperature is 67.5 °C. Stable pulsed cesium laser with a maximum average output power of 2.6 W is obtained at a repetition rate of 76 Hz, and the pulse repetition rate can be extend to 1 kHz with a pulse width of 18 μs.

Selection of reference genes for expression studies with fish myogenic cell cultures

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Johnston Ian A

2009-08-01

Full Text Available Abstract Background Relatively few studies have used cell culture systems to investigate gene expression and the regulation of myogenesis in fish. To produce robust data from quantitative real-time PCR mRNA levels need to be normalised using internal reference genes which have stable expression across all experimental samples. We have investigated the expression of eight candidate genes to identify suitable reference genes for use in primary myogenic cell cultures from Atlantic salmon (Salmo salar L.. The software analysis packages geNorm, Normfinder and Best keeper were used to rank genes according to their stability across 42 samples during the course of myogenic differentiation. Results Initial results showed several of the candidate genes exhibited stable expression throughout myogenic culture while Sdha was identified as the least stable gene. Further analysis with geNorm, Normfinder and Bestkeeper identified Ef1α, Hprt1, Ppia and RNApolII as stably expressed. Comparison of data normalised with the geometric average obtained from combinations of any three of these genes showed no significant differences, indicating that any combination of these genes is valid. Conclusion The geometric average of any three of Hprt1, Ef1α, Ppia and RNApolII is suitable for normalisation of gene expression data in primary myogenic cultures from Atlantic salmon.

Selection of reference genes for expression studies with fish myogenic cell cultures.

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Bower, Neil I; Johnston, Ian A

2009-08-10

Relatively few studies have used cell culture systems to investigate gene expression and the regulation of myogenesis in fish. To produce robust data from quantitative real-time PCR mRNA levels need to be normalised using internal reference genes which have stable expression across all experimental samples. We have investigated the expression of eight candidate genes to identify suitable reference genes for use in primary myogenic cell cultures from Atlantic salmon (Salmo salar L.). The software analysis packages geNorm, Normfinder and Best keeper were used to rank genes according to their stability across 42 samples during the course of myogenic differentiation. Initial results showed several of the candidate genes exhibited stable expression throughout myogenic culture while Sdha was identified as the least stable gene. Further analysis with geNorm, Normfinder and Bestkeeper identified Ef1alpha, Hprt1, Ppia and RNApolII as stably expressed. Comparison of data normalised with the geometric average obtained from combinations of any three of these genes showed no significant differences, indicating that any combination of these genes is valid. The geometric average of any three of Hprt1, Ef1alpha, Ppia and RNApolII is suitable for normalisation of gene expression data in primary myogenic cultures from Atlantic salmon.

Human fatigue expression recognition through image-based dynamic multi-information and bimodal deep learning

Science.gov (United States)

Zhao, Lei; Wang, Zengcai; Wang, Xiaojin; Qi, Yazhou; Liu, Qing; Zhang, Guoxin

2016-09-01

Human fatigue is an important cause of traffic accidents. To improve the safety of transportation, we propose, in this paper, a framework for fatigue expression recognition using image-based facial dynamic multi-information and a bimodal deep neural network. First, the landmark of face region and the texture of eye region, which complement each other in fatigue expression recognition, are extracted from facial image sequences captured by a single camera. Then, two stacked autoencoder neural networks are trained for landmark and texture, respectively. Finally, the two trained neural networks are combined by learning a joint layer on top of them to construct a bimodal deep neural network. The model can be used to extract a unified representation that fuses landmark and texture modalities together and classify fatigue expressions accurately. The proposed system is tested on a human fatigue dataset obtained from an actual driving environment. The experimental results demonstrate that the proposed method performs stably and robustly, and that the average accuracy achieves 96.2%.

Gating mechanism of Kv11.1 (hERG) K+ channels without covalent connection between voltage sensor and pore domains.

Science.gov (United States)

de la Peña, Pilar; Domínguez, Pedro; Barros, Francisco

2018-03-01

Kv11.1 (hERG, KCNH2) is a voltage-gated potassium channel crucial in setting the cardiac rhythm and the electrical behaviour of several non-cardiac cell types. Voltage-dependent gating of Kv11.1 can be reconstructed from non-covalently linked voltage sensing and pore modules (split channels), challenging classical views of voltage-dependent channel activation based on a S4-S5 linker acting as a rigid mechanical lever to open the gate. Progressive displacement of the split position from the end to the beginning of the S4-S5 linker induces an increasing negative shift in activation voltage dependence, a reduced z g value and a more negative ΔG 0 for current activation, an almost complete abolition of the activation time course sigmoid shape and a slowing of the voltage-dependent deactivation. Channels disconnected at the S4-S5 linker near the S4 helix show a destabilization of the closed state(s). Furthermore, the isochronal ion current mode shift magnitude is clearly reduced in the different splits. Interestingly, the progressive modifications of voltage dependence activation gating by changing the split position are accompanied by a shift in the voltage-dependent availability to a methanethiosulfonate reagent of a Cys introduced at the upper S4 helix. Our data demonstrate for the first time that alterations in the covalent connection between the voltage sensor and the pore domains impact on the structural reorganizations of the voltage sensor domain. Also, they support the hypothesis that the S4-S5 linker integrates signals coming from other cytoplasmic domains that constitute either an important component or a crucial regulator of the gating machinery in Kv11.1 and other KCNH channels.

Rehabilitating drug-induced long-QT promoters: in-silico design of hERG-neutral cisapride analogues with retained pharmacological activity.

Science.gov (United States)

Durdagi, Serdar; Randall, Trevor; Duff, Henry J; Chamberlin, Adam; Noskov, Sergei Y

2014-03-08

The human ether-a-go-go related gene 1 (hERG1), which codes for a potassium ion channel, is a key element in the cardiac delayed rectified potassium current, IKr, and plays an important role in the normal repolarization of the heart's action potential. Many approved drugs have been withdrawn from the market due to their prolongation of the QT interval. Most of these drugs have high potencies for their principal targets and are often irreplaceable, thus "rehabilitation" studies for decreasing their high hERG1 blocking affinities, while keeping them active at the binding sites of their targets, have been proposed to enable these drugs to re-enter the market. In this proof-of-principle study, we focus on cisapride, a gastroprokinetic agent withdrawn from the market due to its high hERG1 blocking affinity. Here we tested an a priori strategy to predict a compound's cardiotoxicity using de novo drug design with molecular docking and Molecular Dynamics (MD) simulations to generate a strategy for the rehabilitation of cisapride. We focused on two key receptors, a target interaction with the (adenosine) receptor and an off-target interaction with hERG1 channels. An analysis of the fragment interactions of cisapride at human A2A adenosine receptors and hERG1 central cavities helped us to identify the key chemical groups responsible for the drug activity and hERG1 blockade. A set of cisapride derivatives with reduced cardiotoxicity was then proposed using an in-silico two-tier approach. This set was compared against a large dataset of commercially available cisapride analogs and derivatives. An interaction decomposition of cisapride and cisapride derivatives allowed for the identification of key active scaffolds and functional groups that may be responsible for the unwanted blockade of hERG1.

Measurements of density profile evolution during the stably-stratified filling of an open enclosure

International Nuclear Information System (INIS)

Tarawneh, Constantine M.; Homan, K.O.

2008-01-01

The stably-stratified filling of an open enclosure produces an interfacial gradient layer which is transported through the enclosure with the bulk flow. The evolution of this interfacial layer is strongly time-dependent and is driven by the nature of the interaction between the internal gravity waves and the inlet-driven interfacial shear. Measurements of density profile evolution have been completed for a rectangular enclosure with a single corner inlet and density variation produced by saline concentration. This system serves as a mass transfer analog to large-scale, thermally-stratified energy storage devices, preserving dynamic similitude in a laboratory-scale system. The experiments covered jet Reynolds numbers of 200-2200 and Froude numbers of 0.06-0.6 in an enclosure with a width 23 times the jet inlet height. The density profiles are seen to be strongly asymmetric and exhibit growth rates significantly different than due to simple one-dimensional molecular diffusion. In addition, shadowgraph and hydrogen bubble visualizations of the density and velocity fields in the gradient layer show the persistence of complex multi-dimensional flow structure even at relatively late stages of the filling process when the gradient layer has been transported well away from the enclosure inlet. The evolution of the vertical density profile has been compared quantitatively to a quasi one-dimensional model based upon empirical diffusivity coefficients

Pildiseiklusi maal ja merel / Neeme Korv

Index Scriptorium Estoniae

Korv, Neeme, 1974-

2010-01-01

Arvustus: Hergé, pseud. Kuldsõrgadega krabi / [prantsuse keelest tõlkinud Katrin Kaarep. Tallinn] : Tänapäev, 2010. (Tintini seiklused) ; Hergé, pseud. Must last / [prantsuse keelest tõlkinud Katrin Kaarep. Tallinn] : Tänapäev, 2010. (Tintini seiklused)

Suppression of the lipopolysaccharide-induced expression of MARCKS-related protein (MRP) affects transmigration in activated RAW264.7 cells.

Science.gov (United States)

Chun, Kwang-Rok; Bae, Eun Mi; Kim, Jae-Kwan; Suk, Kyoungho; Lee, Won-Ha

2009-01-01

The molecular action mechanism of MRP, one of the protein kinase C (PKC) substrates, has been under intense investigation, but reports on its role in macrophage function remain controversial. The treatment of macrophage cell lines with bacterial lipopolysaccharide (LPS) induced a high level of MRP expression suggesting that MRP plays a role in the function of activated macrophages. In order to investigate the role of MRP in activated RAW264.7 cells, we stably transfected MRP-specific shRNA expression constructs and tested for alterations in macrophage-related functions. The down-regulation of MRP expression resulted in a marked reduction in chemotaxis toward MCP-1 or extracellular matrix proteins. Furthermore, pharmacological inhibitors of PKC significantly inhibited the chemotaxis in RAW264.7 cells. These data reveals the pivotal role of MRP in the transmigration of activated RAW264.7 cells.

Identification and validation of reference genes for quantification of target gene expression with quantitative real-time PCR for tall fescue under four abiotic stresses.

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Zhimin Yang

Full Text Available Tall fescue (Festuca arundinacea Schreb. is widely utilized as a major forage and turfgrass species in the temperate regions of the world and is a valuable plant material for studying molecular mechanisms of grass stress tolerance due to its superior drought and heat tolerance among cool-season species. Selection of suitable reference genes for quantification of target gene expression is important for the discovery of molecular mechanisms underlying improved growth traits and stress tolerance. The stability of nine potential reference genes (ACT, TUB, EF1a, GAPDH, SAND, CACS, F-box, PEPKR1 and TIP41 was evaluated using four programs, GeNorm, NormFinder, BestKeeper, and RefFinder. The combinations of SAND and TUB or TIP41 and TUB were most stably expressed in salt-treated roots or leaves. The combinations of GAPDH with TIP41 or TUB were stable in roots and leaves under drought stress. TIP41 and PEPKR1 exhibited stable expression in cold-treated roots, and the combination of F-box, TIP41 and TUB was also stable in cold-treated leaves. CACS and TUB were the two most stable reference genes in heat-stressed roots. TIP41 combined with TUB and ACT was stably expressed in heat-stressed leaves. Finally, quantitative real-time polymerase chain reaction (qRT-PCR assays of the target gene FaWRKY1 using the identified most stable reference genes confirmed the reliability of selected reference genes. The selection of suitable reference genes in tall fescue will allow for more accurate identification of stress-tolerance genes and molecular mechanisms conferring stress tolerance in this stress-tolerant species.

A dimer of the lymphoid protein RAG1 recognizes the recombination signal sequence and the complex stably incorporates the high mobility group protein HMG2.

Science.gov (United States)

Rodgers, K K; Villey, I J; Ptaszek, L; Corbett, E; Schatz, D G; Coleman, J E

1999-07-15

RAG1 and RAG2 are the two lymphoid-specific proteins required for the cleavage of DNA sequences known as the recombination signal sequences (RSSs) flanking V, D or J regions of the antigen-binding genes. Previous studies have shown that RAG1 alone is capable of binding to the RSS, whereas RAG2 only binds as a RAG1/RAG2 complex. We have expressed recombinant core RAG1 (amino acids 384-1008) in Escherichia coli and demonstrated catalytic activity when combined with RAG2. This protein was then used to determine its oligomeric forms and the dissociation constant of binding to the RSS. Electrophoretic mobility shift assays show that up to three oligomeric complexes of core RAG1 form with a single RSS. Core RAG1 was found to exist as a dimer both when free in solution and as the minimal species bound to the RSS. Competition assays show that RAG1 recognizes both the conserved nonamer and heptamer sequences of the RSS. Zinc analysis shows the core to contain two zinc ions. The purified RAG1 protein overexpressed in E.coli exhibited the expected cleavage activity when combined with RAG2 purified from transfected 293T cells. The high mobility group protein HMG2 is stably incorporated into the recombinant RAG1/RSS complex and can increase the affinity of RAG1 for the RSS in the absence of RAG2.

Strategies to reduce the risk of drug-induced QT interval prolongation: a pharmaceutical company perspective.

Science.gov (United States)

Pollard, C E; Valentin, J-P; Hammond, T G

2008-08-01

Drug-induced prolongation of the QT interval is having a significant impact on the ability of the pharmaceutical industry to develop new drugs. The development implications for a compound causing a significant effect in the 'Thorough QT/QTc Study' -- as defined in the clinical regulatory guidance (ICH E14) -- are substantial. In view of this, and the fact that QT interval prolongation is linked to direct inhibition of the hERG channel, in the early stages of drug discovery the focus is on testing for and screening out hERG activity. This has led to understanding of how to produce low potency hERG blockers whilst retaining desirable properties. Despite this, a number of factors mean that when an integrated risk assessment is generated towards the end of the discovery phase (by conducting at least an in vivo QT assessment) a QT interval prolongation risk is still often apparent; inhibition of hERG channel trafficking and partitioning into cardiac tissue are just two confounding factors. However, emerging information suggests that hERG safety margins have high predictive value and that when hERG and in vivo non-clinical data are combined, their predictive value to man, whilst not perfect, is >80%. Although understanding the anomalies is important and is being addressed, of greater importance is developing a better understanding of TdP, with the aim of being able to predict TdP rather than using an imperfect surrogate marker (QT interval prolongation). Without an understanding of how to predict TdP risk, high-benefit drugs for serious indications may never be marketed.

Real-time quantitative reverse transcription-PCR analysis of expression stability of Actinobacillus pleuropneumoniae housekeeping genes during in vitro growth under iron-depleted conditions

DEFF Research Database (Denmark)

Nielsen, K. K.; Boye, Mette

2005-01-01

up-regulation under iron-restricted conditions compared to bacteria grown in medium with sufficient iron. The observed expression patterns of the genes of interest were consistent with previous observations. This study therefore lends further support to the use of real-time quantitative RT...... controls, as such controls have not been defined yet for this bacterium. Bacterial gene expression was studied during in vitro exponential and early stationary growth in medium with and without sufficient iron, respectively. First, the stability of expression of five genes, the glyA, tpiA, pykA, rec......F, and rhoAP genes involved in basic housekeeping, was evaluated on the basis of the mean pairwise variation. All the housekeeping genes included were stably expressed under the conditions investigated and consequently were included in the normalization procedure. Next, the geometric mean of the internal...

Clone and expression of human transferrin receptor gene: a marker gene for magnetic resonance imaging

International Nuclear Information System (INIS)

Li Li; Liu Lizhi; Lv Yanchun; Liu Xuewen; Cui Chunyan; Wu Peihong; Liu Qicai; Ou Shanxing

2007-01-01

Objective: To clone human transferrin receptor (hTfR) gene and construct expression vector producing recombination protein. Methods: Human transferrin receptor gene cDNA was amplified by RT-PCR from human embryonic liver and lung tissue. Recombinant pcDNA3-hTfR and pEGFP-Cl-hTfR plasmids were constructed and confirmed by DNA sequencing. These plasmids were stably transfected into the HEK293 cells. The protein expression in vitro was confirmed by Western Blot. The efficiency of expression and the location of hTfR were also investigated by fluorescence microscopy and confocal fluorescence microscopy. Results: The full length cDNA of hTfR gene (2332 bp) was cloned and sequenced. The hTfR (190 000) was overexpressed in transfected HEK293 cells by Western blot analysis. Fluorescence micrographs displayed that the hTfR was expressed at high level and located predominantly in the cell surface. Conclusions: Human transferrin receptor (hTfR) gene has been successfully cloned and obtained high-level expression in HEK293 cells, and the recombination protein of hTfR distributed predominantly in the cell membrane. (authors)

A recombinant canine distemper virus expressing a modified rabies virus glycoprotein induces immune responses in mice.

Science.gov (United States)

Li, Zhili; Wang, Jigui; Yuan, Daoli; Wang, Shuang; Sun, Jiazeng; Yi, Bao; Hou, Qiang; Mao, Yaping; Liu, Weiquan

2015-06-01

Canine distemper virus (CDV) and rabies virus (RV) are two important pathogens of the dog. CDV, a member of the morbillivirus genus, has shown promise as an expression vector. The glycoprotein from RV is a main contributor to protective immunity and capable of eliciting the production of virus-neutralizing antibodies. In this study, we recovered an attenuated strain of canine distemper virus and constructed a recombinant virus, rCDV-RV-G, expressing a modified (R333Q) rabies virus glycoprotein (RV-G) of RV Flury strain LEP. RV-G expression by the recombinant viruses was confirmed. Furthermore, G was proved to be incorporated into the surface of CDV particles. While replication of the recombinant virus was slightly reduced compared with the parental CDV, it stably expressed the RV-G over ten serial passages. Inoculation of mice induced specific neutralizing antibodies against both RV-G and CDV. Therefore, the rCDV-RV-G has the potential as a vaccine that may be used to control rabies virus infection in dogs and other animals.

Selection of reliable reference genes for gene expression studies in Trichoderma afroharzianum LTR-2 under oxalic acid stress.

Science.gov (United States)

Lyu, Yuping; Wu, Xiaoqing; Ren, He; Zhou, Fangyuan; Zhou, Hongzi; Zhang, Xinjian; Yang, Hetong

2017-10-01

An appropriate reference gene is required to get reliable results from gene expression analysis by quantitative real-time reverse transcription PCR (qRT-PCR). In order to identify stable and reliable reference genes in Trichoderma afroharzianum under oxalic acid (OA) stress, six commonly used housekeeping genes, i.e., elongation factor 1, ubiquitin, ubiquitin-conjugating enzyme, glyceraldehyde-3-phosphate dehydrogenase, α-tubulin, actin, from the effective biocontrol isolate T. afroharzianum strain LTR-2 were tested for their expression during growth in liquid culture amended with OA. Four in silico programs (comparative ΔCt, NormFinder, geNorm and BestKeeper) were used to evaluate the expression stabilities of six candidate reference genes. The elongation factor 1 gene EF-1 was identified as the most stably expressed reference gene, and was used as the normalizer to quantify the expression level of the oxalate decarboxylase coding gene OXDC in T. afroharzianum strain LTR-2 under OA stress. The result showed that the expression of OXDC was significantly up-regulated as expected. This study provides an effective method to quantify expression changes of target genes in T. afroharzianum under OA stress. Copyright © 2017 Elsevier B.V. All rights reserved.

Heterologous expression in Tritrichomonas foetus of functional Trichomonas vaginalis AP65 adhesin

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Alderete JF

2005-03-01

Full Text Available Abstract Background Trichomonosis, caused by Trichomonas vaginalis, is the number one, nonviral sexually transmitted infection that has adverse consequences for the health of women and children. The interaction of T. vaginalis with vaginal epithelial cells (VECs, a step preparatory to infection, is mediated in part by the prominent surface protein AP65. The bovine trichomonad, Tritrichomonas foetus, adheres poorly to human VECs. Thus, we established a transfection system for heterologous expression of the T. vaginalis AP65 in T. foetus, as an alternative approach to confirm adhesin function for this virulence factor. Results In this study, we show stable transfection and expression of the T. vaginalis ap65 gene in T. foetus from an episomal pBS-ap65-neo plasmid. Expression of the gene and protein was confirmed by RT-PCR and immunoblots, respectively. AP65 in transformed T. foetus bound to host cells. Specific mAbs revealed episomally-expressed AP65 targeted to the parasite surface and hydrogenosome organelles. Importantly, surface-expression of AP65 in T. foetus paralleled increased levels of adherence of transfected bovine trichomonads to human VECs. Conclusion The T. vaginalis AP65 adhesin was stably expressed in T. foetus, and the data obtained using this heterologous system strongly supports the role of AP65 as a prominent adhesin for T. vaginalis. In addition, the heterologous expression in T. foetus of a T. vaginalis gene offers an important, new approach for confirming and characterizing virulence factors.

Regulation and expression of Lcr plasmid-mediated peptides in pesticinogenic Yersinia pestis

International Nuclear Information System (INIS)

Sample, A.K.

1987-01-01

It is shown in this thesis that cells of Lcr + , Pst - Y. pestis KIM are able to express Yops at levels comparable to that of Lcr + Yersinia pseudotuberculosis. Pulse-chase radiolabeling with 35 S-methionine was used to demonstrate that Lcr + , Pst + Y. pestis synthesized at least 11 distinct peptides during the low calcium response and that seven of the labeled peptides were rapidly degraded. These seven peptides were stably expressed in Lcr + , Pst - Y. pestis and were of identical molecular weights as the Yops expressed by that strain. Radiolabeled fragments of low molecular weight accumulated in the extracellular medium of Pst + cultures and were assumed to be stable degradation fragments derived from Yops. It was also shown that the set of stable peptides, including V antigen, were made during restriction by both Pst + and Pst - Y. pestis KIM and were located primarily within the cytoplasm. Those radiolabeled peptides which underwent proteolytic degradation in Pst + Y. pestis were localized to the outer membrane and extracellular medium in the Pst - strain. It is concluded that the failure of Lcr + , Pst + Y. pestis to express Yops is the result of post-translational degradation and is not a block in the synthesis of Yops

Characterization of a new Lactobacillus salivarius strain engineered to express IBV multi-epitope antigens by chromosomal integration.

Science.gov (United States)

Ma, Bing-cun; Yang, Xin; Wang, Hong-ning; Cao, Hai-peng; Xu, Peng-wei; Ding, Meng-die; Liu, Hui

2016-01-01

To obtain adhesive and safe lactic acid bacteria (LAB) strains for expressing heterologous antigens, we screened LAB inhabitants in intestine of Tibetan chickens by analyzing their adhesion and safety properties and the selected LAB was engineered to express heterologous antigen (UTEpi C-A) based on chromosomal integration strategy. We demonstrated that a new Lactobacillu salivarius TCMM17 strain is strongly adhesive to chicken intestinal epithelial cells, contains no endogenous plasmids, is susceptible to tested antimicrobials, and shows no toxicities. In order to examine the potential of TCMM17 strain as heterogenous antigen delivering vehicle, we introduced a UTEpi C-A expression cassette in its chromosome by constructing a non-replicative plasmid (pORI280-UUTEpi C-AD). The recombinant TCMM17 strain (∆TCMM17) stably was found to keep the gene cassette through 50 generations, and successfully displayed EpiC encoded by the cassette on its surface. This work provides a universal platform for development of novel oral vaccines and expression of further antigens of avian pathogens.

Ha-ras oncogene expression directed by a milk protein gene promoter: tissue specificity, hormonal regulation, and tumor induction in transgenic mice

International Nuclear Information System (INIS)

Andres, A.C.; Schoenenberger, C.A.; Groner, B.; Henninghausen, L.; LeMeur, M.; Gelinger, P.

1987-01-01

The activated human Ha-ras oncogene was subjected to the control of the promoter region of the murine whey acidic protein (Wap) gene, which is expressed in mammary epithelial cells in response to lactogenic hormones. The Wap-ras gene was stably introduced into the mouse germ line of five transgenic mice (one male and four females). Wap-ras expression was observed in the mammary glands of lactating females in two lines derived from female founders. The tissue-directed and hormone-dependent Wap expression was conferred on the Ha-ras oncogene. The signals governing Wap expression are located within 2.5 kilobases of 5' flanking sequence. The other two lines derived from female founders did not express the chimeric gene. In the line derived from the male founder the Wap-ras gene is integrated into the Y chromosome. Expression was found in the salivary gland of male animals only. After a long latency, Wap-ras-expressing mice developed tumors. The tumors arose in tissues expressing Wap-ras - i.e., mammary or salivary glands. Compared to the corresponding nonmalignant tissues, Wap-ras expression was enhanced in the tumors

Characterization of cell lines stably transfected with rubella virus replicons

International Nuclear Information System (INIS)

Tzeng, Wen-Pin; Xu, Jie; Frey, Teryl K.

2012-01-01

Rubella virus (RUBV) replicons expressing a drug resistance gene and a gene of interest were used to select cell lines uniformly harboring the replicon. Replicons expressing GFP and a virus capsid protein GFP fusion (C-GFP) were compared. Vero or BHK cells transfected with either replicon survived drug selection and grew into a monolayer. However, survival was ∼9-fold greater following transfection with the C-GFP-replicon than with the GFP-expressing replicon and while the C-GFP-replicon cells grew similarly to non-transfected cells, the GFP-replicon cells grew slower. Neither was due to the ability of the CP to enhance RNA synthesis but survival during drug selection was correlated with the ability of CP to inhibit apoptosis. Additionally, C-GFP-replicon cells were not cured of the replicon in the absence of drug selection. Interferon-alpha suppressed replicon RNA and protein synthesis, but did not cure the cells, explaining in part the ability of RUBV to establish persistent infections.

Characterization of cell lines stably transfected with rubella virus replicons

Energy Technology Data Exchange (ETDEWEB)

Tzeng, Wen-Pin; Xu, Jie [Department of Biology, Georgia State University, P.O. Box 4010, Atlanta GA 30302-4010 (United States); Frey, Teryl K., E-mail: tfrey@gsu.edu [Department of Biology, Georgia State University, P.O. Box 4010, Atlanta GA 30302-4010 (United States)

2012-07-20

Rubella virus (RUBV) replicons expressing a drug resistance gene and a gene of interest were used to select cell lines uniformly harboring the replicon. Replicons expressing GFP and a virus capsid protein GFP fusion (C-GFP) were compared. Vero or BHK cells transfected with either replicon survived drug selection and grew into a monolayer. However, survival was {approx}9-fold greater following transfection with the C-GFP-replicon than with the GFP-expressing replicon and while the C-GFP-replicon cells grew similarly to non-transfected cells, the GFP-replicon cells grew slower. Neither was due to the ability of the CP to enhance RNA synthesis but survival during drug selection was correlated with the ability of CP to inhibit apoptosis. Additionally, C-GFP-replicon cells were not cured of the replicon in the absence of drug selection. Interferon-alpha suppressed replicon RNA and protein synthesis, but did not cure the cells, explaining in part the ability of RUBV to establish persistent infections.

A Case Study of Offshore Advection of Boundary Layer Rolls over a Stably Stratified Sea Surface

Directory of Open Access Journals (Sweden)

Nina Svensson

2017-01-01

Full Text Available Streaky structures of narrow (8-9 km high wind belts have been observed from SAR images above the Baltic Sea during stably stratified conditions with offshore winds from the southern parts of Sweden. Case studies using the WRF model and in situ aircraft observations indicate that the streaks originate from boundary layer rolls generated over the convective air above Swedish mainland, also supported by visual satellite images showing the typical signature cloud streets. The simulations indicate that the rolls are advected and maintained at least 30–80 km off the coast, in agreement with the streaks observed by the SAR images. During evening when the convective conditions over land diminish, the streaky structures over the sea are still seen in the horizontal wind field; however, the vertical component is close to zero. Thus advected feature from a land surface can affect the wind field considerably for long times and over large areas in coastal regions. Although boundary layer rolls are a well-studied feature, no previous study has presented results concerning their persistence during situations with advection to a strongly stratified boundary layer. Such conditions are commonly encountered during spring in coastal regions at high latitudes.

[Construction of the eukaryotic recombinant vector and expression of the outer membrane protein LipL32 gene from Leptospira serovar Lai].

Science.gov (United States)

Huang, Bi; Bao, Lang; Zhong, Qi; Shang, Zheng-ling; Zhang, Hui-dong; Zhang, Ying

2008-02-01

To construct the eukaryotic experssion vector of LipL32 gene from Leptospira serovar Lai and express the recombinant plasmid in COS-7 cell. The LipL32 gene was amplified from Leptospira strain 017 genomic DNA by PCR and cloned into pcDNA3.1, through restriction nuclease enzyme digestion. Then the recombinant plasmid was transformed into E.coli DH5alpha. After identified by nuclease digestion, PCR and sequencing analysis, the recombinant vector was transfected into COS-7 cell with lipsome. The expression of the target gene was detected by RT-PCR and Western blot. The eukaryotic experssion vector pcDNA3.1-LipL32 was successfully constructed and stably expressed in COS-7 cell. The eukaryotic recombinant vector of outer membrane protein LipL32 gene from Leptospira serovar Lai can be expressed in mammalian cell, which provides an experimental basis for the application of the Leptospira DNA vaccine.

Noncytotoxic orange and red/green derivatives of DsRed-Express2 for whole-cell labeling

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Glick Benjamin S

2009-04-01

Full Text Available Abstract Background Whole-cell labeling is a common application of fluorescent proteins (FPs, but many red and orange FPs exhibit cytotoxicity that limits their use as whole-cell labels. Recently, a tetrameric red FP called DsRed-Express2 was engineered for enhanced solubility and was shown to be noncytotoxic in bacterial and mammalian cells. Our goal was to create derivatives of this protein with different spectral properties. Results Building on previous studies of DsRed mutants, we created two DsRed-Express2 derivatives: E2-Orange, an orange FP, and E2-Red/Green, a dual-color FP with both red and green emission. We show that these new FPs retain the low cytotoxicity of DsRed-Express2. In addition, we show that these new FPs are useful as second or third colors for flow cytometry and fluorescence microscopy. Conclusion E2-Orange and E2-Red/Green will facilitate the production of healthy, stably fluorescent cell lines and transgenic organisms for multi-color labeling studies.

Endogenous and ectopic expression of telomere regulating genes in chicken embryonic fibroblasts

International Nuclear Information System (INIS)

Michailidis, Georgios; Saretzki, Gabriele; Hall, Judith

2005-01-01

In this study, we compared the endogenous expression of genes encoding telomere regulating proteins in cultured chicken embryonic fibroblasts (CEFs) and 10-day-old chicken embryos. CEFs maintained in vitro senesced and senescence was accompanied by reduced telomere length, telomerase activity, and expression of the chicken (c) TRF1 gene. There was no change in TRF2 gene expression although the major TRF2 transcript identified in 10-day-old chicken embryos encoded a truncated TRF2 protein (TRF2'), containing an N-terminal dimerisation domain but lacking a myb-related DNA binding domain and nuclear localisation signal. Senescence of the CEFs in vitro was associated with the loss of the TRF2' transcript, indicative of a novel function for the encoded protein. Senescence was also coupled with decreased expression of RAD51, but increased RAD52 expression. These data support that RAD51 independent recombination mechanisms do not function in vitro to maintain chicken telomeres. To attempt to rescue the CEFs from replicative senescence, we stably transfected passage 3 CEFs with the human telomerase reverse transcriptase (hTERT) catalytic subunit. While hTERT expression was detected in the stable transfectants neither telomerase activity nor the stabilisation of telomere length was observed, and the transfectant cells senesced at the same passage number as the untransfected cells. These data indicate that the human TERT is incompatible with the avian telomere maintenance apparatus and suggest the functioning of a species specific telomere system in the avian

Recovery of NIS expression in thyroid cancer cells by overexpression of Pax8 gene

International Nuclear Information System (INIS)

Presta, Ivan; Filetti, Sebastiano; Russo, Diego; Arturi, Franco; Ferretti, Elisabetta; Mattei, Tiziana; Scarpelli, Daniela; Tosi, Emanuele; Scipioni, Angela; Celano, Marilena; Gulino, Alberto

2005-01-01

Recovery of iodide uptake in thyroid cancer cells by means of obtaining the functional expression of the sodium/iodide symporter (NIS) represents an innovative strategy for the treatment of poorly differentiated thyroid cancer. However, the NIS gene expression alone is not always sufficient to restore radioiodine concentration ability in these tumour cells. In this study, the anaplastic thyroid carcinoma ARO cells were stably transfected with a Pax8 gene expression vector. A quantitative RT-PCR was performed to assess the thyroid specific gene expression in selected clones. The presence of NIS protein was detected by Western blot and localized by immunofluorescence. A iodide uptake assay was also performed to verify the functional effect of NIS induction and differentiation switch. The clones overexpressing Pax8 showed the re-activation of several thyroid specific genes including NIS, Pendrin, Thyroglobulin, TPO and TTF1. In ARO-Pax8 clones NIS protein was also localized both in cell cytoplasm and membrane. Thus, the ability to uptake the radioiodine was partially restored, associated to a high rate of efflux. In addition, ARO cells expressing Pax8 presented a lower rate of cell growth. These finding demonstrate that induction of Pax8 expression may determine a re-differentiation of thyroid cancer cells, including a partial recovery of iodide uptake, fundamental requisite for a radioiodine-based therapeutic approach for thyroid tumours

Validation of visualized transgenic zebrafish as a high throughput model to assay bradycardia related cardio toxicity risk candidates.

Science.gov (United States)

Wen, Dingsheng; Liu, Aiming; Chen, Feng; Yang, Julin; Dai, Renke

2012-10-01

Drug-induced QT prolongation usually leads to torsade de pointes (TdP), thus for drugs in the early phase of development this risk should be evaluated. In the present study, we demonstrated a visualized transgenic zebrafish as an in vivo high-throughput model to assay the risk of drug-induced QT prolongation. Zebrafish larvae 48 h post-fertilization expressing green fluorescent protein in myocardium were incubated with compounds reported to induce QT prolongation or block the human ether-a-go-go-related gene (hERG) K⁺ current. The compounds sotalol, indapaminde, erythromycin, ofoxacin, levofloxacin, sparfloxacin and roxithromycin were additionally administrated by microinjection into the larvae yolk sac. The ventricle heart rate was recorded using the automatic monitoring system after incubation or microinjection. As a result, 14 out of 16 compounds inducing dog QT prolongation caused bradycardia in zebrafish. A similar result was observed with 21 out of 26 compounds which block hERG current. Among the 30 compounds which induced human QT prolongation, 25 caused bradycardia in this model. Thus, the risk of compounds causing bradycardia in this transgenic zebrafish correlated with that causing QT prolongation and hERG K⁺ current blockage in established models. The tendency that high logP values lead to high risk of QT prolongation in this model was indicated, and non-sensitivity of this model to antibacterial agents was revealed. These data suggest application of this transgenic zebrafish as a high-throughput model to screen QT prolongation-related cardio toxicity of the drug candidates. Copyright © 2012 John Wiley & Sons, Ltd.

Expression of human oxoguanine glycosylase 1 or formamidopyrimidine glycosylase in human embryonic kidney 293 cells exacerbates methylmercury toxicity in vitro

Energy Technology Data Exchange (ETDEWEB)

Ondovcik, Stephanie L.; Preston, Thomas J.; McCallum, Gordon P. [Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario M5S 3M2 (Canada); Wells, Peter G., E-mail: pg.wells@utoronto.ca [Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario M5S 3M2 (Canada); Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario M5S 1A8 (Canada)

2013-08-15

Exposure to methylmercury (MeHg) acutely at high levels, or via chronic low-level dietary exposure from daily fish consumption, can lead to adverse neurological effects in both the adult and developing conceptus. To determine the impact of variable DNA repair capacity, and the role of reactive oxygen species (ROS) and oxidatively damaged DNA in the mechanism of toxicity, transgenic human embryonic kidney (HEK) 293 cells that stably express either human oxoguanine glycosylase 1 (hOgg1) or its bacterial homolog, formamidopyrimidine glycosylase (Fpg), which primarily repair the oxidative lesion 8-oxo-2′-deoxyguanosine (8-oxodG), were used to assess the in vitro effects of MeHg. Western blotting confirmed the expression of hOgg1 or Fpg in both the nuclear and mitochondrial compartments of their respective cell lines. Following acute (1–2 h) incubations with 0–10 μM MeHg, concentration-dependent decreases in clonogenic survival and cell growth accompanied concentration-dependent increases in lactate dehydrogenase (LDH) release, ROS formation, 8-oxodG levels and apurinic/apyrimidinic (AP) sites, consistent with the onset of cytotoxicity. Paradoxically, hOgg1- and Fpg-expressing HEK 293 cells were more sensitive than wild-type cells stably transfected with the empty vector control to MeHg across all cellular and biochemical parameters, exhibiting reduced clonogenic survival and cell growth, and increased LDH release and DNA damage. Accordingly, upregulation of specific components of the base excision repair (BER) pathway may prove deleterious potentially due to the absence of compensatory enhancement of downstream processes to repair toxic intermediary abasic sites. Thus, interindividual variability in DNA repair activity may constitute an important risk factor for environmentally-initiated, oxidatively damaged DNA and its pathological consequences. - Highlights: • hOgg1 and Fpg repair oxidatively damaged DNA. • hOgg1- and Fpg-expressing cells are more

Manufacture of clinical-grade CD19-specific T cells stably expressing chimeric antigen receptor using Sleeping Beauty system and artificial antigen presenting cells.

Directory of Open Access Journals (Sweden)

Harjeet Singh

Full Text Available Adoptive transfer of T cells expressing a CD19-specific chimeric antigen receptor (CAR is being evaluated in multiple clinical trials. Our current approach to adoptive immunotherapy is based on a second generation CAR (designated CD19RCD28 that signals through a CD28 and CD3-ζ endodomain. T cells are electroporated with DNA plasmids from the Sleeping Beauty (SB transposon/transposase system to express this CAR. Stable integrants of genetically modified T cells can then be retrieved when co-cultured with designer artificial antigen presenting cells (aAPC in the presence of interleukin (IL-2 and 21. Here, we reveal how the platform technologies of SB-mediated transposition and CAR-dependent propagation on aAPC were adapted for human application. Indeed, we have initiated clinical trials in patients with high-risk B-lineage malignancies undergoing autologous and allogeneic hematopoietic stem-cell transplantation (HSCT. We describe the process to manufacture clinical grade CD19-specific T cells derived from healthy donors. Three validation runs were completed in compliance with current good manufacturing practice for Phase I/II trials demonstrating that by 28 days of co-culture on γ-irradiated aAPC ∼10(10 T cells were produced of which >95% expressed CAR. These genetically modified and propagated T cells met all quality control testing and release criteria in support of infusion.

Susceptibility to virus-cell fusion at the plasma membrane is reduced through expression of HIV gp41 cytoplasmic domains

International Nuclear Information System (INIS)

Malinowsky, Katharina; Luksza, Julia; Dittmar, Matthias T.

2008-01-01

The cytoplasmic tail of the HIV transmembrane protein plays an important role in viral infection. In this study we analyzed the role of retroviral cytoplasmic tails in modulating the cytoskeleton and interfering with virus-cell fusion. HeLaP4 cells expressing different HIV cytoplasmic tail constructs showed reduced acetylated tubulin levels whereas the cytoplasmic tail of MLV did not alter microtubule stability indicating a unique function for the lentiviral cytoplasmic tail. The effect on tubulin is mediated through the membrane proximal region of the HIV cytoplasmic tail and was independent of membrane localization. Site-directed mutagenesis identified three motifs in the HIV-2 cytoplasmic tail required to effect the reduction in acetylated tubulin. Both the YxxΦ domain and amino acids 21 to 45 of the HIV-2 cytoplasmic tail need to be present to change the level of acetylated tubulin in transfected cells. T-cells stably expressing one HIV-2 cytoplasmic tail derived construct showed also a reduction in acetylated tubulin thus confirming the importance of this effect not only for HeLaP4 and 293T cells. Challenge experiments using transiently transfected HeLaP4 cells and T cells stably expressing an HIV cytoplasmic tail construct revealed both reduced virus-cell fusion and replication of HIV-1 NL4.3 compared to control cells. In the virus-cell fusion assay only virions pseudotyped with either HIV or MLV envelopes showed reduced fusion efficiency, whereas VSV-G pseudotyped virions where not affected by the expression of HIV derived cytoplasmic tail constructs, indicating that fusion at the plasma but not endosomal membrane is affected. Overexpression of human histone-deacetylase 6 (HDAC6) and constitutively active RhoA resulted in a reduction of acetylated tubulin and reduced virus-cell fusion as significant as that observed following expression of HIV cytoplasmic tail constructs. Inhibition of HDAC6 showed a strong increase in acetylated tubulin and increase of

Root-specific expression of opine genes and opine accumulation in some cultivars of the naturally occurring genetically modified organism Nicotiana tabacum.

Science.gov (United States)

Chen, Ke; de Borne, François Dorlhac; Julio, Emilie; Obszynski, Julie; Pale, Patrick; Otten, Léon

2016-08-01

Previous studies have shown that Nicotiana tabacum contains three Agrobacterium-derived T-DNA sequences inherited from its paternal ancestor Nicotiana tomentosiformis. Among these, the TB locus carries an intact mannopine synthase 2' gene (TB-mas2'). This gene is similar to the Agrobacterium rhizogenes A4-mas2' gene that encodes the synthesis of the Amadori compound deoxyfructosyl-glutamine (DFG or santhopine). In this study we show that TB-mas2' is expressed at very low levels in N. tomentosiformis and in most N. tabacum cultivars; however, some cultivars show high TB-mas2' expression levels. The TB-mas2' promoter sequences of low- and high-expressing cultivars are identical. The low/high level of expression segregates as a single Mendelian factor in a cross between a low- and a high-expression cultivar. pTB-mas2'-GUS and pA4-mas2'-GUS reporter genes were stably introduced in N. benthamiana. Both were mainly expressed in the root expansion zone and leaf vasculature. Roots of tobacco cultivars with high TB-mas2' expression contain detectable levels of DFG. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

The antagonistic effect of antipsychotic drugs on a HEK293 cell line stably expressing human alpha(1A1)-adrenoceptors

DEFF Research Database (Denmark)

Nourian, Zahra; Mulvany, Michael J; Nielsen, Karsten Bork

2008-01-01

challenged with phenylephrine (EC(50)=1.61x10(-8) M). From Schild analysis, prazosin, sertindole, risperidone, and haloperidol caused a concentration-dependent, rightward shift of the cumulative concentration-response curves for phenylephrine in cells expressing human recombinant alpha(1A1)-adrenoceptors...... human alpha(1A1)-adrenoceptors in competition binding studies confirmed much higher antagonist affinity of sertindole and risperidone than haloperidol for these receptors. In summary, it can be concluded that there is an approximately 10-fold higher adrenoceptor affinity of risperidone and sertindole...... for human alpha(1A1)-adrenoceptors compared to haloperidol. These findings are consistent with the observation that risperidone and sertindole have a higher incidence of orthostatic hypotension than haloperidol....

Expression of insulin-like growth factor-2 receptors on EL4 lymphoma cells overexpressing growth hormone.

Science.gov (United States)

Farmer, John T; Weigent, Douglas A

2007-01-01

In the present study, we report the upregulation of functional IGF-2Rs in cells overexpressing growth hormone (GH). EL4 lymphoma cells stably transfected with an rGH cDNA overexpression vector (GHo) exhibited an increase in the binding of (125)I-IGF-2 with no change in the binding affinity compared to vector alone controls. An increase in the expression of the insulin-like growth factor-2 receptor (IGF-2R) in cells overexpressing GH was confirmed by Western blot analysis and IGF-2R promoter luciferase assays. EL4 cells produce insulin-like growth factor-2 (IGF-2) as detected by the reverse transcription-polymerase chain reaction (RT-PCR); however, no IGF-2 protein was detected by Western analysis. The increase in the expression of the IGF-2R resulted in greater levels of IGF-2 uptake in GHo cells compared to vector alone controls. The data suggest that one of the consequences of the overexpression of GH is an increase in the expression of the IGF-2R.

Epstein-Barr virus nuclear antigen 2 specifically induces expression of the B-cell activation antigen CD23

International Nuclear Information System (INIS)

Wang, F.; Gregory, C.D.; Rowe, M.; Rickinson, A.B.; Wang, D.; Birkenbach, M.; Kikutani, H.; Kishimoto, T.; Kieff, E.

1987-01-01

Epstein-Barr virus (EBV) infection of EBV-negative Burkitt lymphoma (BL) cells includes some changes similar to those seen in normal B lymphocytes that have been growth transformed by EBV. The role of individual EBV genes in this process was evaluated by introducing each of the viral genes that are normally expressed in EBV growth-transformed and latently infected lymphoblasts into an EBV-negative BL cell line, using recombinant retrovirus-mediated transfer. Clones of cells were derived that stably express the EBV nuclear antigen 1 (EBNA-1), EBNA-2, EBNA-3, EBNA-leader protein, or EBV latent membrane protein (LMP). These were compared with control clones infected with the retrovirus vector. All 10 clones converted to EBNA-2 expression differed from control clones or clones expressing other EBV proteins by growth in tight clumps and by markedly increased expression of one particular surface marker of B-cell activation, CD23. Other activation antigens were unaffected by EBNA-2 expression, as were markers already expressed on the parent BL cell line. The results indicate that EBNA-2 is a specific direct or indirect trans-activator of CD23. This establishes a link between an EBV gene and cell gene expression. Since CD23 has been implicated in the transduction of B-cell growth signals, its specific induction by EBNA-2 could be important in EBV induction of B-lymphocyte transformation

NF45/ILF2 tissue expression, promoter analysis, and interleukin-2 transactivating function

International Nuclear Information System (INIS)

Zhao Guohua; Shi Lingfang; Qiu Daoming; Hu Hong; Kao, Peter N.

2005-01-01

NF45/ILF2 associates with NF90/ILF3 in the nucleus and regulates IL-2 gene transcription at the antigen receptor response element (ARRE)/NF-AT DNA target sequence (P.N. Kao, L. Chen, G. Brock, J. Ng, A.J. Smith, B. Corthesy, J. Biol. Chem. 269 (1994) 20691-20699). NF45 is widely expressed in normal tissues, especially testis, brain, and kidney, with a predominantly nuclear distribution. NF45 mRNA expression is increased in lymphoma and leukemia cell lines. The human and murine NF45 proteins differ only by substitution of valine by isoleucine at amino acid 142. Fluorescence in situ hybridization localized the human NF45 gene to chromosome 1q21.3, and mouse NF45 gene to chromosome 3F1. Promoter analysis of 2.5 kB of the murine NF45 gene reveals that significant activation is conferred by factors, possible including NF-Y, that bind to the CCAAT-box sequence. The function of human NF45 in regulating IL-2 gene expression was characterized in Jurkat T-cells stably transfected with plasmids directing expression of NF45 cDNA in sense or antisense orientations. NF45 sense expression increased IL-2 luciferase reporter gene activity 120-fold, and IL-2 protein expression 2-fold compared to control cells. NF45 is a highly conserved, regulated transcriptional activator, and one target gene is IL-2

Evaluation of nefazodone-induced cardiotoxicity in human induced pluripotent stem cell-derived cardiomyocytes

Energy Technology Data Exchange (ETDEWEB)

Lee, Sujeong, E-mail: crystalee@gmail.com [Next-generation Pharmaceutical Research Center, Korea Institute of Toxicology, Korea Research Institute of Chemical Technology, 141 Gajeong-ro, Yuseong-gu, Daejeon 305-343 (Korea, Republic of); Lee, Hyang-Ae, E-mail: hyangaelee@gmail.com [Next-generation Pharmaceutical Research Center, Korea Institute of Toxicology, Korea Research Institute of Chemical Technology, 141 Gajeong-ro, Yuseong-gu, Daejeon 305-343 (Korea, Republic of); Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, 110-799 (Korea, Republic of); Human and Environmental Toxicology Program, University of Science and Technology, 217 Gajeong-ro, Yuseong-gu, Daejeon 305-350 (Korea, Republic of); Choi, Sung Woo, E-mail: djmaya@snu.ac.kr [Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, 110-799 (Korea, Republic of); Kim, Sung Joon, E-mail: sjoonkim@snu.ac.kr [Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, 110-799 (Korea, Republic of); Kim, Ki-Suk, E-mail: idkks00@gmail.com [Next-generation Pharmaceutical Research Center, Korea Institute of Toxicology, Korea Research Institute of Chemical Technology, 141 Gajeong-ro, Yuseong-gu, Daejeon 305-343 (Korea, Republic of); Human and Environmental Toxicology Program, University of Science and Technology, 217 Gajeong-ro, Yuseong-gu, Daejeon 305-350 (Korea, Republic of)

2016-04-01

The recent establishment of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), which express the major cardiac ion channels and recapitulate spontaneous mechanical and electrical activities, may provide a possible solution for the lack of in vitro human-based cardiotoxicity testing models. Cardiotoxicity induced by the antidepressant nefazodone was previously revealed to cause an acquired QT prolongation by hERG channel blockade. To elucidate the cellular mechanisms underlying the cardiotoxicity of nefazodone beyond hERG, its effects on cardiac action potentials (APs) and ion channels were investigated using hiPSC-CMs with whole-cell patch clamp techniques. In a proof of principle study, we examined the effects of cardioactive channel blockers on the electrophysiological profile of hiPSC-CMs in advance of the evaluation of nefazodone. Nefazodone dose-dependently prolonged the AP duration at 90% (APD{sub 90}) and 50% (APD{sub 50}) repolarization, reduced the maximum upstroke velocity (dV/dt{sub max}) and induced early after depolarizations. Voltage-clamp studies of hiPSC-CMs revealed that nefazodone inhibited various voltage-gated ion channel currents including I{sub Kr}, I{sub Ks}, I{sub Na}, and I{sub Ca}. Among them, I{sub Kr} and I{sub Na} showed relatively higher sensitivity to nefazodone, consistent with the changes in the AP parameters. In summary, hiPSC-CMs enabled an integrated approach to evaluate the complex interactions of nefazodone with cardiac ion channels. These results suggest that hiPSC-CMs can be an effective model for detecting drug-induced arrhythmogenicity beyond the current standard assay of heterologously expressed hERG K{sup +} channels. - Highlights: • Nefazodone prolonged APD and decreased upstroke velocity of APs in hiPSC-CMs. • Nefazodone inhibited cardiac ion channels, especially I{sub Kr} and I{sub Na}, in hiPSC-CMs. • Nefazodone-induced AP changes are mainly the result of I{sub Kr} and I{sub Na} inhibition

Active RNA replication of hepatitis C virus downregulates CD81 expression.

Science.gov (United States)

Ke, Po-Yuan; Chen, Steve S-L

2013-01-01

So far how hepatitis C virus (HCV) replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS) protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp) infection and downregulated cell surface level of CD81, a critical HCV entry (co)receptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81.

Active RNA replication of hepatitis C virus downregulates CD81 expression.

Directory of Open Access Journals (Sweden)

Po-Yuan Ke

Full Text Available So far how hepatitis C virus (HCV replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp infection and downregulated cell surface level of CD81, a critical HCV entry (coreceptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81.

Inhibition of muscle-specific gene expression by Id3: requirement of the C-terminal region of the protein for stable expression and function.

Science.gov (United States)

Chen, B; Han, B H; Sun, X H; Lim, R W

1997-01-15

We have examined the role of an Id-like protein, Id3 (also known as HLH462), in the regulation of muscle-specific gene expression. Id proteins are believed to block expression of muscle-specific genes by preventing the dimerization between ubiquitous bHLH proteins (E proteins) and myogenic bHLH proteins such as MyoD. Consistent with its putative role as an inhibitor of differentiation, Id3 mRNA was detected in proliferating skeletal muscle cells, was further induced by basic fibroblast growth factor (bFGF) and was down-regulated in differentiated muscle cultures. Overexpression of Id3 efficiently inhibited the MyoD-mediated activation of the muscle-specific creatine kinase (MCK) reporter gene. Deletion analysis indicated that the C-terminal 15 amino acids of Id3 are critical for the full inhibitory activity while deleting up to 42 residues from the C-terminus of the related protein, Id2, did not affect its ability to inhibit the MCK reporter gene. Chimeric protein containing the N-terminal region of Id3 and the C-terminus of Id2 was also non-functional in transfected cells. In contrast, wild-type Id3, the C-terminal mutants, and the Id3/Id2 chimera could all interact with the E-protein E47in vitro. Additional studies indicated that truncation of the Id3 C-terminus might have adversely affected the expression level of the mutant proteins but the Id3/Id2 chimera was stably expressed. Taken together, our results revealed a more complex requirement for the expression and proper function of the Id family proteins than was hitherto expected.

Staphylococcus epidermidis polysaccharide intercellular adhesin induces IL-8 expression in human astrocytes via a mechanism involving TLR2.

LENUS (Irish Health Repository)

Stevens, Niall T

2009-03-01

Staphylococcus epidermidis is an opportunistic biofilm-forming pathogen associated with neurosurgical device-related meningitis. Expression of the polysaccharide intercellular adhesin (PIA) on its surface promotes S. epidermidis biofilm formation. Here we investigated the pro-inflammatory properties of PIA against primary and transformed human astrocytes. PIA induced IL-8 expression in a dose- and\\/or time-dependent manner from U373 MG cells and primary normal human astrocytes. This effect was inhibited by depletion of N-acetyl-beta-d-glucosamine polymer from the PIA preparation with Lycopersicon esculentum lectin or sodium meta-periodate. Expression of dominant-negative versions of the TLR2 and TLR4 adaptor proteins MyD88 and Mal in U373 MG cells inhibited PIA-induced IL-8 production. Blocking IL-1 had no effect. PIA failed to induce IL-8 production from HEK293 cells stably expressing TLR4. However, in U373 MG cells which express TLR2, neutralization of TLR2 impaired PIA-induced IL-8 production. In addition to IL-8, PIA also induced expression of other cytokines from U373 MG cells including IL-6 and MCP-1. These data implicate PIA as an important immunogenic component of the S. epidermidis biofilm that can regulate pro-inflammatory cytokine production from human astrocytes, in part, via TLR2.

Production of cloned pigs with targeted attenuation of gene expression.

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Vilceu Bordignon

Full Text Available The objective of this study was to demonstrate that RNA interference (RNAi and somatic cell nuclear transfer (SCNT technologies can be used to attenuate the expression of specific genes in tissues of swine, a large animal species. Apolipoprotein E (apoE, a secreted glycoprotein known for its major role in lipid and lipoprotein metabolism and transport, was selected as the target gene for this study. Three synthetic small interfering RNAs (siRNA targeting the porcine apoE mRNA were tested in porcine granulosa cells in primary culture and reduced apoE mRNA abundance ranging from 45-82% compared to control cells. The most effective sequence was selected for cloning into a short hairpin RNA (shRNA expression vector under the control of RNA polymerase III (U6 promoter. Stably transfected fetal porcine fibroblast cells were generated and used to produce embryos with in vitro matured porcine oocytes, which were then transferred into the uterus of surrogate gilts. Seven live and one stillborn piglet were born from three gilts that became pregnant. Integration of the shRNA expression vector into the genome of clone piglets was confirmed by PCR and expression of the GFP transgene linked to the expression vector. Analysis showed that apoE protein levels in the liver and plasma of the clone pigs bearing the shRNA expression vector targeting the apoE mRNA was significantly reduced compared to control pigs cloned from non-transfected fibroblasts of the same cell line. These results demonstrate the feasibility of applying RNAi and SCNT technologies for introducing stable genetic modifications in somatic cells for eventual attenuation of gene expression in vivo in large animal species.

B7.1 expression on tumor cells circumvents the need of professional antigen presentation for in vitro propagation of cytotoxic T cell lines.

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Iezzi, G; Protti, M P; Rugarli, C; Bellone, M

1996-01-01

In vitro propagation of tumor-specific CTLs, to be used for identification of tumor antigens (Ag) and/or adoptive immunotherapy, is hampered by the need of large amounts of professional antigen-presenting cells (APC) used for periodical cycles of restimulation. We evaluated whether RMA T lymphoma cells, stably transfected with the cDNA encoding for the B7.1 costimulatory molecule, provided the activation signals to CD8+ T lymphocytes in the absence of professional APC and CD4+ helper cells. We demonstrate here that long-term CD8+ cell lines can be efficiently propagated in vitro by repeated cycles of stimulation with tumor cells stably expressing B7.1. Professional APC and CD4+ helper cells are not required as far as interleukin 2 is exogenously provided. Furthermore, CD8+ blasts needed both signal 1 (Ag in the contest of the MHC molecule) and signal 2 (interaction of costimulatory molecules) for restimulation. T cell blasts in the presence of signal 1 or 2 only still retained their effector potential but did not undergo clonal expansion. These results are very promising for further applications of specific immunotherapies in humans.

Catalase expression impairs oxidative stress-mediated signalling in Trypanosoma cruzi.

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Freire, Anna Cláudia Guimarães; Alves, Ceres Luciana; Goes, Grazielle Ribeiro; Resende, Bruno Carvalho; Moretti, Nilmar Silvio; Nunes, Vinícius Santana; Aguiar, Pedro Henrique Nascimento; Tahara, Erich Birelli; Franco, Glória Regina; Macedo, Andréa Mara; Pena, Sérgio Danilo Junho; Gadelha, Fernanda Ramos; Guarneri, Alessandra Aparecida; Schenkman, Sergio; Vieira, Leda Quercia; Machado, Carlos Renato

2017-09-01

Trypanosoma cruzi is exposed to oxidative stresses during its life cycle, and amongst the strategies employed by this parasite to deal with these situations sits a peculiar trypanothione-dependent antioxidant system. Remarkably, T. cruzi's antioxidant repertoire does not include catalase. In an attempt to shed light on what are the reasons by which this parasite lacks this enzyme, a T. cruzi cell line stably expressing catalase showed an increased resistance to hydrogen peroxide (H2O2) when compared with wild-type cells. Interestingly, preconditioning carried out with low concentrations of H2O2 led untransfected parasites to be as much resistant to this oxidant as cells expressing catalase, but did not induce the same level of increased resistance in the latter ones. Also, presence of catalase decreased trypanothione reductase and increased superoxide dismutase levels in T. cruzi, resulting in higher levels of residual H2O2 after challenge with this oxidant. Although expression of catalase contributed to elevated proliferation rates of T. cruzi in Rhodnius prolixus, it failed to induce a significant increase of parasite virulence in mice. Altogether, these results indicate that the absence of a gene encoding catalase in T. cruzi has played an important role in allowing this parasite to develop a shrill capacity to sense and overcome oxidative stress.

Expression of PprI from Deinococcus radiodurans Improves Lactic Acid Production and Stress Tolerance in Lactococcus lactis.

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Xiangrong Dong

Full Text Available PprI is a general switch protein that regulates the expression of certain proteins involved in pathways of cellular resistance in the extremophilic bacterium Deinococcus radiodurans. In this study, we transformed pprI into Lactococcus lactis strain MG1363 using the lactococcal shuttle vector pMG36e and investigated its effects on the tolerance and lactic acid production of L. lactis while under stress. PprI was stably expressed in L. lactis as confirmed by western blot assays. L. lactis expressing PprI exhibited significantly improved resistance to oxidative stress and high osmotic pressure. This enhanced cellular tolerance to stressors might be due to the regulation of resistance-related genes (e.g., recA, recO, sodA, and nah by pprI. Moreover, transformed L. lactis demonstrated increased lactic acid production, attributed to enhanced lactate dehydrogenase activity. These results suggest that pprI can improve the tolerance of L. lactis to environmental stresses, and this transformed bacterial strain is a promising candidate for industrial applications of lactic acid production.

Expression of PprI from Deinococcus radiodurans Improves Lactic Acid Production and Stress Tolerance in Lactococcus lactis.

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Dong, Xiangrong; Tian, Bing; Dai, Shang; Li, Tao; Guo, Linna; Tan, Zhongfang; Jiao, Zhen; Jin, Qingsheng; Wang, Yanping; Hua, Yuejin

2015-01-01

PprI is a general switch protein that regulates the expression of certain proteins involved in pathways of cellular resistance in the extremophilic bacterium Deinococcus radiodurans. In this study, we transformed pprI into Lactococcus lactis strain MG1363 using the lactococcal shuttle vector pMG36e and investigated its effects on the tolerance and lactic acid production of L. lactis while under stress. PprI was stably expressed in L. lactis as confirmed by western blot assays. L. lactis expressing PprI exhibited significantly improved resistance to oxidative stress and high osmotic pressure. This enhanced cellular tolerance to stressors might be due to the regulation of resistance-related genes (e.g., recA, recO, sodA, and nah) by pprI. Moreover, transformed L. lactis demonstrated increased lactic acid production, attributed to enhanced lactate dehydrogenase activity. These results suggest that pprI can improve the tolerance of L. lactis to environmental stresses, and this transformed bacterial strain is a promising candidate for industrial applications of lactic acid production.

[Establishment of an iRFP and luciferase dual-color fluorescence-traced hepatocellular carcinoma transplantation model in nude mice].

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Li, Hongjun; Yang, Tianhua; Huang, Yanping; Liu, Mingzhu; Qin, Zhongqiang; Chu, Fei; Li, Zhenghong; Li, Yonghai

2017-11-01

Objective To establish a hepatocellular carcinoma xenograft model in nude mice which could stably express gene and be monitored dynamically. Methods We first constructed the lentiviral particles containing luciferase (Luc) and near-infrared fluorescent protein (iRFP) and puromycin resistance gene, and then transduced them into the HepG2 hepatoma cells. The cell line stably expressing Luc and iRFP genes were screened and inoculated into nude mice to establish xenograft tumor model. Tumor growth was monitored using in vivo imaging system. HE staining and immunohistochemistry were used to evaluate the pathological features and tumorigenic ability. Results HepG2 cells stably expressing iRFP and Luc were obtained; with the engineered cell line, xenograft model was successfully established with the features of proper tumor developing time and high rate of tumor formation as well as typical pathological features as showed by HE staining and immunohistochemistry. Conclusion Hepatocellular carcinoma model in nude mice with the features of stable gene expression and dynamical monitoring has been established successfully with the HepG2-iRFP-Luc cell line.

Intercellular adhesion molecule-1 expression by skeletal muscle cells augments myogenesis

International Nuclear Information System (INIS)

Goh, Qingnian; Dearth, Christopher L.; Corbett, Jacob T.; Pierre, Philippe; Chadee, Deborah N.; Pizza, Francis X.

2015-01-01

We previously demonstrated that the expression of intercellular adhesion molecule-1 (ICAM-1) by skeletal muscle cells after muscle overload contributes to ensuing regenerative and hypertrophic processes in skeletal muscle. The objective of the present study is to reveal mechanisms through which skeletal muscle cell expression of ICAM-1 augments regenerative and hypertrophic processes of myogenesis. This was accomplished by genetically engineering C2C12 myoblasts to stably express ICAM-1, and by inhibiting the adhesive and signaling functions of ICAM-1 through the use of a neutralizing antibody or cell penetrating peptide, respectively. Expression of ICAM-1 by cultured skeletal muscle cells augmented myoblast–myoblast adhesion, myotube formation, myonuclear number, myotube alignment, myotube–myotube fusion, and myotube size without influencing the ability of myoblasts to proliferate or differentiate. ICAM-1 augmented myotube formation, myonuclear accretion, and myotube alignment through a mechanism involving adhesion-induced activation of ICAM-1 signaling, as these dependent measures were reduced via antibody and peptide inhibition of ICAM-1. The adhesive and signaling functions of ICAM-1 also facilitated myotube hypertrophy through a mechanism involving myotube–myotube fusion, protein synthesis, and Akt/p70s6k signaling. Our findings demonstrate that ICAM-1 expression by skeletal muscle cells augments myogenesis, and establish a novel mechanism through which the inflammatory response facilitates growth processes in skeletal muscle. - Highlights: • We examined mechanisms through which skeletal muscle cell expression of ICAM-1 facilitates events of in vitro myogenesis. • Expression of ICAM-1 by cultured myoblasts did not influence their ability to proliferate or differentiate. • Skeletal muscle cell expression of ICAM-1 augmented myoblast fusion, myotube alignment, myotube–myotube fusion, and myotube size. • ICAM-1 augmented myogenic processes through

Intercellular adhesion molecule-1 expression by skeletal muscle cells augments myogenesis

Energy Technology Data Exchange (ETDEWEB)

Goh, Qingnian; Dearth, Christopher L.; Corbett, Jacob T. [Department of Kinesiology, The University of Toledo, Toledo, OH (United States); Pierre, Philippe [Centre d’Immunologie de Marseille-Luminy U2M, Aix-Marseille Université, Marseille (France); INSERM U631, Institut National de la Santé et Recherche Médicale, Marseille (France); CNRS UMR6102, Centre National de la Recherche Scientifique, Marseille (France); Chadee, Deborah N. [Department of Biological Sciences, The University of Toledo, Toledo, OH (United States); Pizza, Francis X., E-mail: Francis.Pizza@utoledo.edu [Department of Kinesiology, The University of Toledo, Toledo, OH (United States)

2015-02-15

We previously demonstrated that the expression of intercellular adhesion molecule-1 (ICAM-1) by skeletal muscle cells after muscle overload contributes to ensuing regenerative and hypertrophic processes in skeletal muscle. The objective of the present study is to reveal mechanisms through which skeletal muscle cell expression of ICAM-1 augments regenerative and hypertrophic processes of myogenesis. This was accomplished by genetically engineering C2C12 myoblasts to stably express ICAM-1, and by inhibiting the adhesive and signaling functions of ICAM-1 through the use of a neutralizing antibody or cell penetrating peptide, respectively. Expression of ICAM-1 by cultured skeletal muscle cells augmented myoblast–myoblast adhesion, myotube formation, myonuclear number, myotube alignment, myotube–myotube fusion, and myotube size without influencing the ability of myoblasts to proliferate or differentiate. ICAM-1 augmented myotube formation, myonuclear accretion, and myotube alignment through a mechanism involving adhesion-induced activation of ICAM-1 signaling, as these dependent measures were reduced via antibody and peptide inhibition of ICAM-1. The adhesive and signaling functions of ICAM-1 also facilitated myotube hypertrophy through a mechanism involving myotube–myotube fusion, protein synthesis, and Akt/p70s6k signaling. Our findings demonstrate that ICAM-1 expression by skeletal muscle cells augments myogenesis, and establish a novel mechanism through which the inflammatory response facilitates growth processes in skeletal muscle. - Highlights: • We examined mechanisms through which skeletal muscle cell expression of ICAM-1 facilitates events of in vitro myogenesis. • Expression of ICAM-1 by cultured myoblasts did not influence their ability to proliferate or differentiate. • Skeletal muscle cell expression of ICAM-1 augmented myoblast fusion, myotube alignment, myotube–myotube fusion, and myotube size. • ICAM-1 augmented myogenic processes through

SATB1 regulates SPARC expression in K562 cell line through binding to a specific sequence in the third intron

International Nuclear Information System (INIS)

Li, K.; Cai, R.; Dai, B.B.; Zhang, X.Q.; Wang, H.J.; Ge, S.F.; Xu, W.R.; Lu, J.

2007-01-01

Special AT-rich binding protein 1 (SATB1), a cell type-specific nuclear matrix attachment region (MAR) DNA-binding protein, tethers to a specific DNA sequence and regulates gene expression through chromatin remodeling and HDAC (histone deacetylase complex) recruitment. In this study, a SATB1 eukaryotic expression plasmid was transfected into the human erythroleukemia K562 cell line and individual clones that stably over-expressed the SATB1 protein were isolated. Microarray analysis revealed that hundreds of genes were either up- or down-regulated in the SATB1 over-expressing K562 cell lines. One of these was the extra-cellular matrix glycoprotein, SPARC (human secreted protein acidic and rich in cysteine). siRNA knock-down of SATB1 also reduced SPARC expression, which was consistent with elevated SPARC levels in the SATB1 over-expressing cell line. Bioinformatics software Mat-inspector showed that a 17 bp DNA sequence in the third intron of SPARC possessed a high potential for SATB1 binding; a finding confirmed by Chromatin immunoprecipitation (ChIP) with anti-SATB1 antibody. Our results show for the first time that forced-expression of SATB1 in K562 cells triggers SPARC up-regulation by binding to a 17 bp DNA sequence in the third intron

Purification and characterization of recombinant full-length and protease domain of murine MMP-9 expressed in Drosophila S2 cells

DEFF Research Database (Denmark)

Rasch, Morten G; Lund, Ida K; Illemann, Martin

2010-01-01

MMP-9. Constructs encoding zymogens of full-length murine MMP-9 and a version lacking the O-glycosylated linker region and hemopexin domains were therefore generated and expressed in stably transfected Drosophila S2 insect cells. After 7 days of induction the expression levels of the full......-length and truncated versions were 5 mg/l and 2 mg/l, respectively. The products were >95% pure after gelatin Sepharose chromatography and possessed proteolytic activity when analyzed by gelatin zymography. Using the purified full-length murine MMP-9 we raised polyclonal antibodies by immunizations of rabbits......Matrix metalloproteinase-9 (MMP-9) is a 92-kDa soluble pro-enzyme implicated in pathological events including cancer invasion. It is therefore an attractive target for therapeutic intervention studies in mouse models. Development of inhibitors requires sufficient amounts of correctly folded murine...

The effects of multiple UV exposures on HIV-LTR (long terminal repeat) expression

International Nuclear Information System (INIS)

Schreck, S.; Milton, J.; Panozzo, J.; Libertin, C.R.; Woloschak, G.E.; Loyola Univ., Maywood, IL

1995-01-01

Previous studies have shown that cellular stress agents such as UV radiation induce transcription from the long terminal repeat (LTR) of the human immunodeficiency virus (HIV). Using HeLa cells stably transfected with the HIV-LTR sequence, which transcriptionally drives the chloramphenicol acetyl transferase (CAT) reporter gene, we examined the effects of multiple exposures to UVC (254 nm) on HIV-LTR-CAT expression. Low doses (≤ 5 J m -2 ) had no effect on CAT expression, but up to 29-fold induction was observed with 10 J m -2 when cells were harvested 48 h after completion of the exposure. Little difference was noted in induction levels when cells were exposed to one 25 J m -2 dose, viable cells were harvested at 24 h, 48 h or 72 h, and cell lysates were assayed for CAT expression. Two sequential 12.5 J m -2 exposures, given 24 h apart, resulted in an additive effect on CAT expression; these two exposures produced CAT activity equivalent to that induced following a single 25 J m -2 dose. Our data suggest that HIV-LTR requires a specific threshold UV dose in order to elicit induction; a maximal induction dose is also evident; exposures higher than this maximal dose contribute no more to HIV-LTR induction in viable cells. (author)

Knocking out Ornithine Decarboxylase Antizyme 1 (OAZ1 Improves Recombinant Protein Expression in the HEK293 Cell Line

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Laura Abaandou

2018-06-01

Full Text Available Creating efficient cell lines is a priority for the biopharmaceutical industry, which produces biologicals for various uses. A recent approach to achieving this goal is the use of non-coding RNAs, microRNA (miRNA and small interfering RNA (siRNA, to identify key genes that can potentially improve production or growth. The ornithine decarboxylase antizyme 1 (OAZ1 gene, a negative regulator of polyamine biosynthesis, was identified in a genome-wide siRNA screen as a potential engineering target, because its knock down by siRNA increased recombinant protein expression from human embryonic kidney 293 (HEK293 cells by two-fold. To investigate this further, the OAZ1 gene in HEK293 cells was knocked out using CRISPR genome editing. The OAZ1 knockout cell lines displayed up to four-fold higher expression of both stably and transiently expressed proteins, with comparable growth and metabolic activity to the parental cell line; and an approximately three-fold increase in intracellular polyamine content. The results indicate that genetic inactivation of OAZ1 in HEK293 cells is an effective strategy to improve recombinant protein expression in HEK293 cells.

With Reference to Reference Genes: A Systematic Review of Endogenous Controls in Gene Expression Studies.

Science.gov (United States)

Chapman, Joanne R; Waldenström, Jonas

2015-01-01

The choice of reference genes that are stably expressed amongst treatment groups is a crucial step in real-time quantitative PCR gene expression studies. Recent guidelines have specified that a minimum of two validated reference genes should be used for normalisation. However, a quantitative review of the literature showed that the average number of reference genes used across all studies was 1.2. Thus, the vast majority of studies continue to use a single gene, with β-actin (ACTB) and/or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) being commonly selected in studies of vertebrate gene expression. Few studies (15%) tested a panel of potential reference genes for stability of expression before using them to normalise data. Amongst studies specifically testing reference gene stability, few found ACTB or GAPDH to be optimal, whereby these genes were significantly less likely to be chosen when larger panels of potential reference genes were screened. Fewer reference genes were tested for stability in non-model organisms, presumably owing to a dearth of available primers in less well characterised species. Furthermore, the experimental conditions under which real-time quantitative PCR analyses were conducted had a large influence on the choice of reference genes, whereby different studies of rat brain tissue showed different reference genes to be the most stable. These results highlight the importance of validating the choice of normalising reference genes before conducting gene expression studies.

Expression of sodium/iodide symporter transgene in neural stem cells

International Nuclear Information System (INIS)

Kim, Yun Hui; Lee, Dong Soo; Kang, Joo Hyun; Lee, Yong Jin; Chung, June Key; Lee, Myung Chul

2004-01-01

The ability to noninvasively track the migration of neural progenitor cells would have significant clinical and research implications. We generated stably transfected F3 human neural progenitor cells with human sodium/iodide symporter (hNIS) for noninvasively tracking F3. In this study, the expression patterns of hNIS gene in F3-NIS were examined according to the cultured time and the epigenetic modulation. F3 human neural stem cells had been obtained from Dr. Seung U. Kim (Ajou University, Suwon, Korea). hNIS and hygromycin resistance gene were linked with IRES (internal Ribosome Entry Site) under control of CMV promoter. This construct was transfected to F3 with Liposome. To investigate the restoration of hNIS gene expression in F3-NIS, cells were treated with demethylating agent (5-Azacytidine) and Histone deacetylase inhibitor (Trichostatin A: TSA). The expression of hNIS was measured by I-125 uptake assay and RT-PCR analysis. The iodide uptake of the F3-NIS was higher 12.86 times than F3 cell line. According to the cell passage number, hNIS expression in F3-NIS gradually diminished. After treatment of 5-Azacytidine and TSA with serial doses (up to 20μM, up to 62.5nM, respectively) for 24 hours, I-125 uptake and mRNA of hNIS in F3-NIS were increased. These results suggest that hNIS transfected F3 might undergo a change in its biological characters by cell passage. Therefore, the gene expression of exogenous gene transferred human stem cell might be affected to the epigenetic modulation such as promoter methylation and Histone deacetylation and to the cell culture conditions

A construct with fluorescent indicators for conditional expression of miRNA

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Xia Xugang

2008-10-01

-mediated induction of the miRNA expression. This construct can be used to increase the efficiency of making cell lines or transgenic animals that stably express miRNA targeting specific genes.

Molecular Imaging of Human Embryonic Stem Cells Stably Expressing Human PET Reporter Genes After Zinc Finger Nuclease-Mediated Genome Editing.

Science.gov (United States)

Wolfs, Esther; Holvoet, Bryan; Ordovas, Laura; Breuls, Natacha; Helsen, Nicky; Schönberger, Matthias; Raitano, Susanna; Struys, Tom; Vanbilloen, Bert; Casteels, Cindy; Sampaolesi, Maurilio; Van Laere, Koen; Lambrichts, Ivo; Verfaillie, Catherine M; Deroose, Christophe M

2017-10-01

Molecular imaging is indispensable for determining the fate and persistence of engrafted stem cells. Standard strategies for transgene induction involve the use of viral vectors prone to silencing and insertional mutagenesis or the use of nonhuman genes. Methods: We used zinc finger nucleases to induce stable expression of human imaging reporter genes into the safe-harbor locus adeno-associated virus integration site 1 in human embryonic stem cells. Plasmids were generated carrying reporter genes for fluorescence, bioluminescence imaging, and human PET reporter genes. Results: In vitro assays confirmed their functionality, and embryonic stem cells retained differentiation capacity. Teratoma formation assays were performed, and tumors were imaged over time with PET and bioluminescence imaging. Conclusion: This study demonstrates the application of genome editing for targeted integration of human imaging reporter genes in human embryonic stem cells for long-term molecular imaging. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

N,N'-dinitrosopiperazine--mediated heat-shock protein 70-2 expression is involved in metastasis of nasopharyngeal carcinoma.

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Zhengke Peng

Full Text Available N,N'-Dinitrosopiperazine (DNP is invovled in nasopharyngeal carcinoma (NPC development and metastasis, and it shows organ specificity to the nasopharyngeal epithelium. Herein, we demonstrate that DNP induces heat-shock protein (HSP 70-2 expression in NPC cells (6-10B at a non-cytotoxic concentration. DNP induced HSP70-2 expression in a dose- and time- dependent manner, but showed no effect on other HSP70 family members. Furthermore, DNP also increased HSP70-2 RNA transcription through directly binding to the hypoxia-responsive elements (HRE and heat shock elements (HSE located in the HSP70-2 promoter. DNP-mediated HSP70-2 expression might act through enhancing the transcription of HSP70-2 RNA. Importantly, DNP induced motility and invasion of 6-10B cells dose- and time-dependently, and DNP-mediated NPC metastasis was confirmed in nude mice, which showed high HSP70-2 expression in the metastatic tumor tissue. However, the motility and invasion of NPC cells that were stably transfected using short interfering RNA against HSP70-2 could not effectively induce DNP. These results indicate that DNP induces HSP70-2 expression through increasing HSP70-2 transcription, increases the motility and invasion of cells, and promotes NPC tumor metastasis. Therefore, DNP mediated HSP70-2 expression may be an important factor of NPC-high metastasis.

Resveratrol inhibits Cdk5 activity through regulation of p35 expression

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Kulkarni Ashok B

2011-07-01

Full Text Available Abstract Background We have previously reported that cyclin-dependent kinase 5 (Cdk5 participates in the regulation of nociceptive signaling. Through activation of the ERK1/2 pathway, Tumor Necrosis Factor-α (TNF-α induces expression of Egr-1. This results in the sustained and robust expression of p35, a coactivator of Cdk5, in PC12 cells, thereby increasing Cdk5 kinase activity. The aim of our present study was to test whether resveratrol, a polyphenolic compound with known analgesic activity, can regulate Cdk5/p35 activity. Results Here we used a cell-based assay in which a p35 promoter-luciferase construct was stably transfected in PC12 cells. Our studies demonstrate that resveratrol inhibits p35 promoter activity and also blocks the TNF-α mediated increase in Cdk5 activity in PC12 cells. Resveratrol also inhibits p35 expression and blocks the TNF-α mediated increase in Cdk5 activity in DRG neurons. In the presence of resveratrol, the MEK inhibitor decreased p35 promoter activity, whereas the inhibitors of p38 MAPK, JNK and NF-κB increased p35 promoter activity, indicating that these pathways regulate p35 expression differently. The TNF-α-mediated increase in Egr-1 expression was decreased by resveratrol treatment with a concomitant reduction in p35 expression and protein levels, resulting in reduced Cdk5 kinase activity. Conclusions We demonstrate here that resveratrol regulates p35 promoter activity in PC12 cells and DRG neurons. Most importantly, resveratrol blocks the TNF-α-mediated increase in p35 promoter activity, thereby reducing p35 expression and subsequent Cdk5 kinase activity. This new molecular mechanism adds to the known analgesic effects of resveratrol and confirms the need for identifying new analgesics based on their ability to inhibit Cdk5 activity for effective treatment of pain.

GM-CSF enhances tumor invasion by elevated MMP-2, -9, and -26 expression

International Nuclear Information System (INIS)

Gutschalk, Claudia M; Yanamandra, Archana K; Linde, Nina; Meides, Alice; Depner, Sofia; Mueller, Margareta M

2013-01-01

Granulocyte–macrophage colony-stimulating factor (GM-CSF) promotes tumor progression in different tumor models in an autocrine and paracrine manner. However, at the same time GM-CSF is used in cancer therapies to ameliorate neutropenia. We have previously shown in GM-CSF and G-CSF expressing or negative skin or head and neck squamous cell carcinoma that GM-CSF expression is associated with a highly angiogenic and invasive tumor phenotype. To determine the functional contribution of GM-CSF to tumor invasion, we stably transfected a GM-CSF negative colon adenocarcinoma cell line HT-29 with GM-CSF or treated the same cell line with exogenous GM-CSF. While GM-CSF overexpression and treatment reduced tumor cell proliferation and tumor growth in vitro and in vivo, respectively, it contributed to tumor progression. Together with an enhanced migratory capacity in vitro, we observed a striking increase in tumor cell invasion into the surrounding tissue concomitant with the induction of an activated tumor stroma in GM-CSF overexpressing or GM-CSF treated tumors. In a complex 3D in vitro model, enhanced GM-CSF expression was associated with a discontinued basement membrane deposition that might be mediated by the increased expression and activation of MMP-2, -9, and -26. Treatment with GM-CSF blocking antibodies reversed this effect. The increased presence and activity of these tumor cell derived proteases was confirmed in vivo. Here, expression of MMP-26 protein was predominantly located in pre- and early-invasive areas suggesting MMP-26 expression as an early event in promoting GM-CSF dependent tumor invasion

Process development and economic evaluation of recombinant human lactoferrin expressed in rice grain.

Science.gov (United States)

Nandi, Somen; Yalda, Dorice; Lu, Stephen; Nikolov, Zivko; Misaki, Ryo; Fujiyama, Kazuhito; Huang, Ning

2005-06-01

In this paper, we show that recombinant human lactoferrin (rhLF) has been stably expressed at 0.5% brown rice flour weight for nine generations. Process development indicates that rhLF can be efficiently extracted from rice flour in 20 mM phosphate buffer (pH 7.0) containing up to 0.5 M NaCl and at a ratio of 1 kg flour to 10 L buffer. After solid/liquid separation, the extract can then be loaded directly onto an ion-exchange column and rhLF can be eluted using 0.8 M NaCl. The resulting rhLF is about 95% pure. A range of biochemical and biophysical analyses were carried out and results indicated that the purified rhLF was identical to its native human counterpart other than its glycosylation. Economic analysis shows that at 600 kg/year scale, the cash cost to produce 1 g of rhLF of pharmaceutical grade is US$ 5.90. Analysis also indicates that the expression level has profound impact on costs related to planting, milling, extraction and purification, thus high level expression of recombinant protein in plants is one of the key parameters for the success of plant made pharmaceuticals.

Axl acts as a tumor suppressor by regulating LIGHT expression in T lymphoma

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Young, Kon-Ji; Park, A-Reum; Choi, Ha-Rim; Lee, Hwa-Youn; Kim, Su-Man; Chung, Byung Yeoup; Park, Chul-Hong; Choi, Hyo Jin; Ko, Young-Hyeh; Bai, Hyoung-Woo; Kang, Hyung-Sik

2017-01-01

Axl is an oncogenic receptor tyrosine kinase that plays a role in many cancers. LIGHT (Lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells) is a ligand that induces robust anti-tumor immunity by enhancing the recruitment and activation of effector immune cells at tumor sites. We observed that mouse EL4 and human Jurkat T lymphoma cells that stably overexpressed Axl also showed high expression of LIGHT. When Jurkat-Axl cells were treated with Gas6, a ligand for Axl, LIGHT expression was upregulated through activation of the PI3K/AKT signaling pathway and transcriptional induction by Sp1. The lytic activity of cytotoxic T lymphocytes and natural killer cells was enhanced by EL4-Axl cells. In addition, tumor volume and growth were markedly reduced due to enhanced apoptotic cell death in EL4-Axl tumor-bearing mice as compared to control mice. We also observed upregulated expression of CCL5 and its receptor, CCR5, and enhanced intratumoral infiltration of cytotoxic T lymphocytes and natural killer cells in EL4-Axl-bearing mice as compared to mock controls. These data strongly suggested that Axl exerts novel tumor suppressor effects by inducing upregulation of LIGHT in the tumor microenvironment of T lymphoma. PMID:28423548

CXCL12 chemokine expression suppresses human pancreatic cancer growth and metastasis.

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Ishan Roy

Full Text Available Pancreatic ductal adenocarcinoma is an unsolved health problem with nearly 75% of patients diagnosed with advanced disease and an overall 5-year survival rate near 5%. Despite the strong link between mortality and malignancy, the mechanisms behind pancreatic cancer dissemination and metastasis are poorly understood. Correlative pathological and cell culture analyses suggest the chemokine receptor CXCR4 plays a biological role in pancreatic cancer progression. In vivo roles for the CXCR4 ligand CXCL12 in pancreatic cancer malignancy were investigated. CXCR4 and CXCR7 were consistently expressed in normal and cancerous pancreatic ductal epithelium, established cell lines, and patient-derived primary cancer cells. Relative to healthy exocrine ducts, CXCL12 expression was pathologically repressed in pancreatic cancer tissue specimens and patient-derived cell lines. To test the functional consequences of CXCL12 silencing, pancreatic cancer cell lines stably expressingthe chemokine were engineered. Consistent with a role for CXCL12 as a tumor suppressor, cells producing the chemokine wereincreasingly adherent and migration deficient in vitro and poorly metastatic in vivo, compared to control cells. Further, CXCL12 reintroduction significantly reduced tumor growth in vitro, with significantly smaller tumors in vivo, leading to a pronounced survival advantage in a preclinical model. Together, these data demonstrate a functional tumor suppressive role for the normal expression of CXCL12 in pancreatic ducts, regulating both tumor growth andcellulardissemination to metastatic sites.

Differential Expression of Ccn4 and Other Genes Between Metastatic and Non-metastatic EL4 Mouse Lymphoma Cells.

Science.gov (United States)

Chahal, Manpreet S; Ku, H Teresa; Zhang, Zhihong; Legaspi, Christian M; Luo, Angela; Hopkins, Mandi M; Meier, Kathryn E

Previous work characterized variants of the EL4 murine lymphoma cell line. Some are non-metastatic, and others metastatic, in syngenic mice. In addition, metastatic EL4 cells were stably transfected with phospholipase D2 (PLD2), which further enhanced metastasis. Microarray analyses of mRNA expression was performed for non-metastatic, metastatic, and PLD2-expressing metastatic EL4 cells. Many differences were observed between non-metastatic and metastatic cell lines. One of the most striking new findings was up-regulation of mRNA for the matricellular protein WNT1-inducible signaling pathway protein 1 (CCN4) in metastatic cells; increased protein expression was verified by immunoblotting and immunocytochemistry. Other differentially expressed genes included those for reproductive homeobox 5 (Rhox5; increased in metastatic) and cystatin 7 (Cst7; decreased in metastatic). Differences between PLD2-expressing and parental cell lines were limited but included the signaling proteins Ras guanyl releasing protein 1 (RGS18; increased with PLD2) and suppressor of cytokine signaling 2 (SOCS2; decreased with PLD2). The results provide insights into signaling pathways potentially involved in conferring metastatic ability on lymphoma cells. Copyright© 2016, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

BRCA1 is expressed in uterine serous carcinoma (USC) and controls insulin-like growth factor I receptor (IGF-IR) gene expression in USC cell lines.

Science.gov (United States)

Amichay, Keren; Kidron, Debora; Attias-Geva, Zohar; Schayek, Hagit; Sarfstein, Rive; Fishman, Ami; Werner, Haim; Bruchim, Ilan

2012-06-01

The insulin-like growth factor I receptor (IGF-IR) and BRCA1 affect cell growth and apoptosis. Little information is available about BRCA1 activity on the IGF signaling pathway. This study evaluated the effect of BRCA1 on IGF-IR expression. BRCA1 and IGF-IR immunohistochemistry on archival tissues (35 uterine serous carcinomas [USCs] and 17 metastases) were performed. USPC1 and USPC2 cell lines were transiently cotransfected with an IGF-IR promoter construct driving a luciferase reporter gene and a BRCA1 expression plasmid. Endogenous IGF-IR levels were evaluated by Western immunoblotting. We found high BRCA1 and IGF-IR protein expression in primary and metastatic USC tumors. All samples were immunostained for BRCA1-71% strongly stained; and 33/35 (94%) were stained positive for IGF-IR-2 (6%) strongly stained. No difference in BRCA1 and IGF-IR staining intensity was noted between BRCA1/2 mutation carriers and noncarriers. Metastatic tumors stained more intensely for BRCA1 than did the primary tumor site (P = 0.041) and with borderline significance for IGF-IR (P = 0.069). BRCA1 and IGF-IR staining did not correlate to survival. BRCA1 expression led to 35% and 54% reduction in IGF-IR promoter activity in the USPC1 and USCP2 cell lines, respectively. Western immunoblotting showed a decline in phosphorylated IGF-IR and phosphorylated AKT in both transiently and stably transfected cells. BRCA1 and IGF-IR are highly expressed in USC tumors. BRCA1 suppresses IGF-IR gene expression and activity. These findings suggest a possible biological link between the BRCA1 and the IGF-I signaling pathways in USC. The clinical implications of this association need to be explored.

The expression and activity of thioredoxin reductase 1 splice variants v1 and v2 regulate the expression of genes associated with differentiation and adhesion

Science.gov (United States)

Nalvarte, Ivan; Damdimopoulos, Anastasios E.; Rüegg, Joëlle; Spyrou, Giannis

2015-01-01

The mammalian redox-active selenoprotein thioredoxin reductase (TrxR1) is a main player in redox homoeostasis. It transfers electrons from NADPH to a large variety of substrates, particularly to those containing redox-active cysteines. Previously, we reported that the classical form of cytosolic TrxR1 (TXNRD1_v1), when overexpressed in human embryonic kidney cells (HEK-293), prompted the cells to undergo differentiation [Nalvarte et al. (2004) J. Biol. Chem. 279, 54510–54517]. In the present study, we show that several genes associated with differentiation and adhesion are differentially expressed in HEK-293 cells stably overexpressing TXNRD1_v1 compared with cells expressing its splice variant TXNRD1_v2. Overexpression of these two splice forms resulted in distinctive effects on various aspects of cellular functions including gene regulation patterns, alteration of growth rate, migration and morphology and susceptibility to selenium-induced toxicity. Furthermore, differentiation of the neuroblastoma cell line SH-SY5Y induced by all-trans retinoic acid (ATRA) increased both TXNRD1_v1 and TXNRD1_v2 expressions along with several of the identified genes associated with differentiation and adhesion. Selenium supplementation in the SH-SY5Y cells also induced a differentiated morphology and changed expression of the adhesion protein fibronectin 1 and the differentiation marker cadherin 11, as well as different temporal expression of the studied TXNRD1 variants. These data suggest that both TXNRD1_v1 and TXNRD1_v2 have distinct roles in differentiation, possibly by altering the expression of the genes associated with differentiation, and further emphasize the importance in distinguishing each unique action of different TrxR1 splice forms, especially when studying the gene silencing or knockout of TrxR1. PMID:26464515

Targeted surface expression of an exogenous antigen in stably transfected babesia bovis

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Babesia bovis is a tick-borne intraerythocytic protozoan responsible for acute disease in cattle which can be controlled by vaccination with attenuated B. bovis strains. Emerging B. bovis transfection technologies may increase the usefulness of these live vaccines. Here we propose using transfected ...

Defectively N-glycosylated and non-O-glycosylated aminopeptidase N (CD13) is normally expressed at the cell surface and has full enzymatic activity

DEFF Research Database (Denmark)

Norén, K; Hansen, Gert Helge; Clausen, H

1997-01-01

In order to study the effects of the absence of O-glycosylation and modifications of N-glycosylation on a class II membrane protein, pig and human aminopeptidase N (CD13) were stably expressed in the ldl(D) cell line. This cell line carries a UDP-Gal/UDP-GalNAc-epimerase deficiency which blocks...... the conversion of glucose into galactose derivatives. Thus it is possible in the ldl(D) cell line to selectively block O-glycosylation by the omission of N-acetylgalactoseamine from the culture medium and to alter N-glycosylation by the omission of galactose. In this way selectively altered glycosylated forms...

Knockdown of ARK5 Expression Suppresses Invasion and Metastasis of Gastric Cancer

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Dehu Chen

2017-06-01

Full Text Available Background/Aims: Gastric cancer (GC is a common and lethal malignancy, and AMP-activated protein kinase-related kinase 5 (ARK5 has been discovered to promote cancer metastasis in certain types of cancer. In this study, we explored the role of ARK5 in GC invasion and metastasis. Methods: ARK5 and epithelial-mesenchymal transition (EMT-related markers were determined by immunohistochemistry and western blot in GC specimens. Other methods including stably transfected against ARK5 into SGC7901 and AGS cells, western blot, migration and invasion assays in vitro and nude mice tumorigenicity in vivo were also employed. Results: The results demonstrated that ARK5 expression was increased and positively correlated with metastasis, EMT-related markers and poor prognosis in patients with GC. Knockdown of ARK5 expression remarkably suppressed GC cells invasion and metastasis via regulating EMT, rather than proliferation in vitro and in vivo. And knockdown of ARK5 expression in GC cells resulted in the down-regulation of the mTOR/p70S6k signals, Slug and SIP1. Conclusion: The elevated ARK5 expression was closely associated with cancer metastasis and patient survival, and it seemed to function in GC cells migration and invasion via EMT alteration, together with the alteration of the mTOR/p70S6k signals, Slug and SIP1, thus providing a potential therapeutic target for GC.

Development of transgenic wheat (Triticum aestivum L.) expressing avidin gene conferring resistance to stored product insects.

Science.gov (United States)

Abouseadaa, Heba H; Osman, Gamal H; Ramadan, Ahmed M; Hassanein, Sameh E; Abdelsattar, Mohamed T; Morsy, Yasser B; Alameldin, Hussien F; El-Ghareeb, Doaa K; Nour-Eldin, Hanan A; Salem, Reda; Gad, Adel A; Elkhodary, Soheir E; Shehata, Maher M; Mahfouz, Hala M; Eissa, Hala F; Bahieldin, Ahmed

2015-07-22

Wheat is considered the most important cereal crop all over the world. The wheat weevil Sitophilus granarius is a serious insect pests in much of the wheat growing area worldwide and is responsible for significant loss of yield. Avidin proteins has been proposed to function as plant defense agents against insect pests. A synthetic avidin gene was introduced into spring wheat (Triticum aestivum L.) cv. Giza 168 using a biolistic bombardment protocol. The presence and expression of the transgene in six selected T0 transgenic wheat lines were confirmed at the molecular level. Accumulation of avidin protein was detected in transgenic plants compared to non-transgenic plants. Avidin transgene was stably integrated, transcribed and translated as indicated by Southern blot, ELISA, and dot blot analyses, with a high level of expression in transgenic wheat seeds. However, no expression was detected in untransformed wheat seeds. Functional integrity of avidin was confirmed by insect bioassay. The results of bioassay using transgenic wheat plants challenged with wheat weevil revealed 100 % mortality of the insects reared on transgenic plants after 21 days. Transgenic wheat plants had improved resistance to Sitophilus granarius.

The structure of the stably stratified internal boundary layer in offshore flow over the sea

Science.gov (United States)

Garratt, J. R.; Ryan, B. F.

1989-04-01

Observations obtained mainly from a research aircraft are presented of the mean and turbulent structure of the stably stratified internal boundary layer (IBL) over the sea formed by warm air advection from land to sea. The potential temperature and humidity fields reveal the vertical extent of the IBL, for fetches out to several hundred of kilometres, geostrophic winds of 20 25 m s-1, and potential temperature differences between undisturbed continental air and the sea surface of 7 to 17 K. The dependence of IBL depth on these external parameters is discussed in the context of the numerical results of Garratt (1987), and some discrepancies are noted. Wind observations show the development of a low-level wind maximum (wind component normal to the coast) and rotation of the wind to smaller cross-isobar flow angles. Potential temperature (θ) profiles within the IBL reveal quite a different structure to that found in the nocturnal boundary layer (NBL) over land. Over the sea, θ profiles have large positive curvature with vertical gradients increasing monotonically with height; this reflects the dominance of turbulent cooling within the layer. The behaviour is consistent with known behaviour in the NBL over land where curvature becomes negative (vertical gradients of θ decreasing with height) as radiative cooling becomes dominant. Turbulent properties are discussed in terms of non-dimensional quantities, normalised by the surface friction velocity, as functions of normalised height using the IBL depth. Vertical profiles of these and the normalised wavelength of the spectral maximum agree well with known results for the stable boundary layer over land (Caughey et al., 1979).

Ubiquitin fusion expression and tissue-dependent targeting of hG-CSF in transgenic tobacco

Science.gov (United States)

2011-01-01

Background Human granulocyte colony-stimulating factor (hG-CSF) is an important human cytokine which has been widely used in oncology and infection protection. To satisfy clinical needs, expression of recombinant hG-CSF has been studied in several organisms, including rice cell suspension culture and transient expression in tobacco leaves, but there was no published report on its expression in stably transformed plants which can serve as a more economical expression platform with potential industrial application. Results In this study, hG-CSF expression was investigated in transgenic tobacco leaves and seeds in which the accumulation of hG-CSF could be enhanced through fusion with ubiquitin by up to 7 fold in leaves and 2 fold in seeds, leading to an accumulation level of 2.5 mg/g total soluble protein (TSP) in leaves and 1.3 mg/g TSP in seeds, relative to hG-CSF expressed without a fusion partner. Immunoblot analysis showed that ubiquitin was processed from the final protein product, and ubiquitination was up-regulated in all transgenic plants analyzed. Driven by CaMV 35S promoter and phaseolin signal peptide, hG-CSF was observed to be secreted into apoplast in leaves but deposited in protein storage vacuole (PSV) in seeds, indicating that targeting of the hG-CSF was tissue-dependent in transgenic tobacco. Bioactivity assay showed that hG-CSF expressed in both seeds and leaves was bioactive to support the proliferation of NFS-60 cells. Conclusions In this study, the expression of bioactive hG-CSF in transgenic plants was improved through ubiquitin fusion strategy, demonstrating that protein expression can be enhanced in both plant leaves and seeds through fusion with ubiquitin and providing a typical case of tissue-dependent expression of recombinant protein in transgenic plants. PMID:21985646

Expression of the central growth regulator BIG BROTHER is regulated by multiple cis-elements

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Breuninger Holger

2012-03-01

Full Text Available Abstract Background Much of the organismal variation we observe in nature is due to differences in organ size. The observation that even closely related species can show large, stably inherited differences in organ size indicates a strong genetic component to the control of organ size. Despite recent progress in identifying factors controlling organ growth in plants, our overall understanding of this process remains limited, partly because the individual factors have not yet been connected into larger regulatory pathways or networks. To begin addressing this aim, we have studied the upstream regulation of expression of BIG BROTHER (BB, a central growth-control gene in Arabidopsis thaliana that prevents overgrowth of organs. Final organ size and BB expression levels are tightly correlated, implying the need for precise control of its expression. BB expression mirrors proliferative activity, yet the gene functions to limit proliferation, suggesting that it acts in an incoherent feedforward loop downstream of growth activators to prevent over-proliferation. Results To investigate the upstream regulation of BB we combined a promoter deletion analysis with a phylogenetic footprinting approach. We were able to narrow down important, highly conserved, cis-regulatory elements within the BB promoter. Promoter sequences of other Brassicaceae species were able to partially complement the A. thaliana bb-1 mutant, suggesting that at least within the Brassicaceae family the regulatory pathways are conserved. Conclusions This work underlines the complexity involved in precise quantitative control of gene expression and lays the foundation for identifying important upstream regulators that determine BB expression levels and thus final organ size.

The endogenous retroviral insertion in the human complement C4 gene modulates the expression of homologous genes by antisense inhibition.

Science.gov (United States)

Schneider, P M; Witzel-Schlömp, K; Rittner, C; Zhang, L

2001-02-01

Intron 9 contains the complete endogenous retrovirus HERV-K(C4) as a 6.4-kb insertion in 60% of human C4 genes. The retroviral insertion is in reverse orientation to the C4 coding sequence. Therefore, expression of C4 could lead to the transcription of an antisense RNA, which might protect against exogenous retroviral infections. To test this hypothesis, open reading frames from the HERV sequence were subcloned in sense orientiation into a vector allowing expression of a beta-galactosidase fusion protein. Mouse L cells which had been stably transfected with either the human C4A or C4B gene both carrying the HERV insertion (LC4 cells), and L(Tk-) cells without the C4 gene were transiently transfected either with a retroviral construct or with the wild-type vector. Expression was monitored using an enzymatic assay. We demonstrated that (1) HERV-K(C4) antisense mRNA transcripts are present in cells constitutively expressing C4, (2) expression of retroviral-like constructs is significantly downregulated in cells expressing C4, and (3) this downregulation is further modulated in a dose-dependent fashion following interferon-gamma stimulation of C4 expression. These results support the hypothesis of a genomic antisense strategy mediated by the HERV-K(C4) insertion as a possible defense mechanism against exogenous retroviral infections.

A longitudinal study of gene expression in healthy individuals

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Tessier Michel

2009-06-01

Full Text Available Abstract Background The use of gene expression in venous blood either as a pharmacodynamic marker in clinical trials of drugs or as a diagnostic test requires knowledge of the variability in expression over time in healthy volunteers. Here we defined a normal range of gene expression over 6 months in the blood of four cohorts of healthy men and women who were stratified by age (22–55 years and > 55 years and gender. Methods Eleven immunomodulatory genes likely to play important roles in inflammatory conditions such as rheumatoid arthritis and infection in addition to four genes typically used as reference genes were examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR, as well as the full genome as represented by Affymetrix HG U133 Plus 2.0 microarrays. Results Gene expression levels as assessed by qRT-PCR and microarray were relatively stable over time with ~2% of genes as measured by microarray showing intra-subject differences over time periods longer than one month. Fifteen genes varied by gender. The eleven genes examined by qRT-PCR remained within a limited dynamic range for all individuals. Specifically, for the seven most stably expressed genes (CXCL1, HMOX1, IL1RN, IL1B, IL6R, PTGS2, and TNF, 95% of all samples profiled fell within 1.5–2.5 Ct, the equivalent of a 4- to 6-fold dynamic range. Two subjects who experienced severe adverse events of cancer and anemia, had microarray gene expression profiles that were distinct from normal while subjects who experienced an infection had only slightly elevated levels of inflammatory markers. Conclusion This study defines the range and variability of gene expression in healthy men and women over a six-month period. These parameters can be used to estimate the number of subjects needed to observe significant differences from normal gene expression in clinical studies. A set of genes that varied by gender was also identified as were a set of genes with elevated

Water-resistant, monodispersed and stably luminescent CsPbBr3/CsPb2Br5 core-shell-like structure lead halide perovskite nanocrystals

Science.gov (United States)

Qiao, Bo; Song, Pengjie; Cao, Jingyue; Zhao, Suling; Shen, Zhaohui; Gao, Di; Liang, Zhiqin; Xu, Zheng; Song, Dandan; Xu, Xurong

2017-11-01

Lead halide perovskite materials are thriving in optoelectronic applications due to their excellent properties, while their instability due to the fact that they are easily hydrolyzed is still a bottleneck for their potential application. In this work, water-resistant, monodispersed and stably luminescent cesium lead bromine perovskite nanocrystals coated with CsPb2Br5 were obtained using a modified non-stoichiometric solution-phase method. CsPb2Br5 2D layers were coated on the surface of CsPbBr3 nanocrystals and formed a core-shell-like structure in the synthetic processes. The stability of the luminescence of the CsPbBr3 nanocrystals in water and ethanol atmosphere was greatly enhanced by the photoluminescence-inactive CsPb2Br5 coating with a wide bandgap. The water-stable enhanced nanocrystals are suitable for long-term stable optoelectronic applications in the atmosphere.

Prediction of stably stratified homogeneous shear flows with second-order turbulence models

International Nuclear Information System (INIS)

Pereira, J C F; Rocha, J M P

2010-01-01

The present study investigated the role of pressure-correlation second-order turbulence modelling schemes on the predicted behaviour of stably stratified homogeneous vertical-sheared turbulence. The pressure-correlation terms were modelled with a nonlinear formulation (Craft 1991), which was compared with a linear pressure-strain model and the 'isotropization of production' model for the pressure-scalar correlation. Two additional modelling issues were investigated: the influence of the buoyancy term in the kinetic energy dissipation rate equation and the time scale in the thermal production term in the scalar variance dissipation equation. The predicted effects of increasing the Richardson number on turbulence characteristics were compared against a comprehensive set of direct numerical simulation databases. The linear models provide a broadly satisfactory description of the major effects of the Richardson number on stratified shear flow. The buoyancy term in the dissipation equation of the turbulent kinetic energy generates excessively low levels of dissipation. For moderate and large Richardson numbers, the term yields unrealistic linear oscillations in the shear and buoyancy production terms, and therefore should be dropped in this flow (or at least their coefficient c ε3 should be substantially reduced from its standard value). The mechanical dissipation time scale provides marginal improvements in comparison to the scalar time scale in the production. The observed inaccuracy of the linear model in predicting the magnitude of the effects on the velocity anisotropy was demonstrated to be attributed mainly to the defective behaviour of the pressure-correlation model, especially for stronger stratification. The turbulence closure embodying a nonlinear formulation for the pressure-correlations and specific versions of the dissipation equations failed to predict the tendency of the flow to anisotropy with increasing stratification. By isolating the effects of the

Evaluation of Candidate Reference Genes for Quantitative Gene Expression Analysis in Spodoptera exigu a after Long-time Exposure to Cadmium.

Science.gov (United States)

Płachetka-Bożek, Anna; Augustyniak, Maria

2017-08-21

Studies on the transcriptional control of gene expression play an important role in many areas of biology. Reference genes, which are often referred to as housekeeping genes, such as GAPDH, G3PDH, EF2, RpL7A, RpL10, TUBα and Actin, have traditionally been assumed to be stably expressed in all conditions, and they are frequently used to normalize mRNA levels between different samples in qPCR analysis. However, it is known that the expression of these genes is influenced by numerous factors, such as experimental conditions. The difference in gene expression underlies a range of biological processes, including development, reproduction and behavior. The aim of this study was to show the problems associated with using reference genes in the qPCR technique, in a study on inbred strains of Spodoptera exigua selected toward cadmium resistance. We present and discuss our results and observations, and give some recommendations concerning the use and limitations of housekeeping genes as internal standards, especially in research on insects. Our results suggest that holometabolism and poikilothermia, as well as time since metamorphosis and the level of exposure to the selective factor (cadmium in this case), have a significant effect on the expression of reference genes.

Tissue-specific expression of transfected human insulin genes in pluripotent clonal rat insulinoma lines induced during passage in vivo

Energy Technology Data Exchange (ETDEWEB)

Madsen, O.D.; Andersen, L.C.; Michelsen, B.; Owerbach, D.; Larsson, L.I.; Lernmark, A.; Steiner, D.F. (Hagedorn Research Laboratory, Gentofte (Denmark))

1988-09-01

The pluripotent rat islet tumor cell line MSL-G2 expresses primarily glucagon or cholecystokinin and not insulin in vitro but changes phenotype completely after prolonged in vivo cultivation to yield small-sized hypoglycemic tumors composed almost entirely of insulin-producing beta cells. When a genomic DNA fragment containing the coding and upstream regulatory regions of the human insulin gene was stably transfected into MSL-G2 cells no measurable amounts of insulin or insulin mRNA were detected in vitro. However, successive transplantation of two transfected clones resulted in hypoglycemic tumors that efficiently coexpressed human and rat insulin as determined by human C-peptide-specific immunoreagents. These results demonstrate that cis-acting tissue-specific insulin gene enhancer elements are conserved between rat and human insulin genes. The authors propose that the in vivo differentiation of MSL-G2 cells and transfected subclones into insulin-producing cells reflects processes of natural beta-cell ontogeny leading to insulin gene expression.

Tissue-specific expression of transfected human insulin genes in pluripotent clonal rat insulinoma lines induced during passage in vivo

International Nuclear Information System (INIS)

Madsen, O.D.; Andersen, L.C.; Michelsen, B.; Owerbach, D.; Larsson, L.I.; Lernmark, A.; Steiner, D.F.

1988-01-01

The pluripotent rat islet tumor cell line MSL-G2 expresses primarily glucagon or cholecystokinin and not insulin in vitro but changes phenotype completely after prolonged in vivo cultivation to yield small-sized hypoglycemic tumors composed almost entirely of insulin-producing beta cells. When a genomic DNA fragment containing the coding and upstream regulatory regions of the human insulin gene was stably transfected into MSL-G2 cells no measurable amounts of insulin or insulin mRNA were detected in vitro. However, successive transplantation of two transfected clones resulted in hypoglycemic tumors that efficiently coexpressed human and rat insulin as determined by human C-peptide-specific immunoreagents. These results demonstrate that cis-acting tissue-specific insulin gene enhancer elements are conserved between rat and human insulin genes. The authors propose that the in vivo differentiation of MSL-G2 cells and transfected subclones into insulin-producing cells reflects processes of natural beta-cell ontogeny leading to insulin gene expression

The stably stratified internal boundary layer for steady and diurnally varying offshore flow

Science.gov (United States)

Garratt, J. R.

1987-03-01

A two-dimensional numerical mesoscale model is used to investigate the internal structure and growth of the stably stratified internal boundary layer (IBL) beneath warm, continental air flowing over a cooler sea. Two situations are studied — steady-state and diurnally varying offshore flow. In the steady-state case, vertical profiles of mean quantities and eddy diffusion coefficients ( K) within the IBL show small, but significant, changes with increasing distance from the coast. The top of the IBL is well defined, with large vertical gradients within the layer and a maximum in the coast-normal wind component near the top. Well away from the coast, turbulence, identified by non-zero K, decreases to insignificant levels near the top of the IBL; the IBL itself is characterised by a critical value of the layer-flux Richardson number equal to 0.18. The overall behaviour of the mean profiles is similar to that found in the horizontally homogeneous stable boundary layer over land. A simple physical model is used to relate the depth of the layer h to several relevant physical parameters viz., x, the distance from the coast and U, the large-scale wind (both normal to the coastline) and gδθ/θ, Δθ being the temperature difference between continental mixed-layer air and sea surface, θ is the mean potential temperature and g is the acceleration due to gravity. Excellent agreement with the numerical results is found, with h = 0.014 x 1/2 U ( gδθ/θ)-1/2. In the diurnally varying case, the mean profiles within the IBL show only small differences from the steady-state case, although diurnal variations, particularly in the wind maximum, are evident within a few hundred kilometres of the coast. A mesoscale circulation normal to the coast, and superimposed upon the mean offshore flow, develops seawards of the coastline with maximum vertical velocities about sunset, of depth about 2 km and horizontal scale ≈ 500 km. The circulation is related to the advection, and

Molecular basis and drug sensitivity of the delayed rectifier (IKr) in the fish heart.

Science.gov (United States)

Hassinen, Minna; Haverinen, Jaakko; Vornanen, Matti

2015-01-01

Fishes are increasingly used as models for human cardiac diseases, creating a need for a better understanding of the molecular basis of fish cardiac ion currents. To this end we cloned KCNH6 channel of the crucian carp (Carassius carassius) that produces the rapid component of the delayed rectifier K(+) current (IKr), the main repolarising current of the fish heart. KCNH6 (ccErg2) was the main isoform of the Kv11 potassium channel family with relative transcript levels of 98.9% and 99.6% in crucian carp atrium and ventricle, respectively. KCNH2 (ccErg1), an orthologue to human cardiac Erg (Herg) channel, was only slightly expressed in the crucian carp heart. The native atrial IKr and the cloned ccErg2 were inhibited by similar concentrations of verapamil, terfenadine and KB-R7943 (P>0.05), while the atrial IKr was about an order of magnitude more sensitive to E-4031 than ccErg2 (P<0.05) suggesting that some accessory β-subunits may be involved. Sensitivity of the crucian carp atrial IKr to E-4031, terfenadine and KB-R7943 was similar to what has been reported for the Herg channel. In contrast, the sensitivity of the crucian carp IKr to verapamil was approximately 30 times higher than the previously reported values for the Herg current. In conclusion, the cardiac IKr is produced by non-orthologous gene products in fish (Erg2) and mammalian hearts (Erg1) and some marked differences exist in drug sensitivity between fish and mammalian Erg1/2 which need to be taken into account when using fish heart as a model for human heart. Copyright © 2015 Elsevier Inc. All rights reserved.

Design of Tokamak synchronous data acquisition system based on PXI express

International Nuclear Information System (INIS)

Liu Rui; Zheng Wei; Zhang Ming; Weng Chuqiao; Zhuang Ge; Ding Tonghai; Yu Kexun

2014-01-01

With the development of J-TEXT device, the original data acquisition system can't meet the experiment's requirement on stability, modularity and sampling rate, so a new data acquisition system needs to be built. This paper introduces the design and implementation of the distributed Tokamak synchronous high-speed data acquisition system based on PXI Express. The acquisition unit consists of PXIe case Nl PXIe 1062Q, PXIe controller NI PXIe-8133 and high-speed synchronous data acquisition card Nl PXIe-6368, compatible with the latest standard of ITER CODAC, so it has good mechanical sealing, strong modularity and high sampling rate etc. The system takes a synchronous difference acquisition for diagnosis signal. The data storage adopts MDSplus which is the general database in the nuclear fusion field. The test and experimental results show that the system can work continuously and stably at 2 MSps sampling rate, and meet the requirement of experiment device's operation well. (authors)

Selection and validation of endogenous reference genes for qRT-PCR analysis in leafy spurge (Euphorbia esula.

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Wun S Chao

Full Text Available Quantitative real-time polymerase chain reaction (qRT-PCR is the most important tool in measuring levels of gene expression due to its accuracy, specificity, and sensitivity. However, the accuracy of qRT-PCR analysis strongly depends on transcript normalization using stably expressed reference genes. The aim of this study was to find internal reference genes for qRT-PCR analysis in various experimental conditions for seed, adventitious underground bud, and other organs of leafy spurge. Eleven candidate reference genes (BAM4, PU1, TRP-like, FRO1, ORE9, BAM1, SEU, ARF2, KAPP, ZTL, and MPK4 were selected from among 171 genes based on expression stabilities during seed germination and bud growth. The other ten candidate reference genes were selected from three different sources: (1 3 stably expressed leafy spurge genes (60S, bZIP21, and MD-100 identified from the analyses of leafy spurge microarray data; (2 3 orthologs of Arabidopsis "general purpose" traditional reference genes (GAPDH_1, GAPDH_2, and UBC; and (3 4 orthologs of Arabidopsis stably expressed genes (UBC9, SAND, PTB, and F-box identified from Affymetrix ATH1 whole-genome GeneChip studies. The expression stabilities of these 21 genes were ranked based on the C(T values of 72 samples using four different computation programs including geNorm, Normfinder, BestKeeper, and the comparative ΔC(T method. Our analyses revealed SAND, PTB, ORE9, and ARF2 to be the most appropriate reference genes for accurate normalization of gene expression data. Since SAND and PTB were obtained from 4 orthologs of Arabidopsis, while ORE9 and ARF2 were selected from 171 leafy spurge genes, it was more efficient to identify good reference genes from the orthologs of other plant species that were known to be stably expressed than that of randomly testing endogenous genes. Nevertheless, the two newly identified leafy spurge genes, ORE9 and ARF2, can serve as orthologous candidates in the search for reference genes

Initiator of carcinogenesis selectively and stably inhibits stem cell differentiation: a concept that initiation of carcinogenesis involves multiple phases

International Nuclear Information System (INIS)

Scott, R.E.; Maercklein, P.B.

1985-01-01

A concept of carcinogenesis was recently devised in our laboratory that suggests the development of defects in the control of cell differentiation is associated with an early phase of carcinogenesis. To test this proposal directly, the effects of an initiator of carcinogenesis (i.e., UV irradiation) on proadipocyte stem cell differentiation and proliferation was assayed. In this regard, 3T3 T proadipocytes represent a nontransformed mesenchymal stem cell line that possesses the ability to regulate its differentiation at a distinct state in the G 1 phase of the cell cycle as well as the ability to regulate its proliferation at two additional G 1 states. The results establish that a slow dosage of 254 nm UV irradiation selectivity and stably inhibits the differentiation of a high percentage of proadipocyte stem cells without significantly altering their ability to regulate cellular proliferation in growth factor-deficient or nutrient-deficient culture conditions. Differentiation-defect proadipocyte stem cells are demonstrated not to be completely transformed but to show an increased spontaneous transformation rate, as evidenced by the formation of type III foci in high density cell cultures. These data support the role of defects in the control of differentiation in the inhibition of carcinogenesis. These observations support a concept that the initiation of carcinogenesis involves multiple phases

Amyloid precursor protein regulates migration and metalloproteinase gene expression in prostate cancer cells

Energy Technology Data Exchange (ETDEWEB)

Miyazaki, Toshiaki; Ikeda, Kazuhiro; Horie-Inoue, Kuniko [Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Saitama 350-1241 (Japan); Inoue, Satoshi, E-mail: INOUE-GER@h.u-tokyo.ac.jp [Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Saitama 350-1241 (Japan); Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655 (Japan); Department of Anti-Aging Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655 (Japan)

2014-09-26

Highlights: • APP knockdown reduced proliferation and migration of prostate cancer cells. • APP knockdown reduced expression of metalloproteinase and EMT-related genes. • APP overexpression promoted LNCaP cell migration. • APP overexpression increased expression of metalloproteinase and EMT-related genes. - Abstract: Amyloid precursor protein (APP) is a type I transmembrane protein, and one of its processed forms, β-amyloid, is considered to play a central role in the development of Alzheimer’s disease. We previously showed that APP is a primary androgen-responsive gene in prostate cancer and that its increased expression is correlated with poor prognosis for patients with prostate cancer. APP has also been implicated in several human malignancies. Nevertheless, the mechanism underlying the pro-proliferative effects of APP on cancers is still not well-understood. In the present study, we explored a pathophysiological role for APP in prostate cancer cells using siRNA targeting APP (siAPP). The proliferation and migration of LNCaP and DU145 prostate cancer cells were significantly suppressed by siAPP. Differentially expressed genes in siAPP-treated cells compared to control siRNA-treated cells were identified by microarray analysis. Notably, several metalloproteinase genes, such as ADAM10 and ADAM17, and epithelial–mesenchymal transition (EMT)-related genes, such as VIM, and SNAI2, were downregulated in siAPP-treated cells as compared to control cells. The expression of these genes was upregulated in LNCaP cells stably expressing APP when compared with control cells. APP-overexpressing LNCaP cells exhibited enhanced migration in comparison to control cells. These results suggest that APP may contribute to the proliferation and migration of prostate cancer cells by modulating the expression of metalloproteinase and EMT-related genes.

Evaluation of endogenous control gene(s) for gene expression studies in human blood exposed to 60Co γ-rays ex vivo

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Vaiphei, S. Thangminlal; Keppen, Joshua; Nongrum, Saibadaiahun; Sharan, R.N.; Chaubey, R.C.; Kma, L.

2015-01-01

In gene expression studies, it is critical to normalize data using a stably expressed endogenous control gene in order to obtain accurate and reliable results. However, we currently do not have a universally applied endogenous control gene for normalization of data for gene expression studies, particularly those involving 60 Co γ-ray-exposed human blood samples. In this study, a comparative assessment of the gene expression of six widely used housekeeping endogenous control genes, namely 18S, ACTB, B2M, GAPDH, MT-ATP6 and CDKN1A, was undertaken for a range of 60 Co γ-ray doses (0.5, 1.0, 2.0 and 4.0 Gy) at 8.4 Gy min -1 at 0 and 24 h post-irradiation time intervals. Using the NormFinder algorithm, real-time PCR data obtained from six individuals (three males and three females) were analyzed with respect to the threshold cycle (Ct) value and abundance, ΔCt pair-wise comparison, intra- and inter-group variability assessments, etc. GAPDH, either alone or in combination with 18S, was found to be the most suitable endogenous control gene and should be used in gene expression studies, especially those involving qPCR of γ-ray-exposed human blood samples. (author)

Pharmacological profile of DA-6886, a novel 5-HT4 receptor agonist to accelerate colonic motor activity in mice.

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Lee, Min Jung; Cho, Kang Hun; Park, Hyun Min; Sung, Hyun Jung; Choi, Sunghak; Im, Weonbin

2014-07-15

DA-6886, the gastrointestinal prokinetic benzamide derivative is a novel 5-HT4 receptor agonist being developed for the treatment of constipation-predominant irritable bowel syndrome (IBS-C). The purpose of this study was to characterize in vitro and in vivo pharmacological profile of DA-6886. We used various receptor binding assay, cAMP accumulation assay, organ bath experiment and colonic transit assay in normal and chemically constipated mice. DA-6886 exhibited high affinity and selectivity to human 5-HT4 receptor splice variants, with mean pKi of 7.1, 7.5, 7.9 for the human 5-HT4a, 5-HT4b and 5-HT4d, respectively. By contrast, DA-6886 did not show significant affinity for several receptors including dopamine D2 receptor, other 5-HT receptors except for 5-HT2B receptor (pKi value of 6.2). The affinity for 5-HT4 receptor was translated into functional agonist activity in Cos-7 cells expressing 5-HT4 receptor splice variants. Furthermore, DA-6886 induced relaxation of the rat oesophagus preparation (pEC50 value of 7.4) in a 5-HT4 receptor antagonist-sensitive manner. The evaluation of DA-6886 in CHO cells expressing hERG channels revealed that it inhibited hERG channel current with an pIC50 value of 4.3, indicating that the compound was 1000-fold more selective for the 5-HT4 receptor over hERG channels. In the normal ICR mice, oral administration of DA-6886 (0.4 and 2mg/kg) resulted in marked stimulation of colonic transit. Furthermore, in the loperamide-induced constipation mouse model, 2mg/kg of DA-6886 significantly improved the delay of colonic transit, similar to 10mg/kg of tegaserod. Taken together, DA-6886 is a highly potent and selective 5-HT4 receptor agonist to accelerate colonic transit in mice, which might be therapeutic agent having a favorable safety profile in the treatment of gastrointestinal motor disorders such as IBS-C and chronic constipation. Copyright © 2014 Elsevier B.V. All rights reserved.

Quantitative monitoring of the Chlamydia trachomatis developmental cycle using GFP-expressing bacteria, microscopy and flow cytometry.

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François Vromman

Full Text Available Chlamydiae are obligate intracellular bacteria. These pathogens develop inside host cells through a biphasic cycle alternating between two morphologically distinct forms, the infectious elementary body and the replicative reticulate body. Recently, C. trachomatis strains stably expressing fluorescent proteins were obtained. The fluorochromes are expressed during the intracellular growth of the microbe, allowing bacterial visualization by fluorescence microscopy. Whether they are also present in the infectious form, the elementary body, to a detectable level has not been studied. Here, we show that a C. trachomatis strain transformed with a plasmid expressing the green fluorescent protein (GFP accumulates sufficient quantities of the probe in elementary bodies for detection by microscopy and flow cytometry. Adhesion of single bacteria was detected. The precise kinetics of bacterial entry were determined by microscopy using automated procedures. We show that during the intracellular replication phase, GFP is a convenient read-out for bacterial growth with several advantages over current methods. In particular, infection rates within a non-homogenous cell population are easily quantified. Finally, in spite of their small size, individual elementary bodies are detected by flow cytometers, allowing for direct enumeration of a bacterial preparation. In conclusion, GFP-expressing chlamydiae are suitable to monitor, in a quantitative manner, progression throughout the developmental cycle. This will facilitate the identification of the developmental steps targeted by anti-chlamydial drugs or host factors.

LyGDI expression in HeLa cells increased its sensitivity to radiation-induced apoptosis

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Zhou Xinwen; Xu Yaxiang

2006-01-01

Objective: In order to confirm whether LyGDI has apoptotic signal transduction function and can increase the apoptotic rate of radiation-induced cell death, the lyGDI and mutant D19lyGDI gene, which constructed with the pCDNA3. 1 His A, were transfected into no-endogenous lyGDI HeLa cells. Methods Transient expressions of lyGDI and D19lyGDI in HeLa cells were analyzed by Western blot using anti-mono antibody of LyGDI and Xpress tag. Cell apoptosis was assayed with Annexin V-FITC apoptosis kit. To select stable clone, the transferred HeLa cells had been maintained in G418 medium for 3 weeks, then a cell line, which stably expressed LyGDI and mutant D19lyGDI, was selected. The selected cell line was irradiated with 12 Gy 60 Co y-rays. Caspase-3 activity of the cells was determined by Western blot and cell viability by clone-forming assay after 48 hours post-irradiation culture. Results: Western blot and Annexin V-FITC apoptotic analysis revealed that lyGDI and D19lyGDI transient expressions in HeLa cells induced apoptosis; Caspase-3 activity measurement and clone-forming assay showed that lyGDI increased sensitivity to radiation-induced cell apoptosis. Conclusions: lyGDI performs function in apoptosis signal transduction, its expression in HeLa cells can increase the sensitivity to radiation-induced cell apoptosis. (authors)

Selection and validation of a set of reliable reference genes for quantitative sod gene expression analysis in C. elegans

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Vandesompele Jo

2008-01-01

Full Text Available Abstract Background In the nematode Caenorhabditis elegans the conserved Ins/IGF-1 signaling pathway regulates many biological processes including life span, stress response, dauer diapause and metabolism. Detection of differentially expressed genes may contribute to a better understanding of the mechanism by which the Ins/IGF-1 signaling pathway regulates these processes. Appropriate normalization is an essential prerequisite for obtaining accurate and reproducible quantification of gene expression levels. The aim of this study was to establish a reliable set of reference genes for gene expression analysis in C. elegans. Results Real-time quantitative PCR was used to evaluate the expression stability of 12 candidate reference genes (act-1, ama-1, cdc-42, csq-1, eif-3.C, mdh-1, gpd-2, pmp-3, tba-1, Y45F10D.4, rgs-6 and unc-16 in wild-type, three Ins/IGF-1 pathway mutants, dauers and L3 stage larvae. After geNorm analysis, cdc-42, pmp-3 and Y45F10D.4 showed the most stable expression pattern and were used to normalize 5 sod expression levels. Significant differences in mRNA levels were observed for sod-1 and sod-3 in daf-2 relative to wild-type animals, whereas in dauers sod-1, sod-3, sod-4 and sod-5 are differentially expressed relative to third stage larvae. Conclusion Our findings emphasize the importance of accurate normalization using stably expressed reference genes. The methodology used in this study is generally applicable to reliably quantify gene expression levels in the nematode C. elegans using quantitative PCR.

Mechanisms of IhERG/IKr Modulation by α1-Adrenoceptors in HEK293 Cells and Cardiac Myocytes

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Janire Urrutia

2016-12-01

Full Text Available Background: The rapid delayed rectifier K+ current (IKr, carried by the hERG protein, is one of the main repolarising currents in the human heart and a reduction of this current increases the risk of ventricular fibrillation. α1-adrenoceptors (α1-AR activation reduces IKr but, despite the clear relationship between an increase in the sympathetic tone and arrhythmias, the mechanisms underlying the α1-AR regulation of the hERG channel are controversial. Thus, we aimed to investigate the mechanisms by which α1-AR stimulation regulates IKr. Methods: α1-adrenoceptors, hERG channels, auxiliary subunits minK and MIRP1, the non PIP2-interacting mutant D-hERG (with a deletion of the 883-894 amino acids in the C-terminal and the non PKC-phosphorylable mutant N-terminal truncated-hERG (NTK-hERG were transfected in HEK293 cells. Cell membranes were extracted by centrifugation and the different proteins were visualized by Western blot. Potassium currents were recorded by the patch-clamp technique. IKr was recorded in isolated feline cardiac myocytes. Results: Activation of the α1-AR reduces the amplitude of IhERG and IKr through a positive shift in the activation half voltage, which reduces the channel availability at physiological membrane potentials. The intracellular pathway connecting the α1-AR to the hERG channel in HEK293 cells includes activation of the Gαq protein, PLC activation and PIP2 hydrolysis, activation of PKC and direct phosphorylation of the hERG channel N-terminal. The PKC-mediated IKr channel phosphorylation and subsequent IKr reduction after α1-AR stimulation was corroborated in feline cardiac myocytes. Conclusions: These findings clarify the link between sympathetic nervous system hyperactivity and IKr reduction, one of the best characterized causes of torsades de pointes and ventricular fibrillation.

Directed chromosomal integration and expression of porcine rotavirus outer capsid protein VP4 in Lactobacillus casei ATCC393.

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Yin, Ji-Yuan; Guo, Chao-Qun; Wang, Zi; Yu, Mei-Ling; Gao, Shuai; Bukhari, Syed M; Tang, Li-Jie; Xu, Yi-Gang; Li, Yi-Jing

2016-11-01

Using two-step plasmid integration in the presence of 5-fluorouracil (5-FU), we developed a stable and markerless Lactobacillus casei strain for vaccine antigen expression. The upp of L. casei, which encodes uracil phosphoribosyltransferase (UPRTase), was used as a counterselection marker. We employed the Δupp isogenic mutant, which is resistant to 5-FU, as host and a temperature-sensitive suicide plasmid bearing upp expression cassette as counterselectable integration vector. Extrachromosomal expression of UPRTase complemented the mutated chromosomal upp allele and restored sensitivity to 5-FU. The resultant genotype can either be wild type or recombinant. The efficacy of the system was demonstrated by insertion and expression of porcine rotavirus (PRV) VP4. To improve VP4 expression, we analyzed L. casei transcriptional profiles and selected the constitutive highly expressed enolase gene (eno). The VP4 inserted after the eno termination codon were screened in the presence of 5-FU. Using genomic PCR amplification, we confirmed that VP4 was successfully integrated and stably inherited for at least 50 generations. Western blot demonstrated that VP4 was steadily expressed in medium with different carbohydrates. RT-qPCR and ELISA analysis showed that VP4 expression from the chromosomal location was similar to that achieved by a plasmid expression system. Applying the recombinant strain to immunize BALB/c mice via oral administration revealed that the VP4-expressing L. casei could induce both specific local and systemic humoral immune responses in mice. Overall, the improved gene replacement system represents an efficient method for chromosome recombination in L. casei and provides a safe tool for vaccine production.

Molecular Characterization, Tissue Distribution and Expression, and Potential Antiviral Effects of TRIM32 in the Common Carp (Cyprinus carpio)

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Wang, Yeda; Li, Zeming; Lu, Yuanan; Hu, Guangfu; Lin, Li; Zeng, Lingbing; Zhou, Yong; Liu, Xueqin

2016-01-01

Tripartite motif-containing protein 32 (TRIM32) belongs to the tripartite motif (TRIM) family, which consists of a large number of proteins containing a RING (Really Interesting New Gene) domain, one or two B-box domains, and coiled coil motif followed by different C-terminal domains. The TRIM family is known to be implicated in multiple cellular functions, including antiviral activity. However, it is presently unknown whether TRIM32 of common carp (Cyprinus carpio) has the antiviral effect. In this study, the sequence, expression, and antiviral function of TRIM32 homolog from common carp were analyzed. The full-length coding sequence region of trim32 was cloned from common carp. The results showed that the expression of TRIM32 (mRNA) was highest in the brain, remained stably expressed during embryonic development, and significantly increased following spring viraemia of carp virus (SVCV) infection. Transient overexpression of TRIM32 in affected Epithelioma papulosum cyprinid cells led to significant decrease of SVCV production as compared to the control group. These results suggested a potentially important role of common carp TRIM32 in enhancing host immune response during SVCV infection both in vivo and in vitro. PMID:27735853

Role and expression of FRS2 and FRS3 in prostate cancer

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Valencia, Tania; Joseph, Ajay; Kachroo, Naveen; Darby, Steve; Meakin, Susan; Gnanapragasam, Vincent J

2011-01-01

FGF receptor substrates (FRS2 and FRS3) are key adaptor proteins that mediate FGF-FGFR signalling in benign as well as malignant tissue. Here we investigated FRS2 and FRS3 as a means of disrupting global FGF signalling in prostate cancer. FRS2 and FRS3 manipulation was investigated in vitro using over-expression, knockdown and functional assays. FRS2 and FRS3 expression was profiled in cell lines and clinical tumors of different grades. In a panel of cell lines we observed ubiquitous FRS2 and FRS3 transcript and protein expression in both benign and malignant cells. We next tested functional redundancy of FRS2 and FRS3 in prostate cancer cells. In DU145 cells, specific FRS2 suppression inhibited FGF induced signalling. This effect was not apparent in cells stably over-expressing FRS3. Indeed FRS3 over-expression resulted in enhanced proliferation (p = 0.005) compared to control cells. Given this functional redundancy, we tested the therapeutic principle of dual targeting of FRS2 and FRS3 in prostate cancer. Co-suppression of FRS2 and FRS3 significantly inhibited ERK activation with a concomitant reduction in cell proliferation (p < 0.05), migration and invasion (p < 0.05). Synchronous knockdown of FRS2 and FRS3 with exposure to cytotoxic irradiation resulted in a significant reduction in prostate cancer cell survival compared to irradiation alone (p < 0.05). Importantly, this synergistic effect was not observed in benign cells. Finally, we investigated expression of FRS2 and FRS3 transcript in a cohort of micro-dissected tumors of different grades as well as by immunohistochemistry in clinical biopsies. Here, we did not observe any difference in expression between benign and malignant biopsies. These results suggest functional overlap of FRS2 and FRS3 in mediating mitogenic FGF signalling in the prostate. FRS2 and FRS3 are not over-expressed in tumours but targeted dual inhibition may selectively adversely affect malignant but not benign prostate cells

YB-1 facilitates basal and 5-fluorouracil-inducible expression of the human major vault protein (MVP) gene.

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Stein, Ulrike; Bergmann, Stephan; Scheffer, George L; Scheper, Rik J; Royer, Hans-Dieter; Schlag, Peter M; Walther, Wolfgang

2005-05-19

Vaults have been suggested to play a direct role in multidrug resistance (MDR) to anticancer drugs. The human major vault protein (MVP) also known as lung resistance-related protein (LRP) represents the predominant component of vaults that may be involved in the defense against xenobiotics. Here, we demonstrate that besides MDR-related cytostatics, also the non-MDR-related drug 5-fluorouracil (5-FU) was able to induce MVP mRNA and protein expression. Treatment with 5-FU amplified the binding activity and interaction of the transcription factor Y-box binding protein-1 (YB-1) with the Y-box of the human MVP gene promoter in a time-dependent manner. 5-FU also induced reporter expressions driven by a panel of newly generated MVP promoter deletion mutants. Interestingly, stably YB-1 overexpressing cell clones showed enhanced binding of YB-1 to the Y-box motif, associated with enhanced basal as well as 5-FU-inducible MVP promoter-driven reporter expressions. Moreover, transduction of YB-1 cDNA led to increased expression of endogenous MVP protein. Under physiological conditions, we observed a strong coexpression of MVP and YB-1 in human colon carcinoma specimen. In summary, our data demonstrate a direct involvement of YB-1 in controlling basal and 5-FU-induced MVP promoter activity. Therefore, YB-1 is directly linked to MVP-mediated drug resistance.

Identification of rat Rosa26 locus enables generation of knock-in rat lines ubiquitously expressing tdTomato.

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Kobayashi, Toshihiro; Kato-Itoh, Megumi; Yamaguchi, Tomoyuki; Tamura, Chihiro; Sanbo, Makoto; Hirabayashi, Masumi; Nakauchi, Hiromitsu

2012-11-01

Recent discovery of a method for derivation and culture of germline-competent rat pluripotent stem cells (PSCs) enables generation of transgenic rats or knock-out rats via genetic modification of such PSCs. This opens the way to use rats, as is routine in mice, for analyses of gene functions or physiological features. In mouse or human, one widely used technique to express a gene of interest stably and ubiquitously is to insert that gene into the Rosa26 locus via gene targeting of PSCs. Rosa26 knock-in mice conditionally expressing a reporter or a toxin gene have contributed to tracing or ablation of specific cell lineages. We successfully identified a rat orthologue of the mouse Rosa26 locus. Insertion of tdTomato, a variant of red fluorescent protein, into the Rosa26 locus of PSCs of various rat strains allows ubiquitous expression of tdTomato. Through germline transmission of one Rosa26-tdTomato knock-in embryonic stem cell line, we also obtained tdTomato knock-in rats. These expressed tdTomato ubiquitously throughout their bodies, which indicates that the rat Rosa26 locus conserves functions of its orthologues in mouse and human. The new tools described here (targeting vectors, knock-in PSCs, and rats) should be useful for a variety of research using rats.

SIRT1 sensitizes hepatocellular carcinoma cells expressing hepatitis B virus X protein to oxidative stress-induced apoptosis

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Srisuttee, Ratakorn; Koh, Sang Seok; Malilas, Waraporn; Moon, Jeong; Cho, Il-Rae; Jhun, Byung Hak; Horio, Yoshiyuki; Chung, Young-Hwa

2012-01-01

Highlights: ► Up-regulation of SIRT1 protein and activity sensitizes Hep3B-HBX cells to oxidative stress-induced apoptosis. ► Nuclear localization of SIRT1 is not required for oxidation-induced apoptosis. ► Ectopic expression and enhanced activity of SIRT1 attenuate JNK phosphorylation. ► Inhibition of SIRT1 activity restores resistance to oxidation-induced apoptosis through JNK activation. -- Abstract: We previously showed that SIRT1 deacetylase inhibits proliferation of hepatocellular carcinoma cells expressing hepatitis B virus (HBV) X protein (HBX), by destabilization of β-catenin. Here, we report another role for SIRT1 in HBX-mediated resistance to oxidative stress. Ectopic expression and enhanced activity of SIRT1 sensitize Hep3B cells stably expressing HBX to oxidative stress-induced apoptosis. SIRT1 mutant analysis showed that nuclear localization of SIRT1 is not required for sensitization of oxidation-mediated apoptosis. Furthermore, ectopic expression of SIRT1 and treatment with resveratrol (a SIRT1 activator) attenuated JNK phosphorylation, which is a prerequisite for resistance to oxidative stress-induced apoptosis. Conversely, suppression of SIRT1 activity with nicotinamide inhibited the effect of resveratrol on JNK phosphorylation, leading to restoration of resistance to oxidation-induced apoptosis. Taken together, these results suggest that up-regulation of SIRT1 under oxidative stress may be a therapeutic strategy for treatment of hepatocellular carcinoma cells related to HBV through inhibition of JNK activation.

SIRT1 sensitizes hepatocellular carcinoma cells expressing hepatitis B virus X protein to oxidative stress-induced apoptosis

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Srisuttee, Ratakorn [WCU, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-735 (Korea, Republic of); Koh, Sang Seok [Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology, University of Science and Technology, Daejeon 305-333 (Korea, Republic of); Department of Functional Genomics, University of Science and Technology, Daejeon 305-333 (Korea, Republic of); Malilas, Waraporn; Moon, Jeong; Cho, Il-Rae [WCU, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-735 (Korea, Republic of); Jhun, Byung Hak [Department of Applied Nanoscience, Pusan National University, Busan 609-735 (Korea, Republic of); Horio, Yoshiyuki [Department of Pharmacology, Sapporo Medical University, Sapporo 060-8556 (Japan); Chung, Young-Hwa, E-mail: younghc@pusan.ac.kr [WCU, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-735 (Korea, Republic of)

2012-12-07

Highlights: Black-Right-Pointing-Pointer Up-regulation of SIRT1 protein and activity sensitizes Hep3B-HBX cells to oxidative stress-induced apoptosis. Black-Right-Pointing-Pointer Nuclear localization of SIRT1 is not required for oxidation-induced apoptosis. Black-Right-Pointing-Pointer Ectopic expression and enhanced activity of SIRT1 attenuate JNK phosphorylation. Black-Right-Pointing-Pointer Inhibition of SIRT1 activity restores resistance to oxidation-induced apoptosis through JNK activation. -- Abstract: We previously showed that SIRT1 deacetylase inhibits proliferation of hepatocellular carcinoma cells expressing hepatitis B virus (HBV) X protein (HBX), by destabilization of {beta}-catenin. Here, we report another role for SIRT1 in HBX-mediated resistance to oxidative stress. Ectopic expression and enhanced activity of SIRT1 sensitize Hep3B cells stably expressing HBX to oxidative stress-induced apoptosis. SIRT1 mutant analysis showed that nuclear localization of SIRT1 is not required for sensitization of oxidation-mediated apoptosis. Furthermore, ectopic expression of SIRT1 and treatment with resveratrol (a SIRT1 activator) attenuated JNK phosphorylation, which is a prerequisite for resistance to oxidative stress-induced apoptosis. Conversely, suppression of SIRT1 activity with nicotinamide inhibited the effect of resveratrol on JNK phosphorylation, leading to restoration of resistance to oxidation-induced apoptosis. Taken together, these results suggest that up-regulation of SIRT1 under oxidative stress may be a therapeutic strategy for treatment of hepatocellular carcinoma cells related to HBV through inhibition of JNK activation.

Validation of suitable reference genes for quantitative gene expression analysis in Panax ginseng

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Meizhen eWang

2016-01-01

Full Text Available Reverse transcription-qPCR (RT-qPCR has become a popular method for gene expression studies. Its results require data normalization by housekeeping genes. No single gene is proved to be stably expressed under all experimental conditions. Therefore, systematic evaluation of reference genes is necessary. With the aim to identify optimum reference genes for RT-qPCR analysis of gene expression in different tissues of Panax ginseng and the seedlings grown under heat stress, we investigated the expression stability of eight candidate reference genes, including elongation factor 1-beta (EF1-β, elongation factor 1-gamma (EF1-γ, eukaryotic translation initiation factor 3G (IF3G, eukaryotic translation initiation factor 3B (IF3B, actin (ACT, actin11 (ACT11, glyceraldehyde-3-phosphate dehydrogenase (GAPDH and cyclophilin ABH-like protein (CYC, using four widely used computational programs: geNorm, Normfinder, BestKeeper, and the comparative ΔCt method. The results were then integrated using the web-based tool RefFinder. As a result, EF1-γ, IF3G and EF1-β were the three most stable genes in different tissues of P. ginseng, while IF3G, ACT11 and GAPDH were the top three-ranked genes in seedlings treated with heat. Using three better reference genes alone or in combination as internal control, we examined the expression profiles of MAR, a multiple function-associated mRNA-like non-coding RNA (mlncRNA in P. ginseng. Taken together, we recommended EF1-γ/IF3G and IF3G/ACT11 as the suitable pair of reference genes for RT-qPCR analysis of gene expression in different tissues of P. ginseng and the seedlings grown under heat stress, respectively. The results serve as a foundation for future studies on P. ginseng functional genomics.

Stably engineered nanobubbles and ultrasound - An effective platform for enhanced macromolecular delivery to representative cells of the retina.

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Sachin S Thakur

Full Text Available Herein we showcase the potential of ultrasound-responsive nanobubbles in enhancing macromolecular permeation through layers of the retina, ultimately leading to significant and direct intracellular delivery; this being effectively demonstrated across three relevant and distinct retinal cell lines. Stably engineered nanobubbles of a highly homogenous and echogenic nature were fully characterised using dynamic light scattering, B-scan ultrasound and transmission electron microscopy (TEM. The nanobubbles appeared as spherical liposome-like structures under TEM, accompanied by an opaque luminal core and darkened corona around their periphery, with both features indicative of efficient gas entrapment and adsorption, respectively. A nanobubble +/- ultrasound sweeping study was conducted next, which determined the maximum tolerated dose for each cell line. Detection of underlying cellular stress was verified using the biomarker heat shock protein 70, measured before and after treatment with optimised ultrasound. Next, with safety to nanobubbles and optimised ultrasound demonstrated, each human or mouse-derived cell population was incubated with biotinylated rabbit-IgG in the presence and absence of ultrasound +/- nanobubbles. Intracellular delivery of antibody in each cell type was then quantified using Cy3-streptavidin. Nanobubbles and optimised ultrasound were found to be negligibly toxic across all cell lines tested. Macromolecular internalisation was achieved to significant, yet varying degrees in all three cell lines. The results of this study pave the way towards better understanding mechanisms underlying cellular responsiveness to ultrasound-triggered drug delivery in future ex vivo and in vivo models of the posterior eye.

An Archaea 5S rRNA analog is stably expressed in Escherichia coli

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Yang, Y.; Fox, G. E.

1996-01-01

Mini-genes for 5S-like rRNA were constructed. These genes had a sequence which largely resembles that of the naturally occurring 5S rRNA of a bacterium, Halococcus morrhuae, which phylogenetically belongs to the Archaea. Plasmids carrying the mini-genes were transformed into Escherichia coli (Ec). Ribosomal incorporation was not a prerequisite for stable accumulation of the RNA product. However, only those constructs with a well-base-paired helix I accumulated RNA product. This result strongly implies that this aspect of the structure is likely to be an important condition for stabilizing 5S rRNA-like products. The results are consistent with our current understanding of 5S rRNA processing in Ec. When used in conjunction with rRNA probe technology, the resulting chimeric RNA may be useful as a monitoring tool for genetically engineered microorganisms or naturally occurring organisms that are released into the environment.

Expression of acid-sensing ion channels and selection of reference genes in mouse and naked mole rat.

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Schuhmacher, Laura-Nadine; Smith, Ewan St John

2016-12-13

Acid-sensing ion channels (ASICs) are a family of ion channels comprised of six subunits encoded by four genes and they are expressed throughout the peripheral and central nervous systems. ASICs have been implicated in a wide range of physiological and pathophysiological processes: pain, breathing, synaptic plasticity and excitotoxicity. Unlike mice and humans, naked mole-rats do not perceive acid as a noxious stimulus, even though their sensory neurons express functional ASICs, likely an adaptation to living in a hypercapnic subterranean environment. Previous studies of ASIC expression in the mammalian nervous system have often not examined all subunits, or have failed to adequately quantify expression between tissues; to date there has been no attempt to determine ASIC expression in the central nervous system of the naked mole-rat. Here we perform a geNorm study to identify reliable housekeeping genes in both mouse and naked mole-rat and then use quantitative real-time PCR to estimate the relative amounts of ASIC transcripts in different tissues of both species. We identify RPL13A (ribosomal protein L13A) and CANX (calnexin), and β-ACTIN and EIF4A (eukaryotic initiation factor 4a) as being the most stably expressed housekeeping genes in mouse and naked mole-rat, respectively. In both species, ASIC3 was most highly expressed in dorsal root ganglia (DRG), and ASIC1a, ASIC2b and ASIC3 were more highly expressed across all brain regions compared to the other subunits. We also show that ASIC4, a proton-insensitive subunit of relatively unknown function, was highly expressed in all mouse tissues apart from DRG and hippocampus, but was by contrast the lowliest expressed ASIC in all naked mole-rat tissues.

Benzylglucosinolate Derived Isothiocyanate from Tropaeolum majus Reduces Gluconeogenic Gene and Protein Expression in Human Cells.

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Valentina Guzmán-Pérez

Full Text Available Nasturtium (Tropaeolum majus L. contains high concentrations of benzylglcosinolate. We found that a hydrolysis product of benzyl glucosinolate-the benzyl isothiocyanate (BITC-modulates the intracellular localization of the transcription factor Forkhead box O 1 (FOXO1. FoxO transcription factors can antagonize insulin effects and trigger a variety of cellular processes involved in tumor suppression, longevity, development and metabolism. The current study evaluated the ability of BITC-extracted as intact glucosinolate from nasturtium and hydrolyzed with myrosinase-to modulate i the insulin-signaling pathway, ii the intracellular localization of FOXO1 and, iii the expression of proteins involved in gluconeogenesis, antioxidant response and detoxification. Stably transfected human osteosarcoma cells (U-2 OS with constitutive expression of FOXO1 protein labeled with GFP (green fluorescent protein were used to evaluate the effect of BITC on FOXO1. Human hepatoma HepG2 cell cultures were selected to evaluate the effect on gluconeogenic, antioxidant and detoxification genes and protein expression. BITC reduced the phosphorylation of protein kinase B (AKT/PKB and FOXO1; promoted FOXO1 translocation from cytoplasm into the nucleus antagonizing the insulin effect; was able to down-regulate the gene and protein expression of gluconeogenic enzymes; and induced the gene expression of antioxidant and detoxification enzymes. Knockdown analyses with specific siRNAs showed that the expression of gluconeogenic genes was dependent on nuclear factor (erythroid derived-like2 (NRF2 and independent of FOXO1, AKT and NAD-dependent deacetylase sirtuin-1 (SIRT1. The current study provides evidence that BITC might have a role in type 2 diabetes T2D by reducing hepatic glucose production and increasing antioxidant resistance.

h5-HT(1B) receptor-mediated constitutive Galphai3-protein activation in stably transfected Chinese hamster ovary cells: an antibody capture assay reveals protean efficacy of 5-HT.

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Newman-Tancredi, Adrian; Cussac, Didier; Marini, Laetitia; Touzard, Manuelle; Millan, Mark J

2003-03-01

1. Serotonin 5-HT(1B) receptors couple to G-proteins of the Gi/o family. However, their activation of specific G-protein subtypes is poorly characterised. Using an innovative antibody capture/guanosine-5'-0-(3-[(35)S]thio)-triphosphate ([(35)S]GTPgammaS) binding strategy, we characterised Galpha(i3) subunit activation by h5-HT(1B) receptors stably expressed in Chinese hamster ovary (CHO) cells. 2. The agonists, 5-HT, alniditan and BMS181,101, stimulated Galpha(i3), whereas methiothepin and SB224,289 behaved as inverse agonists. The selective 5-HT(1B) receptor ligand, S18127, modestly stimulated Galpha(i3) and reversed the actions of both 5-HT and methiothepin. S18127 (1 micro M) also produced parallel, dextral shifts of the 5-HT and methiothepin isotherms. 3. Isotopic dilution experiments ([(35)S]GTPgammaS versus GTPgammaS) revealed high-affinity [(35)S]GTPgammaS binding to Galpha(i3) subunits in the absence of receptor ligands indicating constitutive activity. High-affinity [(35)S]GTPgammaS binding was increased 2.8-fold by 5-HT with an increase in the affinity of GTPgammaS for Galpha(i3) subunits. In contrast, methiothepin halved the number of high-affinity binding sites and decreased their affinity. 4. h5-HT(1B) receptor-mediated Galpha(i3) subunit activation was dependent on the concentration of NaCl. At 300 mM, 5-HT stimulated [(35)S]GTPgammaS binding, basal Galpha(i3) activation was low and methiothepin was inactive. In contrast, at 10 mM NaCl, basal activity was enhanced and the inverse agonist activity of methiothepin was accentuated. Under these conditions, 5-HT decreased Galpha(i3) activation. 5. In conclusion, at h5-HT(1B) receptors expressed in CHO cells: (i) inverse agonist induced inhibition of Galpha(i3), and its reversal by S18127, reveals constitutive activation of this Galpha subunit; (ii) constitutive Galpha(i3) activation can be quantified by isotopic dilution [(35)S]GTPgammaS binding and (iii) decreasing NaCl concentrations enhances Galpha(i3

Protein kinase B/Akt1 inhibits autophagy by down-regulating UVRAG expression

Energy Technology Data Exchange (ETDEWEB)

Yang, Wonseok; Ju, Ji-hyun; Lee, Kyung-min; Nam, KeeSoo; Oh, Sunhwa [Department of Life Science, College of Natural Science, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791 (Korea, Republic of); Shin, Incheol, E-mail: incheol@hanyang.ac.kr [Department of Life Science, College of Natural Science, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791 (Korea, Republic of)

2013-02-01

Autophagy, or autophagocytosis, is a selective intracellular degradative process involving the cell's own lysosomal apparatus. An essential component in cell development, homeostasis, repair and resistance to stress, autophagy may result in either cell death or survival. The targeted region of the cell is sequestered within a membrane structure, the autophagosome, for regulation of the catabolic process. A key factor in both autophagosome formation and autophagosome maturation is a protein encoded by the ultraviolet irradiation resistance-associated gene (UVRAG). Conversely, the serine/threonine-specific protein kinase B (PKB, also known as Akt), which regulates survival in various cancers, inhibits autophagy through mTOR activation. We found that Akt1 may also directly inhibit autophagy by down-regulating UVRAG both in a 293T transient transfection system and breast cancer cells stably expressing Akt1. The UVRAG with mutations at putative Akt1-phosphorylation sites were still inhibited by Akt1, and dominant-negative Akt1 also inhibited UVRAG expression, suggesting that Akt1 down-regulates UVRAG by a kinase activity-independent mechanism. We showed that Akt1 overexpression in MDA-MB-231 breast cancer cells down-regulated UVRAG transcription. Cells over-expressing Akt1 were more resistant than control cells to ultraviolet light-induced autophagy and exhibited the associated reduction in cell viability. Levels of the autophagosome indicator protein LC3B-II and mRFP-GFP-LC3 were reduced in cells that over-expressing Akt1. Inhibiting Akt1 by siRNA or reintroducing UVRAG gene rescued the level of LC3B-II in UV-irradiation. Altogether, these data suggest that Akt1 may inhibit autophagy by decreasing UVRAG expression, which also sensitizes cancer cells to UV irradiation.

Extensive characterization of a lentiviral-derived stable cell line expressing rabbit hemorrhagic disease virus VPg protein.

Science.gov (United States)

Zhu, Jie; Miao, Qiuhong; Tan, Yonggui; Guo, Huimin; Li, Chuanfeng; Chen, Zongyan; Liu, Guangqing

2016-11-01

Rabbit hemorrhagic disease virus (RHDV) is an important member of the caliciviridae family. Currently, no suitable tissue culture system is available for proliferating RHDV, which limits the study of its pathogenesis. To bypass this obstacle, we established a cell line, RK13-VPg, stably expressing the VPg gene with a lentivirus packaging system in this study. In addition, the recently constructed RHDV replicon in our laboratory provided an appropriate model for studying the pathogenesis of RHDV without in vitro RHDV propagation and culture. Using this RHDV replicon and RK13-VPg cell line, we further demonstrated that the presence of VPg protein is essential for efficient translation of an RHDV replicon. Therefore, the RK13-VPg cell line is a powerful tool for studying the replication and translation mechanisms of RHDV. Copyright © 2016 Elsevier B.V. All rights reserved.

Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone marrow stromal cells

DEFF Research Database (Denmark)

Simonsen, Janne Lytoft; Rosada, Cecilia; Serakinci, Nedime

2002-01-01

Human bone marrow stromal cells (hMSCs) were stably transduced by a retroviral vector containing the gene for the catalytic subunit of human telomerase (hTERT). Transduced cells (hMSC-TERTs) had telomerase activity, and the mean telomere length was increased as compared with that of control cells....... The transduced cells have now undergone more than 260 population doublings (PD) and continue to proliferate, whereas control cells underwent senescence-associated proliferation arrest after 26 PD. The cells maintained production of osteoblastic markers and differentiation potential during continuous subculturing......, did not form tumors, and had a normal karyotype. When implanted subcutaneously in immunodeficient mice, the transduced cells formed more bone than did normal cells. These results suggest that ectopic expression of telomerase in hMSCs prevents senescence-associated impairment of osteoblast functions....

Toward Personalized Medicine: Using Cardiomyocytes Differentiated From Urine-Derived Pluripotent Stem Cells to Recapitulate Electrophysiological Characteristics of Type 2 Long QT Syndrome.

Science.gov (United States)

Jouni, Mariam; Si-Tayeb, Karim; Es-Salah-Lamoureux, Zeineb; Latypova, Xenia; Champon, Benoite; Caillaud, Amandine; Rungoat, Anais; Charpentier, Flavien; Loussouarn, Gildas; Baró, Isabelle; Zibara, Kazem; Lemarchand, Patricia; Gaborit, Nathalie

2015-09-01

Human genetically inherited cardiac diseases have been studied mainly in heterologous systems or animal models, independent of patients' genetic backgrounds. Because sources of human cardiomyocytes (CMs) are extremely limited, the use of urine samples to generate induced pluripotent stem cell-derived CMs would be a noninvasive method to identify cardiac dysfunctions that lead to pathologies within patients' specific genetic backgrounds. The objective was to validate the use of CMs differentiated from urine-derived human induced pluripotent stem (UhiPS) cells as a new cellular model for studying patients' specific arrhythmia mechanisms. Cells obtained from urine samples of a patient with long QT syndrome who harbored the HERG A561P gene mutation and his asymptomatic noncarrier mother were reprogrammed using the episomal-based method. UhiPS cells were then differentiated into CMs using the matrix sandwich method.UhiPS-CMs showed proper expression of atrial and ventricular myofilament proteins and ion channels. They were electrically functional, with nodal-, atrial- and ventricular-like action potentials recorded using high-throughput optical and patch-clamp techniques. Comparison of HERG expression from the patient's UhiPS-CMs to the mother's UhiPS-CMs showed that the mutation led to a trafficking defect that resulted in reduced delayed rectifier K(+) current (IKr). This phenotype gave rise to action potential prolongation and arrhythmias. UhiPS cells from patients carrying ion channel mutations can be used as novel tools to differentiate functional CMs that recapitulate cardiac arrhythmia phenotypes. © 2015 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Turbulent jet erosion of a stably stratified gas layer in a nuclear reactor test containment

Energy Technology Data Exchange (ETDEWEB)

Ishay, Liel [Department of Mechanical Engineering, Ben-Gurion University of the Negev, Beer-Sheva 84105 (Israel); Bieder, Ulrich [Commissariat à l’énergie atomique et aux énergies alternatives, Centre de SACLAY DEN/SAC/DANS/DM2S/STMF/LMSF, F-91191 Gif-sur-Yvette (France); Ziskind, Gennady [Department of Mechanical Engineering, Ben-Gurion University of the Negev, Beer-Sheva 84105 (Israel); Rashkovan, Alex, E-mail: rashbgu@gmail.com [Physics Department, Nuclear Research Center Negev (NRCN), PO Box 9001, Beer-Sheva 84190 (Israel)

2015-10-15

Highlights: • We model stably stratified layer erosion by vertical turbulent round jet. • Separate effect studies are performed as a platform for choosing modeling approach. • A test performed in MISTRA facility, CEA, Saclay is modeled using Fluent and Trio-U codes. • The proposed modeling approach showed good agreement with the MISTRA facility LOWMA-3 test. - Abstract: A number of integral and separate effect experiments were performed in the last two decades for validation of containment computational tools. The main goal of these benchmark experiments was to assess the ability of turbulence models and computational fluid dynamics codes to predict hydrogen concentration distribution and steam condensation rate in a nuclear reactor containment in the course of severe accidents. It appears from the published literature that the predictive capability of the existing computational tools still needs to be improved. This work examines numerically the temporal evolution of helium concentration in the experiment called LOWMA-3, performed in the MISTRA facility of CEA-Saclay, France. In the experiment, helium is used to mimic hydrogen of a real-case accident. The aim of this separate effect experiment, where steam condensation was not involved, is to predict helium concentration field. The conditions of the experiment are such that both the momentum transport and molecular diffusion contributions to the mixing process are of the same order of magnitude (Fr ∼ 1). A commercial CFD code, Fluent, and a CEA in-house code, Trio-U, are used for flow and helium concentration fields temporal evolution prediction in the present study. The preliminary separate effect studies provide guidance to an optimal modeling approach for the LOWMA-3 experiment. Temporal evolution of helium concentration in the stratification layer is shown, and a comparison to the experiment is discussed. It is shown that correct modeling of the round jet flowfield is essential for a reliable

Turbulent jet erosion of a stably stratified gas layer in a nuclear reactor test containment

International Nuclear Information System (INIS)

Ishay, Liel; Bieder, Ulrich; Ziskind, Gennady; Rashkovan, Alex

2015-01-01

Highlights: • We model stably stratified layer erosion by vertical turbulent round jet. • Separate effect studies are performed as a platform for choosing modeling approach. • A test performed in MISTRA facility, CEA, Saclay is modeled using Fluent and Trio-U codes. • The proposed modeling approach showed good agreement with the MISTRA facility LOWMA-3 test. - Abstract: A number of integral and separate effect experiments were performed in the last two decades for validation of containment computational tools. The main goal of these benchmark experiments was to assess the ability of turbulence models and computational fluid dynamics codes to predict hydrogen concentration distribution and steam condensation rate in a nuclear reactor containment in the course of severe accidents. It appears from the published literature that the predictive capability of the existing computational tools still needs to be improved. This work examines numerically the temporal evolution of helium concentration in the experiment called LOWMA-3, performed in the MISTRA facility of CEA-Saclay, France. In the experiment, helium is used to mimic hydrogen of a real-case accident. The aim of this separate effect experiment, where steam condensation was not involved, is to predict helium concentration field. The conditions of the experiment are such that both the momentum transport and molecular diffusion contributions to the mixing process are of the same order of magnitude (Fr ∼ 1). A commercial CFD code, Fluent, and a CEA in-house code, Trio-U, are used for flow and helium concentration fields temporal evolution prediction in the present study. The preliminary separate effect studies provide guidance to an optimal modeling approach for the LOWMA-3 experiment. Temporal evolution of helium concentration in the stratification layer is shown, and a comparison to the experiment is discussed. It is shown that correct modeling of the round jet flowfield is essential for a reliable

Evaluation of endogenous control gene(s) for gene expression studies in human blood exposed to 60Co γ-rays ex vivo.

Science.gov (United States)

Vaiphei, S Thangminlal; Keppen, Joshua; Nongrum, Saibadaiahun; Chaubey, R C; Kma, L; Sharan, R N

2015-01-01

In gene expression studies, it is critical to normalize data using a stably expressed endogenous control gene in order to obtain accurate and reliable results. However, we currently do not have a universally applied endogenous control gene for normalization of data for gene expression studies, particularly those involving (60)Co γ-ray-exposed human blood samples. In this study, a comparative assessment of the gene expression of six widely used housekeeping endogenous control genes, namely 18S, ACTB, B2M, GAPDH, MT-ATP6 and CDKN1A, was undertaken for a range of (60)Co γ-ray doses (0.5, 1.0, 2.0 and 4.0 Gy) at 8.4 Gy min(-1) at 0 and 24 h post-irradiation time intervals. Using the NormFinder algorithm, real-time PCR data obtained from six individuals (three males and three females) were analyzed with respect to the threshold cycle (Ct) value and abundance, ΔCt pair-wise comparison, intra- and inter-group variability assessments, etc. GAPDH, either alone or in combination with 18S, was found to be the most suitable endogenous control gene and should be used in gene expression studies, especially those involving qPCR of γ-ray-exposed human blood samples. © The Author 2014. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

Validation of endogenous normalizing genes for expression analyses in adult human testis and germ cell neoplasms.

Science.gov (United States)

Svingen, T; Jørgensen, A; Rajpert-De Meyts, E

2014-08-01

The measurement of gene expression levels in cells and tissues typically depends on a suitable point of reference for inferring biological relevance. For quantitative (or real-time) RT-PCR assays, the method of choice is often to normalize gene expression data to an endogenous gene that is stably expressed across the samples analysed: a so-called normalizing or housekeeping gene. Although this is a valid strategy, the identification of stable normalizing genes has proved challenging and a gene showing stable expression across all cells or tissues is unlikely to exist. Therefore, it is necessary to define suitable normalizing genes for specific cells and tissues. Here, we report on the performance of a panel of nine commonly employed normalizing genes in adult human testis and testicular pathologies. Our analyses revealed significant variability in transcript abundance for commonly used normalizers, highlighting the importance of selecting appropriate normalizing genes as comparative measurements can yield variable results when different normalizing genes are employed. Based on our results, we recommend using RPS20, RPS29 or SRSF4 when analysing relative gene expression levels in human testis and associated testicular pathologies. OCT4 and SALL4 can be used with caution as second-tier normalizers when determining changes in gene expression in germ cells and germ cell tumour components, but the relative transcript abundance appears variable between different germ cell tumour types. We further recommend that such studies should be accompanied by additional assessment of histology and cellularity of each sample. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

El cómic como fuente histórica : el falso testimonio de Tintín en el Congo Belga

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Óscar Gual Boronat

2011-01-01

Full Text Available A través del estudio de la obra Tintin au Congo del dibujante belga Hergé, tratamos de probar la capacidad del cómic para ser utilizado como fuente histórica. La actual definición de fuente puede englobarlo perfectamente pero es necesaria una demostración práctica. Tras contextualizar dicha historieta pasamos a realizar una lectura atenta de la misma, así como de sus aspectos más polémicos, para a continuación describir el proceso de trabajo del propio Hergé y las influencias de su entorno. Por último, entraremos a debatir cuál es el origen de la historieta como medio y cuál es el método de análisis a la hora de interrogarla históricamente.Through the study of the Belgian cartoonist Hergé's work Tintin au Congo we try to test the ability of comics to be used as a historical source. The current source definition can cover it very well but requires a practical demonstration. After we contextualize this story we make a careful reading of it and of its most controversial features, then we describe the process of Hergé's own work and the influences of their environment. Finally we enter into a discussion about which is the origin of comics as a medium and which is the method of analysis when questioning historically.

h5-HT1B receptor-mediated constitutive Gαi3-protein activation in stably transfected Chinese hamster ovary cells: an antibody capture assay reveals protean efficacy of 5-HT

Science.gov (United States)

Newman-Tancredi, Adrian; Cussac, Didier; Marini, Laetitia; Touzard, Manuelle; Millan, Mark J

2003-01-01

Serotonin 5-HT1B receptors couple to G-proteins of the Gi/o family. However, their activation of specific G-protein subtypes is poorly characterised. Using an innovative antibody capture/guanosine-5′-0-(3-[35S]thio)-triphosphate ([35S]GTPγS) binding strategy, we characterised Gαi3 subunit activation by h5-HT1B receptors stably expressed in Chinese hamster ovary (CHO) cells. The agonists, 5-HT, alniditan and BMS181,101, stimulated Gαi3, whereas methiothepin and SB224,289 behaved as inverse agonists. The selective 5-HT1B receptor ligand, S18127, modestly stimulated Gαi3 and reversed the actions of both 5-HT and methiothepin. S18127 (1 μM) also produced parallel, dextral shifts of the 5-HT and methiothepin isotherms. Isotopic dilution experiments ([35S]GTPγS versus GTPγS) revealed high-affinity [35S]GTPγS binding to Gαi3 subunits in the absence of receptor ligands indicating constitutive activity. High-affinity [35S]GTPγS binding was increased 2.8-fold by 5-HT with an increase in the affinity of GTPγS for Gαi3 subunits. In contrast, methiothepin halved the number of high-affinity binding sites and decreased their affinity. h5-HT1B receptor-mediated Gαi3 subunit activation was dependent on the concentration of NaCl. At 300 mM, 5-HT stimulated [35S]GTPγS binding, basal Gαi3 activation was low and methiothepin was inactive. In contrast, at 10 mM NaCl, basal activity was enhanced and the inverse agonist activity of methiothepin was accentuated. Under these conditions, 5-HT decreased Gαi3 activation. In conclusion, at h5-HT1B receptors expressed in CHO cells: (i) inverse agonist induced inhibition of Gαi3, and its reversal by S18127, reveals constitutive activation of this Gα subunit; (ii) constitutive Gαi3 activation can be quantified by isotopic dilution [35S]GTPγS binding and (iii) decreasing NaCl concentrations enhances Gαi3 activation and leads to protean agonist properties of 5-HT: that is a switch to inhibition of Gαi3. PMID:12684263

Deleted in malignant brain tumors 1 (DMBT1) elicits increased VEGF and decreased IL-6 production in type II lung epithelial cells

DEFF Research Database (Denmark)

Müller, Hanna; Nagel, Christian; Weiss, Christel

2015-01-01

between VEGF and IL-6 levels to DMBT1 expression in the lungs of preterm and term infants and in lung epithelial cells in vitro. METHODS: We examined by ELISA VEGF levels in 120 tracheal aspirates of 57 preterm and term infants and tested for correlation with different perinatal factors as well...... as with DMBT1 levels. To examine the effect of DMBT1 on VEGF and IL-6 expression we compared type II lung epithelial A549 cells stably transfected with a DMBT1 expression plasmid (DMBT1+ cells) to A549 cells stably transfected with an empty expression plasmid (DMBT1- cells). The concentrations of VEGF and IL-6...... that DMBT1 promotes VEGF and suppresses IL-6 production in alveolar tissues, which could point to DMBT1 having a possible role in the transition from inflammation to regeneration and being a potentially useful clinical marker....

Identification of valid reference genes for the normalization of RT qPCR gene expression data in human brain tissue

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Ravid Rivka

2008-05-01

Full Text Available Abstract Background Studies of gene expression in post mortem human brain can contribute to understanding of the pathophysiology of neurodegenerative diseases, including Alzheimer's disease (AD, Parkinson's disease (PD and dementia with Lewy bodies (DLB. Quantitative real-time PCR (RT qPCR is often used to analyse gene expression. The validity of results obtained using RT qPCR is reliant on accurate data normalization. Reference genes are generally used to normalize RT qPCR data. Given that expression of some commonly used reference genes is altered in certain conditions, this study aimed to establish which reference genes were stably expressed in post mortem brain tissue from individuals with AD, PD or DLB. Results The present study investigated the expression stability of 8 candidate reference genes, (ubiquitin C [UBC], tyrosine-3-monooxygenase [YWHAZ], RNA polymerase II polypeptide [RP II], hydroxymethylbilane synthase [HMBS], TATA box binding protein [TBP], β-2-microglobulin [B2M], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], and succinate dehydrogenase complex-subunit A, [SDHA] in cerebellum and medial temporal gyrus of 6 AD, 6 PD, 6 DLB subjects, along with 5 matched controls using RT qPCR (TaqMan® Gene Expression Assays. Gene expression stability was analysed using geNorm to rank the candidate genes in order of decreasing stability in each disease group. The optimal number of genes recommended for accurate data normalization in each disease state was determined by pairwise variation analysis. Conclusion This study identified validated sets of mRNAs which would be appropriate for the normalization of RT qPCR data when studying gene expression in brain tissue of AD, PD, DLB and control subjects.

Evaluation of suitable reference genes for gene expression studies in bovine muscular tissue

Directory of Open Access Journals (Sweden)

Dunner Susana

2008-09-01

Full Text Available Abstract Background Real-time reverse transcriptase quantitative polymerase chain reaction (real-time RTqPCR is a technique used to measure mRNA species copy number as a way to determine key genes involved in different biological processes. However, the expression level of these key genes may vary among tissues or cells not only as a consequence of differential expression but also due to different factors, including choice of reference genes to normalize the expression levels of the target genes; thus the selection of reference genes is critical for expression studies. For this purpose, ten candidate reference genes were investigated in bovine muscular tissue. Results The value of stability of ten candidate reference genes included in three groups was estimated: the so called 'classical housekeeping' genes (18S, GAPDH and ACTB, a second set of genes used in expression studies conducted on other tissues (B2M, RPII, UBC and HMBS and a third set of novel genes (SF3A1, EEF1A2 and CASC3. Three different statistical algorithms were used to rank the genes by their stability measures as produced by geNorm, NormFinder and Bestkeeper. The three methods tend to agree on the most stably expressed genes and the least in muscular tissue. EEF1A2 and HMBS followed by SF3A1, ACTB, and CASC3 can be considered as stable reference genes, and B2M, RPII, UBC and GAPDH would not be appropriate. Although the rRNA-18S stability measure seems to be within the range of acceptance, its use is not recommended because its synthesis regulation is not representative of mRNA levels. Conclusion Based on geNorm algorithm, we propose the use of three genes SF3A1, EEF1A2 and HMBS as references for normalization of real-time RTqPCR in muscle expression studies.

BDNF regulates the expression and distribution of vesicular glutamate transporters in cultured hippocampal neurons.

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Carlos V Melo

Full Text Available BDNF is a pro-survival protein involved in neuronal development and synaptic plasticity. BDNF strengthens excitatory synapses and contributes to LTP, presynaptically, through enhancement of glutamate release, and postsynaptically, via phosphorylation of neurotransmitter receptors, modulation of receptor traffic and activation of the translation machinery. We examined whether BDNF upregulated vesicular glutamate receptor (VGLUT 1 and 2 expression, which would partly account for the increased glutamate release in LTP. Cultured rat hippocampal neurons were incubated with 100 ng/ml BDNF, for different periods of time, and VGLUT gene and protein expression were assessed by real-time PCR and immunoblotting, respectively. At DIV7, exogenous application of BDNF rapidly increased VGLUT2 mRNA and protein levels, in a dose-dependent manner. VGLUT1 expression also increased but only transiently. However, at DIV14, BDNF stably increased VGLUT1 expression, whilst VGLUT2 levels remained low. Transcription inhibition with actinomycin-D or α-amanitine, and translation inhibition with emetine or anisomycin, fully blocked BDNF-induced VGLUT upregulation. Fluorescence microscopy imaging showed that BDNF stimulation upregulates the number, integrated density and intensity of VGLUT1 and VGLUT2 puncta in neurites of cultured hippocampal neurons (DIV7, indicating that the neurotrophin also affects the subcellular distribution of the transporter in developing neurons. Increased VGLUT1 somatic signals were also found 3 h after stimulation with BDNF, further suggesting an increased de novo transcription and translation. BDNF regulation of VGLUT expression was specifically mediated by BDNF, as no effect was found upon application of IGF-1 or bFGF, which activate other receptor tyrosine kinases. Moreover, inhibition of TrkB receptors with K252a and PLCγ signaling with U-73122 precluded BDNF-induced VGLUT upregulation. Hippocampal neurons express both isoforms during

Transplantation of periodontal ligament cell sheets expressing human β-defensin-3 promotes anti-inflammation in a canine model of periodontitis

Science.gov (United States)

Zhu, Minwen; Miao, Bo; Zhu, Jianhua; Wang, Haiyan; Zhou, Zengtong

2017-01-01

Periodontitis is a chronic oral inflammatory disease caused by microorganisms. Human β-defensin-3 (HBD-3) is an endogenous antimicrobial peptide that inhibits a broad spectrum of microorganisms. Cell sheet technology has been widely applied in tissue and organ reconstructions. In the current study, it was aimed to investigate the anti-inflammatory effect of periodontal tissue engineered by HBD-3 gene-modified periodontal ligament cell (PDLC) sheets, and to identify a suitable method of promoting the regeneration of periodontal tissues. Western blot analysis and antimicrobial tests were used to confirm the expression of HBD-3. The effect of the cell sheets on anti-inflammatory activity and bone remodeling in a dog model of periodontitis was demonstrated by immunohistochemistry. The results demonstrated that the transfected PDLCs stably expressed HBD-3. Periodontal pathogens were susceptible to the antimicrobial activity of the cell sheets. In addition, the cell sheets relieved the bone resorption caused by inflammation in the in vivo model. HBD-3 may potentially be applied in the treatment of periodontitis and may function as osteogenic promoter via its anti-inflammatory effect. PMID:28944821

Dnmt1 Expression in Pre- and Postimplantation Embryogenesis and the Maintenance of IAP Silencing

Science.gov (United States)

Gaudet, F.; Rideout, W. M.; Meissner, A.; Dausman, J.; Leonhardt, H.; Jaenisch, R.

2004-01-01

The methylation of intracisternal A-type particle (IAP) sequences is maintained during mouse embryogenesis. Methylation suppresses IAP expression and the potential for mutagenesis by retrotransposition, but it is not clear how methylation of these elements is maintained during the embryonic stages when the bulk of the genome is being demethylated. It has been suggested that the high levels of DNA methyltransferase-1 (Dnmt1) present during cleavage could be important for keeping IAPs methylated. To test this hypothesis, we combined mutant alleles of Dnmt1 with an agouti allele (Aiapy), which provided a coat color readout for the methylation status of the IAP insertion in the agouti locus. We found that reduction in Dnmt1 levels directly impacted methylation at this locus, leading to stable transcriptional activation of the agouti gene in the adult. Specifically, the short maternal Dnmt1 protein was important in maintaining methylation at the Aiapy locus in cleavage embryos, whereas the longer Dnmt1 isoform found in somatic cells was important in maintaining IAP methylation during the postimplantation stage. These results underscore the importance of maintaining proper maintenance of methylation patterns during gestation and suggest that interference with this process may stably affect gene expression patterns in the adult and may have profound phenotypic consequences. PMID:14749379

The tumor suppressor PTEN inhibits EGF-induced TSP-1 and TIMP-1 expression in FTC-133 thyroid carcinoma cells

International Nuclear Information System (INIS)

Soula-Rothhut, Mahdhia; Coissard, Cyrille; Sartelet, Herve; Boudot, Cedric; Bellon, Georges; Martiny, Laurent; Rothhut, Bernard

2005-01-01

Thrombospondin-1 (TSP-1) is a multidomain extracellular macromolecule that was first identified as natural modulator of angiogenesis and tumor growth. In the present study, we found that epidermal growth factor (EGF) up-regulated TSP-1 expression in FTC-133 (primary tumor) but not in FTC-238 (lung metastasis) thyroid cancer cells. Both EGF and TSP-1 induced expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) in a mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner. In FTC-133 cells, EGF induced proliferation in a TSP-1- and TIMP-1-dependent manner. In addition, we determined that re-expression of the tumor suppressor protein PTEN induced cell death, an effect that correlated with a block of Akt kinase phosphorylation. EGF-induced TSP-1 and TIMP-1 promoter activity and protein expression were inhibited in FTC-133 cells stably expressing wtPTEN but not in cells expressing mutant PTEN. Furthermore, we found that wtPTEN inhibited EGF-but not TSP-1-stimulated FTC-133 cell migration and also inhibited invasion induced by EGF and by TSP-1. Finally, an antibody against TSP-1 reversed EGF-stimulated FTC-133 cell invasion as well as the constitutive invasive potential of FTC-238 cells. Overall, our results suggest that PTEN can function as an important modulator of extracellular matrix proteins in thyroid cancer. Therefore, analyzing differential regulation of TSP-1 by growth factors such as EGF can be helpful in understanding thyroid cancer development

Trichostatin A inhibits beta-casein expression in mammary epithelial cells

International Nuclear Information System (INIS)

Pujuguet, Philippe; Radisky, Derek; Levy, Dinah; Lacza, Charlemagne; Bissell, Mina J.

2002-01-01

Many aspects of cellular behavior are affected by information derived from association of the extracellular matrix (ECM) and with cell membrane receptors. When cultured in the presence of laminin-containing ECM and prolactin (Prl), normal mammary epithelial cells express the milk protein beta-casein. Previously, we defined the minimal ECM- and Prl-responsive enhancer element BCE-1 from the upstream region of the beta-casein gene. We also found that BCE-1 was only active when stably integrated into chromatin, and that trichostatin A (TSA), a reagent that leads to alterations in chromatin structure, was able to activate the integrated enhancer element. We now show that endogenous b-casein gene, which is controlled by a genetic assembly that is highly similar to that of BCE-1 and which is also activated by incubation in ECM and Prl, is instead inhibited by TSA. We provide evidence that the differing response of b-casein and BCE-1 to TSA is neither due to an unusual effect of TSA on mammary epithelial cells, nor to secondary consequences from the expression of a separate gene, nor to a particular property of the BCE-1 construct. As a component of this investigation, we also showed that ECM could mediate rapid histone deacetylation in mammary epithelial cells. These results are discussed in combination with previous work showing that TSA mediates the differentiation of many types of cancer cells but inhibits differentiation of some nonmalignant cell types

Molecular phylogenetic and expression analysis of the complete WRKY transcription factor family in maize.

Science.gov (United States)

Wei, Kai-Fa; Chen, Juan; Chen, Yan-Feng; Wu, Ling-Juan; Xie, Dao-Xin

2012-04-01

The WRKY transcription factors function in plant growth and development, and response to the biotic and abiotic stresses. Although many studies have focused on the functional identification of the WRKY transcription factors, much less is known about molecular phylogenetic and global expression analysis of the complete WRKY family in maize. In this study, we identified 136 WRKY proteins coded by 119 genes in the B73 inbred line from the complete genome and named them in an orderly manner. Then, a comprehensive phylogenetic analysis of five species was performed to explore the origin and evolutionary patterns of these WRKY genes, and the result showed that gene duplication is the major driving force for the origin of new groups and subgroups and functional divergence during evolution. Chromosomal location analysis of maize WRKY genes indicated that 20 gene clusters are distributed unevenly in the genome. Microarray-based expression analysis has revealed that 131 WRKY transcripts encoded by 116 genes may participate in the regulation of maize growth and development. Among them, 102 transcripts are stably expressed with a coefficient of variation (CV) value of WRKY genes with the CV value of >15% are further analysed to discover new organ- or tissue-specific genes. In addition, microarray analyses of transcriptional responses to drought stress and fungal infection showed that maize WRKY proteins are involved in stress responses. All these results contribute to a deep probing into the roles of WRKY transcription factors in maize growth and development and stress tolerance.

Characterization of global microRNA expression reveals oncogenic potential of miR-145 in metastatic colorectal cancer

International Nuclear Information System (INIS)

Arndt, Greg M; Retzlaff, Kathy; Bittner, Anton; Raponi, Mitch; Dossey, Lesley; Cullen, Lara M; Lai, Angela; Druker, Riki; Eisbacher, Michael; Zhang, Chunyan; Tran, Nham; Fan, Hongtao

2009-01-01

MicroRNAs (MiRNAs) are short non-coding RNAs that control protein expression through various mechanisms. Their altered expression has been shown to be associated with various cancers. The aim of this study was to profile miRNA expression in colorectal cancer (CRC) and to analyze the function of specific miRNAs in CRC cells. MirVana miRNA Bioarrays were used to determine the miRNA expression profile in eight CRC cell line models, 45 human CRC samples of different stages, and four matched normal colon tissue samples. SW620 CRC cells were stably transduced with miR-143 or miR-145 expression vectors and analyzed in vitro for cell proliferation, cell differentiation and anchorage-independent growth. Signalling pathways associated with differentially expressed miRNAs were identified using a gene set enrichment analysis. The expression analysis of clinical CRC samples identified 37 miRNAs that were differentially expressed between CRC and normal tissue. Furthermore, several of these miRNAs were associated with CRC tumor progression including loss of miR-133a and gain of miR-224. We identified 11 common miRNAs that were differentially expressed between normal colon and CRC in both the cell line models and clinical samples. In vitro functional studies indicated that miR-143 and miR-145 appear to function in opposing manners to either inhibit or augment cell proliferation in a metastatic CRC model. The pathways targeted by miR-143 and miR-145 showed no significant overlap. Furthermore, gene expression analysis of metastatic versus non-metastatic isogenic cell lines indicated that miR-145 targets involved in cell cycle and neuregulin pathways were significantly down-regulated in the metastatic context. MiRNAs showing altered expression at different stages of CRC could be targets for CRC therapies and be further developed as potential diagnostic and prognostic analytes. The identified biological processes and signalling pathways collectively targeted by co-expressed miRNAs in

Global analysis of gene expression mediated by OX1 orexin receptor signaling in a hypothalamic cell line.

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Eric Koesema

Full Text Available The orexins and their cognate G-protein coupled receptors have been widely studied due to their associations with various behaviors and cellular processes. However, the detailed downstream signaling cascades that mediate these effects are not completely understood. We report the generation of a neuronal model cell line that stably expresses the OX1 orexin receptor (OX1 and an RNA-Seq analysis of changes in gene expression seen upon receptor activation. Upon treatment with orexin, several families of related transcription factors are transcriptionally regulated, including the early growth response genes (Egr, the Kruppel-like factors (Klf, and the Nr4a subgroup of nuclear hormone receptors. Furthermore, some of the transcriptional effects observed have also been seen in data from in vivo sleep deprivation microarray studies, supporting the physiological relevance of the data set. Additionally, inhibition of one of the most highly regulated genes, serum and glucocorticoid-regulated kinase 1 (Sgk1, resulted in the diminished orexin-dependent induction of a subset of genes. These results provide new insight into the molecular signaling events that occur during OX1 signaling and support a role for orexin signaling in the stimulation of wakefulness during sleep deprivation studies.

Ctip2-, Satb2-, Prox1-, and GAD65-Expressing Neurons in Rat Cultures: Preponderance of Single- and Double-Positive Cells, and Cell Type-Specific Expression of Neuron-Specific Gene Family Members, Nsg-1 (NEEP21) and Nsg-2 (P19).

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Digilio, Laura; Yap, Chan Choo; Winckler, Bettina

2015-01-01

The brain consists of many distinct neuronal cell types, but which cell types are present in widely used primary cultures of embryonic rodent brain is often not known. We characterized how abundantly four cell type markers (Ctip2, Satb2, Prox1, GAD65) were represented in cultured rat neurons, how easily neurons expressing different markers can be transfected with commonly used plasmids, and whether neuronal-enriched endosomal proteins Nsg-1 (NEEP21) and Nsg-2 (P19) are ubiquitously expressed in all types of cultured neurons. We found that cultured neurons stably maintain cell type identities that are reflective of cell types in vivo. This includes neurons maintaining simultaneous expression of two transcription factors, such as Ctip2+/Satb2+ or Prox1+/Ctip2+ double-positive cells, which have also been described in vivo. Secondly, we established the superior efficiency of CAG promoters for both Lipofectamine-mediated transfection as well as for electroporation. Thirdly, we discovered that Nsg-1 and Nsg-2 were not expressed equally in all neurons: whereas high levels of both Nsg-1 and Nsg-2 were found in Satb2-, Ctip2-, and GAD65-positive neurons, Prox1-positive neurons in hippocampal cultures expressed low levels of both. Our findings thus highlight the importance of identifying neuronal cell types for doing cell biology in cultured neurons: Keeping track of neuronal cell type might uncover effects in assays that might otherwise be masked by the mixture of responsive and non-responsive neurons in the dish.

Interferon-inducible MyD88 protein inhibits hepatitis B virus replication

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Xiong Wei; Wang Xun; Liu Xiaoying; Xiang Li; Zheng Lingjie; Yuan Zhenghong

2004-01-01

Myeloid differential primary response protein (MyD88) is a critical component in the signaling cascade through Toll-like receptors (TLRs) and is induced by α interferon (IFN-α). To examine the role of MyD88 in the antiviral activity of IFN-α against hepatitis B virus (HBV), we established MyD88 stably expressing cell lines and studied HBV replication in these lines after transient transfection. The levels of HBV proteins and viral replicative intermediates were effectively reduced in MyD88-expressing cells. A significant reduction of total and cytoplasmic viral RNAs in MyD88 stably expressing cells was also observed. Using a nuclear factor-κB (NF-κB) dependent reporter assay, it was shown that activation of NF-κB was moderately increased in the presence of expression of MyD88, and further significantly increased by co-expression of HBV. These results suggest a novel mechanism for the inhibition of HBV replication by IFN-α via expression of MyD88 protein involving activation of NF-κB signaling pathway and downregulation of viral transcription

Construction of stably maintained non-mobilizable derivatives of RSF1010 lacking all known elements essential for mobilization

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Tokmakova Irina L

2007-11-01

Full Text Available Abstract Background RSF1010 is a well-studied broad-host-range plasmid able to be mobilized to different bacteria and plants. RSF1010-derived plasmid vectors are widely used in both basic research and industrial applications. In the latter case, exploiting of mobilizable plasmids or even the plasmids possessing negligible mobilization frequency, but containing DNA fragments that could promote conjugal transfer, is undesirable because of biosafety considerations. Previously, several mutations significantly decreasing efficiency of RSF1010 mobilization have been selected. Nevertheless, construction of the RSF1010 derivative lacking all known loci involved in the conjugal transfer has not been reported yet. Results Novel non-mobilizable derivatives of RSF1010 lacking all known DNA sequences involved in the mobilization process have been obtained due to the exploiting of λRed-driven recombination between the plasmid and a constructed in vitro linear DNA fragment. To provide auto-regulated transcription of the essential replication gene, repB, the plasmid loci oriT, mobC and mobA were substituted by the DNA fragment containing PlacUV5→lacI. Mobilization of the obtained RSFmob plasmid was not detected in standard tests. The derivative of RSFmob with increased copy number has been obtained after lacI elimination. High stability of both constructed plasmids has been demonstrated in Escherichia coli and Pantoea ananatis. Design of RSFmob allows easy substitution of PlacUV5 by any desirable promoter for construction of novel derivatives with changed copy number or host range. Conclusion Novel non-mobilizable derivatives of RSF1010 lacking all known DNA sequences involved in the mobilization process and stably maintained at least in E. coli and P. ananatis have been constructed. The obtained plasmids became the progenitors of new cloning vectors answering all biosafety requirements of genetically modified organisms used in scale-up production.

Intrinsic and Extrinsic Regulation of PD-L2 Expression in Oncogene-Driven Non-Small Cell Lung Cancer.

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Shibahara, Daisuke; Tanaka, Kentaro; Iwama, Eiji; Kubo, Naoki; Ota, Keiichi; Azuma, Koichi; Harada, Taishi; Fujita, Jiro; Nakanishi, Yoichi; Okamoto, Isamu

2018-03-27

The interaction of programmed cell death ligand 2 (PD-L2) with programmed cell death 1 is implicated in tumor immune escape. The regulation of PD-L2 expression in tumor cells has remained unclear, however. We here examined intrinsic and extrinsic regulation of PD-L2 expression in NSCLC. PD-L2 expression was evaluated by reverse transcription and real-time polymerase chain reaction analysis and by flow cytometry. BEAS-2B cells stably expressing an activated mutant form of EGFR or the echinoderm microtubule associated protein like 4 (EML4)-ALK receptor tyrosine kinase fusion oncoprotein manifested increased expression of PD-L2 at both the mRNA and protein levels. Furthermore, treatment of NSCLC cell lines that harbor such driver oncogenes with corresponding EGFR or ALK tyrosine kinase inhibitors or depletion of EGFR or ALK by small interfering RNA transfection suppressed expression of PD-L2, demonstrating that activating EGFR mutations or echinoderm microtubule associated protein like 4 gene (EML4)-ALK receptor tyrosine kinase gene (ALK) fusion intrinsically induce PD-L2 expression. We also found that interferon gamma (IFN-γ) extrinsically induced expression of PD-L2 through signal transducer and activator of transcription 1 signaling in NSCLC cells. Oncogene-driven expression of PD-L2 in NSCLC cells was inhibited by knockdown of the transcription factors signal transducer and activator of transcription 3 (STAT3) or c-FOS. IFN-γ also activated STAT3 and c-FOS, suggesting that these proteins may also contribute to the extrinsic induction of PD-L2 expression. Expression of PD-L2 is induced intrinsically by activating EGFR mutations or EML4-ALK fusion and extrinsically by IFN-γ, with STAT3 and c-FOS possibly contributing to both intrinsic and extrinsic pathways. Our results thus provide insight into the complexity of tumor immune escape in NSCLC. Copyright © 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

TNFα-Induced Mucin 4 Expression Elicits Trastuzumab Resistance in HER2-Positive Breast Cancer.

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Mercogliano, María F; De Martino, Mara; Venturutti, Leandro; Rivas, Martín A; Proietti, Cecilia J; Inurrigarro, Gloria; Frahm, Isabel; Allemand, Daniel H; Deza, Ernesto Gil; Ares, Sandra; Gercovich, Felipe G; Guzmán, Pablo; Roa, Juan C; Elizalde, Patricia V; Schillaci, Roxana

2017-02-01

Although trastuzumab administration improved the outcome of HER2-positive breast cancer patients, resistance events hamper its clinical benefits. We demonstrated that TNFα stimulation in vitro induces trastuzumab resistance in HER2-positive breast cancer cell lines. Here, we explored the mechanism of TNFα-induced trastuzumab resistance and the therapeutic strategies to overcome it. Trastuzumab-sensitive breast cancer cells, genetically engineered to stably overexpress TNFα, and de novo trastuzumab-resistant tumors, were used to evaluate trastuzumab response and TNFα-blocking antibodies effectiveness respectively. Immunohistochemistry and antibody-dependent cell cytotoxicity (ADCC), together with siRNA strategy, were used to explore TNFα influence on the expression and function of its downstream target, mucin 4 (MUC4). The clinical relevance of MUC4 expression was studied in a cohort of 78 HER2-positive breast cancer patients treated with adjuvant trastuzumab. TNFα overexpression turned trastuzumab-sensitive cells and tumors into resistant ones. Histopathologic findings revealed mucin foci in TNFα-producing tumors. TNFα induced upregulation of MUC4 that reduced trastuzumab binding to its epitope and impaired ADCC. Silencing MUC4 enhanced trastuzumab binding, increased ADCC, and overcame trastuzumab and trastuzumab-emtansine antiproliferative effects in TNFα-overexpressing cells. Accordingly, administration of TNFα-blocking antibodies downregulated MUC4 and sensitized de novo trastuzumab-resistant breast cancer cells and tumors to trastuzumab. In HER2-positive breast cancer samples, MUC4 expression was found to be an independent predictor of poor disease-free survival (P = 0.008). We identified TNFα-induced MUC4 expression as a novel trastuzumab resistance mechanism. We propose MUC4 expression as a predictive biomarker of trastuzumab efficacy and a guide to combination therapy of TNFα-blocking antibodies with trastuzumab. Clin Cancer Res; 23(3); 636-48. �

Transplantation of periodontal ligament cell sheets expressing human β‑defensin‑3 promotes anti‑inflammation in a canine model of periodontitis.

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Zhu, Minwen; Miao, Bo; Zhu, Jianhua; Wang, Haiyan; Zhou, Zengtong

2017-11-01

Periodontitis is a chronic oral inflammatory disease caused by microorganisms. Human β‑defensin‑3 (HBD‑3) is an endogenous antimicrobial peptide that inhibits a broad spectrum of microorganisms. Cell sheet technology has been widely applied in tissue and organ reconstructions. In the current study, it was aimed to investigate the anti‑inflammatory effect of periodontal tissue engineered by HBD‑3 gene‑modified periodontal ligament cell (PDLC) sheets, and to identify a suitable method of promoting the regeneration of periodontal tissues. Western blot analysis and antimicrobial tests were used to confirm the expression of HBD‑3. The effect of the cell sheets on anti‑inflammatory activity and bone remodeling in a dog model of periodontitis was demonstrated by immunohistochemistry. The results demonstrated that the transfected PDLCs stably expressed HBD‑3. Periodontal pathogens were susceptible to the antimicrobial activity of the cell sheets. In addition, the cell sheets relieved the bone resorption caused by inflammation in the in vivo model. HBD‑3 may potentially be applied in the treatment of periodontitis and may function as osteogenic promoter via its anti‑inflammatory effect.

Delayed expression of apoptosis in X-irradiated human leukemic MOLT-4 cells transfected with mutant p53

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Nakano, Hisako; Yonekawa, Hiromichi; Shinohara, Kunio

2003-01-01

The effects of X-rays on cell survival, apoptosis, and long-term response in the development of cell death as measured by the dye exclusion test were studied in human leukemic MOLT-4 cells (p53 wild-type) stably transfected with a mutant p53 cDNA expression vector. Cell survival, as determined from colony-forming ability, was increased in an expression level dependent manner, but the increase was partial even with the highest-expressing clone (B3). This contrasts with the prior observation that cell death and apoptosis in B3 are completely inhibited at 24 h after irradiation with 1.8 Gy of X-rays. The examination of B3 cells incubated for longer than 24 h after X-irradiation showed a delay in the induction of cell death and apoptosis. Western blot analysis revealed that the time required to reach the highest level of wild-type p53 protein in B3 was longer than the time in MOLT-4 and that the p53 may be stabilized by the phosphorylation at Ser-15. These results suggest that the introduction of mutant p53 into MOLT-4 merely delays the development of apoptosis, during which the cells could repair the damage induced by X-rays, and results in the partial increase in cell survival. (author)

Plant bioreactors for the antigenic hook-associated flgK protein expression

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Luciana Rossi

2014-01-01

Full Text Available Plants engineered with genes encoding for the antigenic proteins of various microorganisms have shown to correctly express the proteins that elicit the production of antibodies in mammalian hosts. In livestock, plant-based vaccines could represent an innovative strategy for oral vaccination, especially to prevent infection by enteric pathogens. The aim of this study was to evaluate tobacco plants as a seedspecific expression system for the production of the flgK flagellar hook-associated protein from a wild type Salmonella typhimurium strain, as a model of an edible vaccine. The flgK gene is the principal component of bacterial flagella and is recognised as virulence factor by the innate immune system. It was isolated from the Salmonella typhimurium strain by PCR. The encoding sequence of flgK was transferred into a pBI binary vector, under control of soybean basic 7S globulin promoter for the seed-specific. Plant transformation was carried out using recombinant EHA 105 Agrobacterium tumefaciens. A transgenic population was obtained made up of independently kanamycin-resistant transgenic plants, which had a similar morphological appearance to the wild-type plants. Molecular analyses of seeds confirmed the integration of the gene and the average expression level of flgK was estimated to be about 0.6 mg per gram of seeds, corresponding to 0.33% of the total amount of soluble protein in tobacco seeds. This study showed that the foreign flgK gene could be stably incorporated into the tobacco plant genome by transcription through the nuclear apparatus of the plant, and that these genes are inherited by the next generation.

PI3K activation is associated with intracellular sodium/iodide symporter protein expression in breast cancer

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Knostman, Katherine AB; McCubrey, James A; Morrison, Carl D; Zhang, Zhaoxia; Capen, Charles C; Jhiang, Sissy M

2007-01-01

The sodium/iodide symporter (NIS) is a membrane glycoprotein mediating active iodide uptake in the thyroid gland and is the molecular basis for radioiodide imaging and therapeutic ablation of thyroid carcinomas. NIS is expressed in the lactating mammary gland and in many human breast tumors, raising interest in similar use for diagnosis and treatment. However, few human breast tumors have clinically evident iodide uptake ability. We previously identified PI3K signaling as important in NIS upregulation in transgenic mouse models of breast cancer, and the PI3K pathway is commonly activated in human breast cancer. NIS expression, subcellular localization, and function were analyzed in MCF-7 human breast cancer cells and MCF-7 cells stably or transiently expressing PI3K p110alpha subunit using Western blot of whole cell lysate, cell surface biotinylation Western blot and immunofluorescence, and radioiodide uptake assay, respectively. NIS localization was determined in a human breast cancer tissue microarray using immunohistochemical staining (IHC) and was correlated with pre-existing pAkt IHC data. Statistical analysis consisted of Student's t-test (in vitro studies) or Fisher's Exact Test (in vivo correlational studies). In this study, we demonstrate that PI3K activation in MCF-7 human mammary carcinoma cells leads to expression of underglycosylated NIS lacking cell surface trafficking necessary for iodide uptake ability. PI3K activation also appears to interfere with cell surface trafficking of exogenous NIS as well as all-trans retinoic acid-induced endogenous NIS. A correlation between NIS expression and upregulation of PI3K signaling was found in a human breast cancer tissue microarray. Thus, the PI3K pathway likely plays a major role in the discordance between NIS expression and iodide uptake in breast cancer patients. Further study is warranted to realize the application of NIS-mediated radioiodide ablation in breast cancer

Stabilization of Transfected Cells Expressing Low-Incidence Blood Group Antigens: Novel Methods Facilitating Their Use as Reagent-Cells.

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Cecilia González

Full Text Available The identification of erythrocyte antibodies in the serum of patients rely on panels of human red blood cells (RBCs, which coexpress many antigens and are not easily available for low-incidence blood group phenotypes. These problems have been addressed by generating cell lines expressing unique blood group antigens, which may be used as an alternative to human RBCs. However, the use of cell lines implies several drawbacks, like the requirement of cell culture facilities and the high cost of cryopreservation. The application of cell stabilization methods could facilitate their use as reagent cells in clinical laboratories.We generated stably-transfected cells expressing low-incidence blood group antigens (Dia and Lua. High-expresser clones were used to assess the effect of TransFix® treatment and lyophilization as cell preservation methods. Cells were kept at 4°C and cell morphology, membrane permeability and antigenic properties were evaluated at several time-points after treatment.TransFix® addition to cell suspensions allows cell stabilization and proper antigen detection for at least 120 days, despite an increase in membrane permeability and a reduction in antigen expression levels. Lyophilized cells showed minor morphological changes and antigen expression levels were rather conserved at days 1, 15 and 120, indicating a high stability of the freeze-dried product. These stabilized cells have been proved to react specifically with human sera containing alloantibodies.Both stabilization methods allow long-term preservation of the transfected cells antigenic properties and may facilitate their distribution and use as reagent-cells expressing low-incidence antigens, overcoming the limited availability of such rare RBCs.

Seasonal alteration in amounts of lignans and their glucosides and gene expression of the relevant biosynthetic enzymes in the Forsythia suspense leaf.

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Morimoto, Kinuyo; Satake, Honoo

2013-01-01

Lignans of Forsythia spp. are essential components of various Chinese medicines and health diets. However, the seasonal alteration in lignan amounts and the gene expression profile of lignan-biosynthetic enzymes has yet to be investigated. In this study, we have assessed seasonal alteration in amounts of major lignans, such as pinoresinol, matairesinol, and arctigenin, and examined the gene expression profile of pinoresinol/lariciresinol reductase (PLR), pinoresinol-glucosylating enzyme (UGT71A18), and secoisolariciresinol dehydrogenase (SIRD) in the leaf of Forsythia suspense from April to November. All of the lignans in the leaf continuously increased from April to June, reached the maximal level in June, and then decreased. Ninety percent of pinoresinol and matairesinol was converted into glucosides, while approximately 50% of arctigenin was aglycone. PLR was stably expressed from April to August, whereas the PLR expression was not detected from September to November. In contrast, the UGT71A18 expression was found from August to November, but not from April to July. The SIRD expression was prominent from April to May, not detected in June to July, and then increased again from September to November. These expression profiles of the lignan-synthetic enzymes are largely compatible with the alteration in lignan contents. Furthermore, such seasonal lignan profiles are in good agreement with the fact that the Forsythia leaves for Chinese medicinal tea are harvested in June. This is the first report on seasonal alteration in lignans and the relevant biosynthetic enzyme genes in the leaf of Forsythia species.

Antibody expressing pea seeds as fodder for prevention of gastrointestinal parasitic infections in chickens

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Macek Jeanette

2009-09-01

Full Text Available Abstract Background Coccidiosis caused by protozoans of genus Eimeria is a chicken parasitic disease of great economical importance. Conventional disease control strategies depend on vaccination and prophylactic use of anticoccidial drugs. Alternative solution to prevent and treat coccidiosis could be provided by passive immunization using orally delivered neutralizing antibodies. We investigated the possibility to mitigate the parasitic infection by feeding poultry with antibody expressing transgenic crop seeds. Results Using the phage display antibody library, we generated a panel of anti-Eimeria scFv antibody fragments with high sporozoite-neutralizing activity. These antibodies were expressed either transiently in agrobacteria-infiltrated tobacco leaves or stably in seeds of transgenic pea plants. Comparison of the scFv antibodies purified either from tobacco leaves or from the pea seeds demonstrated no difference in their antigen-binding activity and molecular form compositions. Force-feeding experiments demonstrated that oral delivery of flour prepared from the transgenic pea seeds had higher parasite neutralizing activity in vivo than the purified antibody fragments isolated from tobacco. The pea seed content was found to protect antibodies against degradation by gastrointestinal proteases (>100-fold gain in stability. Ad libitum feeding of chickens demonstrated that the transgenic seeds were well consumed and not shunned. Furthermore, feeding poultry with shred prepared from the antibody expressing pea seeds led to significant mitigation of infection caused both by high and low challenge doses of Eimeria oocysts. Conclusion The results suggest that our strategy offers a general approach to control parasitic infections in production animals using cost-effective antibody expression in crop seeds affordable for the animal health market.

Expression Stabilities of Ten Candidate Reference Genes for RT-qPCR in Zanthoxylum bungeanum Maxim.

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Fei, Xitong; Shi, Qianqian; Yang, Tuxi; Fei, Zhaoxue; Wei, Anzhi

2018-03-30

Real-time reverse transcription quantitative PCR has become a common method for studying gene expression, however, the optimal selection of stable reference genes is a prerequisite for obtaining accurate quantification of transcript abundance. Suitable reference genes for RT-qPCR have not yet been identified for Chinese prickly ash ( Zanthoxylum bungeanum Maxim.). Chinese prickly ash is the source of an important food seasoning in China. In recent years, Chinese prickly ash has also been developed as a medicinal plant. The expression stabilities of ten genes ( 18S , 28S , EF , UBA , UBQ , TIF , NTB , TUA , RPS , and TIF5A ) were evaluated in roots, stems, leaves, flowers and fruits at five developmental stages and also under stress from cold, drought, and salt. To do this we used three different statistical algorithms: geNorm, NormFinder and BestKeeper. Among the genes investigated, UBA and UBQ were found to be most stable for the different cultivars and different tissues examined, UBQ and TIF for fruit developmental stage. Meanwhile, EF and TUA were most stable under cold treatment, EF and UBQ under drought treatment and NTB and RPS under salt treatment. UBA and UBQ for all samples evaluated were most stably expressed, but 18S , TUA and RPS were found to be generally unreliable as reference genes. Our results provide a basis for the future selection of reference genes for biological research with Chinese prickly ash, under a variety of conditions.

Epigenomics: dissecting hybridization and polyploidization.

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Jackson, Scott A

2017-06-19

Epigenetic profiling in diploid, allopolyploid, and domesticated cotton shows that despite most DNA methylation being conserved and stably inherited, alterations likely due to hybridization and domestication affect gene expression.

Ectopic expression of Arabidopsis broad-spectrum resistance gene RPW8.2 improves the resistance to powdery mildew in grapevine (Vitis vinifera).

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Hu, Yang; Li, Yajuan; Hou, Fengjuan; Wan, Dongyan; Cheng, Yuan; Han, Yongtao; Gao, Yurong; Liu, Jie; Guo, Ye; Xiao, Shunyuan; Wang, Yuejin; Wen, Ying-Qiang

2018-02-01

Powdery mildew is the most economically important disease of cultivated grapevines worldwide. Here, we report that the Arabidopsis broad-spectrum disease resistance gene RPW8.2 could improve resistance to powdery mildew in Vitis vinifera cv. Thompson Seedless. The RPW8.2-YFP fusion gene was stably expressed in grapevines from either the constitutive 35S promoter or the native promoter (NP) of RPW8.2. The grapevine shoots and plantlets transgenic for 35S::RPW8.2-YFP showed reduced rooting and reduced growth at later development stages in the absence of any pathogens. Infection tests with an adapted grapevine powdery mildew isolate En NAFU1 showed that hyphal growth and sporulation were significantly restricted in transgenic grapevines expressing either of the two constructs. The resistance appeared to be attributable to the ectopic expression of RPW8.2, and associated with the enhanced encasement of the haustorial complex (EHC) and onsite accumulation of H 2 O 2 . In addition, the RPW8.2-YFP fusion protein showed focal accumulation around the fungal penetration sites. Transcriptome analysis revealed that ectopic expression of RPW8.2 in grapevines not only significantly enhanced salicylic acid-dependent defense signaling, but also altered expression of other phytohormone-associated genes. Taken together, our results indicate that RPW8.2 could be utilized as a transgene for improving resistance against powdery mildew in grapevines. Copyright © 2017 Elsevier B.V. All rights reserved.

The etomidate analog ET-26 HCl retains superior myocardial performance: Comparisons with etomidate in vivo and in vitro.

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Liu, Xingxing; Song, Haibo; Yang, Jun; Zhou, Cheng; Kang, Yi; Yang, Linghui; Liu, Jin; Zhang, Wensheng

2018-01-01

(R)-2-methoxyethyl1-(1-phenylethyl)-1H-imidazole-5-carboxylate hydrochloride (ET-26 HCl) is a novel etomidate analogue. The purpose of this study was to characterize whether ET-26 HCl could retain the superior myocardial performance of etomidate in vivo and in vitro. In vivo, the influence of ET-26 HCl and etomidate on the cardiac function of dogs was confirmed using echocardiography and electrocardiogram. In vitro, a Langendorff preparation was used to examine direct myocardial performance in isolated rat hearts, and a whole-cell patch-clamp technique was used to study effects on the human ether-a-go-go-related gene (hERG) channel. In vivo, after a single bolus administration of ET-26 HCl or etomidate, no significant difference in echocardiography and electrocardiogram parameters was observed. No arrhythmia occurred and no QT interval prolongation happened during the study period. In the in vitro Langendorff preparation, none of the cardiac parameters were abnormal, and the hERG recordings showed that ET-26 HCl and etomidate inhibited the tail current of the hERG in a concentration-dependent manner with an IC50 of 742.51 μM and 263.60 μM, respectively. In conclusion, through an in vivo experiment and a whole organ preparation, the current study found that ET-26 HCl can maintain a myocardial performance that is similar to that of etomidate. In addition, the electrophysiology study indicated that ET-26 HCl and etomidate inhibited the hERG at a supra-therapeutic concentration.

Expression and stability of foreign epitopes introduced into 3A nonstructural protein of foot-and-mouth disease virus.

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Pinghua Li

Full Text Available Foot-and-mouth disease virus (FMDV is an aphthovirus that belongs to the Picornaviridae family and causes one of the most important animal diseases worldwide. The capacity of other picornaviruses to express foreign antigens has been extensively reported, however, little is known about FMDV. To explore the potential of FMDV as a viral vector, an 11-amino-acid (aa HSV epitope and an 8 aa FLAG epitope were introduced into the C-terminal different regions of 3A protein of FMDV full-length infectious cDNA clone. Recombinant viruses expressing the HSV or FLAG epitope were successfully rescued after transfection of both modified constructs. Immunofluorescence assay, Western blot and sequence analysis showed that the recombinant viruses stably maintained the foreign epitopes even after 11 serial passages in BHK-21 cells. The 3A-tagged viruses shared similar plaque phenotypes and replication kinetics to those of the parental virus. In addition, mice experimentally infected with the epitope-tagged viruses could induce tag-specific antibodies. Our results demonstrate that FMDV can be used effectively as a viral vector for the delivery of foreign tags.

Expression and Stability of Foreign Epitopes Introduced into 3A Nonstructural Protein of Foot-and-Mouth Disease Virus

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Li, Pinghua; Bai, Xingwen; Cao, Yimei; Han, Chenghao; Lu, Zengjun; Sun, Pu; Yin, Hong; Liu, Zaixin

2012-01-01

Foot-and-mouth disease virus (FMDV) is an aphthovirus that belongs to the Picornaviridae family and causes one of the most important animal diseases worldwide. The capacity of other picornaviruses to express foreign antigens has been extensively reported, however, little is known about FMDV. To explore the potential of FMDV as a viral vector, an 11-amino-acid (aa) HSV epitope and an 8 aa FLAG epitope were introduced into the C-terminal different regions of 3A protein of FMDV full-length infectious cDNA clone. Recombinant viruses expressing the HSV or FLAG epitope were successfully rescued after transfection of both modified constructs. Immunofluorescence assay, Western blot and sequence analysis showed that the recombinant viruses stably maintained the foreign epitopes even after 11 serial passages in BHK-21 cells. The 3A-tagged viruses shared similar plaque phenotypes and replication kinetics to those of the parental virus. In addition, mice experimentally infected with the epitope-tagged viruses could induce tag-specific antibodies. Our results demonstrate that FMDV can be used effectively as a viral vector for the delivery of foreign tags. PMID:22848509

Validation of suitable reference genes for expression normalization in Echinococcus spp. larval stages.

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Espínola, Sergio Martin; Ferreira, Henrique Bunselmeyer; Zaha, Arnaldo

2014-01-01

In recent years, a significant amount of sequence data (both genomic and transcriptomic) for Echinococcus spp. has been published, thereby facilitating the analysis of genes expressed during a specific stage or involved in parasite development. To perform a suitable gene expression quantification analysis, the use of validated reference genes is strongly recommended. Thus, the aim of this work was to identify suitable reference genes to allow reliable expression normalization for genes of interest in Echinococcus granulosus sensu stricto (s.s.) (G1) and Echinococcus ortleppi upon induction of the early pre-adult development. Untreated protoscoleces (PS) and pepsin-treated protoscoleces (PSP) from E. granulosus s.s. (G1) and E. ortleppi metacestode were used. The gene expression stability of eleven candidate reference genes (βTUB, NDUFV2, RPL13, TBP, CYP-1, RPII, EF-1α, βACT-1, GAPDH, ETIF4A-III and MAPK3) was assessed using geNorm, Normfinder, and RefFinder. Our qPCR data showed a good correlation with the recently published RNA-seq data. Regarding expression stability, EF-1α and TBP were the most stable genes for both species. Interestingly, βACT-1 (the most commonly used reference gene), and GAPDH and ETIF4A-III (previously identified as housekeeping genes) did not behave stably in our assay conditions. We propose the use of EF-1α as a reference gene for studies involving gene expression analysis in both PS and PSP experimental conditions for E. granulosus s.s. and E. ortleppi. To demonstrate its applicability, EF-1α was used as a normalizer gene in the relative quantification of transcripts from genes coding for antigen B subunits. The same EF-1α reference gene may be used in studies with other Echinococcus sensu lato species. This report validates suitable reference genes for species of class Cestoda, phylum Platyhelminthes, thus providing a foundation for further validation in other epidemiologically important cestode species, such as those from the

Validation of Suitable Reference Genes for Expression Normalization in Echinococcus spp. Larval Stages

Science.gov (United States)

Espínola, Sergio Martin; Ferreira, Henrique Bunselmeyer; Zaha, Arnaldo

2014-01-01

In recent years, a significant amount of sequence data (both genomic and transcriptomic) for Echinococcus spp. has been published, thereby facilitating the analysis of genes expressed during a specific stage or involved in parasite development. To perform a suitable gene expression quantification analysis, the use of validated reference genes is strongly recommended. Thus, the aim of this work was to identify suitable reference genes to allow reliable expression normalization for genes of interest in Echinococcus granulosus sensu stricto (s.s.) (G1) and Echinococcus ortleppi upon induction of the early pre-adult development. Untreated protoscoleces (PS) and pepsin-treated protoscoleces (PSP) from E. granulosus s.s. (G1) and E. ortleppi metacestode were used. The gene expression stability of eleven candidate reference genes (βTUB, NDUFV2, RPL13, TBP, CYP-1, RPII, EF-1α, βACT-1, GAPDH, ETIF4A-III and MAPK3) was assessed using geNorm, Normfinder, and RefFinder. Our qPCR data showed a good correlation with the recently published RNA-seq data. Regarding expression stability, EF-1α and TBP were the most stable genes for both species. Interestingly, βACT-1 (the most commonly used reference gene), and GAPDH and ETIF4A-III (previously identified as housekeeping genes) did not behave stably in our assay conditions. We propose the use of EF-1α as a reference gene for studies involving gene expression analysis in both PS and PSP experimental conditions for E. granulosus s.s. and E. ortleppi. To demonstrate its applicability, EF-1α was used as a normalizer gene in the relative quantification of transcripts from genes coding for antigen B subunits. The same EF-1α reference gene may be used in studies with other Echinococcus sensu lato species. This report validates suitable reference genes for species of class Cestoda, phylum Platyhelminthes, thus providing a foundation for further validation in other epidemiologically important cestode species, such as those from the

Gene silencing in primary and metastatic tumors by small interfering RNA delivery in mice: quantitative analysis using melanoma cells expressing firefly and sea pansy luciferases.

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Takahashi, Yuki; Nishikawa, Makiya; Kobayashi, Naoki; Takakura, Yoshinobu

2005-07-20

Silencing of oncogenes or other genes contributing to tumor malignancy or progression by RNA interference (RNAi) offers a promising approach to treating tumor patients. To achieve RNAi-based tumor therapy, a small interfering RNA (siRNA) or siRNA-expressing vector needs to be delivered to tumor cells, but little information about its in vivo delivery has been reported. In this study, we examined whether the expression of the target gene in tumor cells can be suppressed by the delivery of RNAi effectors to primary and metastatic tumor cells. To quantitatively evaluate the RNAi effects in tumor cells, mouse melanoma B16-BL6 cells were stably transfected with both firefly (a model target gene) and sea pansy (an internal standard gene) luciferase genes to obtain B16-BL6/dual Luc cells. The target gene expression in subcutaneous primary tumors of B16-BL6/dual Luc cells was significantly suppressed by direct injection of the RNAi effectors followed by electroporation. The expression in metastatic hepatic tumors was also significantly reduced by an intravenous injection of either RNAi effector by the hydrodynamics-based procedure. These results indicate that the both RNAi effectors have a potential to silence target gene in tumor cells in vivo when successfully delivered to tumor cells.

Cloning, Expression, Purification, and Characterization of Glutaredoxin from Antarctic Sea-Ice Bacterium Pseudoalteromonas sp. AN178

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Quanfu Wang

2014-01-01

Full Text Available Glutaredoxins (Grxs are small ubiquitous redox enzymes that catalyze glutathione-dependent reactions to reduce protein disulfide. In this study, a full-length Grx gene (PsGrx with 270 nucleotides was isolated from Antarctic sea-ice bacterium Pseudoalteromonas sp. AN178. It encoded deduced 89 amino acid residues with the molecular weight 9.8 kDa. Sequence analysis of the amino acid sequence revealed the catalytic motif CPYC. Recombinant PsGrx (rPsGrx stably expressed in E. coli BL21 was purified to apparent homogeneity by Ni-affinity chromatography. rPsGrx exhibited optimal activity at 30°C and pH 8.0 and showed 25.5% of the activity at 0°C. It retained 65.0% of activity after incubation at 40°C for 20 min and still exhibited 37.0% activity in 1.0 M NaCl. These results indicated that rPsGrx was a typical cold active protein with low thermostability.

Identification of reference genes for quantitative expression analysis using large-scale RNA-seq data of Arabidopsis thaliana and model crop plants.

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Kudo, Toru; Sasaki, Yohei; Terashima, Shin; Matsuda-Imai, Noriko; Takano, Tomoyuki; Saito, Misa; Kanno, Maasa; Ozaki, Soichi; Suwabe, Keita; Suzuki, Go; Watanabe, Masao; Matsuoka, Makoto; Takayama, Seiji; Yano, Kentaro

2016-10-13

In quantitative gene expression analysis, normalization using a reference gene as an internal control is frequently performed for appropriate interpretation of the results. Efforts have been devoted to exploring superior novel reference genes using microarray transcriptomic data and to evaluating commonly used reference genes by targeting analysis. However, because the number of specifically detectable genes is totally dependent on probe design in the microarray analysis, exploration using microarray data may miss some of the best choices for the reference genes. Recently emerging RNA sequencing (RNA-seq) provides an ideal resource for comprehensive exploration of reference genes since this method is capable of detecting all expressed genes, in principle including even unknown genes. We report the results of a comprehensive exploration of reference genes using public RNA-seq data from plants such as Arabidopsis thaliana (Arabidopsis), Glycine max (soybean), Solanum lycopersicum (tomato) and Oryza sativa (rice). To select reference genes suitable for the broadest experimental conditions possible, candidates were surveyed by the following four steps: (1) evaluation of the basal expression level of each gene in each experiment; (2) evaluation of the expression stability of each gene in each experiment; (3) evaluation of the expression stability of each gene across the experiments; and (4) selection of top-ranked genes, after ranking according to the number of experiments in which the gene was expressed stably. Employing this procedure, 13, 10, 12 and 21 top candidates for reference genes were proposed in Arabidopsis, soybean, tomato and rice, respectively. Microarray expression data confirmed that the expression of the proposed reference genes under broad experimental conditions was more stable than that of commonly used reference genes. These novel reference genes will be useful for analyzing gene expression profiles across experiments carried out under various

Monitoring of phytopathogenic Ralstonia solanacearum cells using green fluorescent protein-expressing plasmid derived from bacteriophage phiRSS1.

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Kawasaki, Takeru; Satsuma, Hideki; Fujie, Makoto; Usami, Shoji; Yamada, Takashi

2007-12-01

A green fluorescent protein (GFP)-expressing plasmid was constructed from a filamentous bacteriophage phiRSS1 that infects the phytopathogen Ralstonia solanacearum. This plasmid designated as pRSS12 (4.7 kbp in size) consists of an approximately 2248 bp region of the phiRSS1 RF DNA, including ORF1-ORF3 and the intergenic region (IG), and a Km cassette in addition to the GFP gene. It was easily introduced by electroporation and stably maintained even without selective pressure in strains of R. solanacearum of different races and biovars. Strong green fluorescence emitted from pRSS12-transformed bacterial cells was easily monitored in tomato tissues (stem, petiole, and root) after infection as well as from soil samples. These results suggest that pRSS12 can serve as an easy-to-use GFP-tagging tool for any given strain of R. solanacearum in cytological as well as field studies.

The effects of 5-fluorouracil and doxorubicin on expression of human immunodeficiency virus type 1 long terminal repeat

International Nuclear Information System (INIS)

Panozzo, J.; Akan, E.; Griffiths, T.D.

1996-01-01

Previous work by many groups has documented induction of the HIV-LTR following exposure of cells to ultraviolet light and other DNA damaging agents. Our experiments set out to determine the relative activation or repression of the HIV-LTR in response to two classes of chemotherapeutic agents: Doxorubicin is a DNA-damage inducing agent, and 5-fluorouracil has an antimetabolic mode of action. Using HeLa cells stably transfected with a construct in which HIV-LTR drives expression of the chloramphenicol acetyl transferase reporter gene, we demonstrated an up to 10-fold induction following doxorubicin treatment in 24 h post-treatment. This induction was repressed by treatment with salicylic acid, suggesting a role for prostaglandin/cyclo-oxygenase pathways and/or NFKB in the inductive response. Induction by 5-fluorouracil, in contrast, was more modest (two-fold at most) though it was consistently elevated over controls

Matrix Metalloproteinase Expression in the Rat Myometrium During Pregnancy, Term Labor, and Postpartum1

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Nguyen, Tina Tu-Thu Ngoc; Shynlova, Oksana; Lye, Stephen J.

2016-01-01

Pregnancy, spontaneous term labor (TL), and postpartum (PP) involution are associated with changes in the cellular and extracellular matrix composition of the uterus. Both the uterine smooth muscle (myometrium) and the infiltrating peripheral blood leukocytes involved in the activation of labor secrete extracellular matrix-degrading enzymes (matrix metalloproteinases, MMPs) that can modulate cellular behavior and barrier function. MMP expression is induced by mechanical stretch in several tissues. We hypothesized that the expression and activity of myometrial MMPs and their tissue inhibitors (TIMPs) are modulated in preparation for TL and PP involution and are regulated by mechanical stretch of uterine walls imposed by the growing fetus. Myometrial tissues were collected from bilaterally and unilaterally pregnant rats across gestation, TL, and PP. Total RNA and proteins were subjected to real-time PCR and immunoblotting, respectively, and tissue localization and activity was examined by immunohistochemistry and in situ zymography. We found that Mmp7, Mmp11, and Mmp12 mRNA levels were upregulated during TL and PP, while Mmp2, Mmp3, Mmp8, Mmp9, Mmp10, and Mmp13 mRNAs were only upregulated during PP. Timp1–Timp4 were stably expressed throughout gestation with some fluctuations PP. Active MMP2 was induced in the empty uterine horn during gestation and in the gravid PP uterus, suggesting negative regulation by biological mechanical stretch. We conclude that specific subsets of uterine MMPs are differentially regulated in the rat myometrium in preparation for two major events: TL and PP uterine involution. PMID:27251092

Selection of reference genes for quantitative gene expression normalization in flax (Linum usitatissimum L.

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Neutelings Godfrey

2010-04-01

Full Text Available Abstract Background Quantitative real-time PCR (qRT-PCR is currently the most accurate method for detecting differential gene expression. Such an approach depends on the identification of uniformly expressed 'housekeeping genes' (HKGs. Extensive transcriptomic data mining and experimental validation in different model plants have shown that the reliability of these endogenous controls can be influenced by the plant species, growth conditions and organs/tissues examined. It is therefore important to identify the best reference genes to use in each biological system before using qRT-PCR to investigate differential gene expression. In this paper we evaluate different candidate HKGs for developmental transcriptomic studies in the economically-important flax fiber- and oil-crop (Linum usitatissimum L. Results Specific primers were designed in order to quantify the expression levels of 20 different potential housekeeping genes in flax roots, internal- and external-stem tissues, leaves and flowers at different developmental stages. After calculations of PCR efficiencies, 13 HKGs were retained and their expression stabilities evaluated by the computer algorithms geNorm and NormFinder. According to geNorm, 2 Transcriptional Elongation Factors (TEFs and 1 Ubiquitin gene are necessary for normalizing gene expression when all studied samples are considered. However, only 2 TEFs are required for normalizing expression in stem tissues. In contrast, NormFinder identified glyceraldehyde-3-phosphate dehydrogenase (GADPH as the most stably expressed gene when all samples were grouped together, as well as when samples were classed into different sub-groups. qRT-PCR was then used to investigate the relative expression levels of two splice variants of the flax LuMYB1 gene (homologue of AtMYB59. LuMYB1-1 and LuMYB1-2 were highly expressed in the internal stem tissues as compared to outer stem tissues and other samples. This result was confirmed with both ge

Selection of reference genes for quantitative gene expression normalization in flax (Linum usitatissimum L.).

Science.gov (United States)

Huis, Rudy; Hawkins, Simon; Neutelings, Godfrey

2010-04-19

Quantitative real-time PCR (qRT-PCR) is currently the most accurate method for detecting differential gene expression. Such an approach depends on the identification of uniformly expressed 'housekeeping genes' (HKGs). Extensive transcriptomic data mining and experimental validation in different model plants have shown that the reliability of these endogenous controls can be influenced by the plant species, growth conditions and organs/tissues examined. It is therefore important to identify the best reference genes to use in each biological system before using qRT-PCR to investigate differential gene expression. In this paper we evaluate different candidate HKGs for developmental transcriptomic studies in the economically-important flax fiber- and oil-crop (Linum usitatissimum L). Specific primers were designed in order to quantify the expression levels of 20 different potential housekeeping genes in flax roots, internal- and external-stem tissues, leaves and flowers at different developmental stages. After calculations of PCR efficiencies, 13 HKGs were retained and their expression stabilities evaluated by the computer algorithms geNorm and NormFinder. According to geNorm, 2 Transcriptional Elongation Factors (TEFs) and 1 Ubiquitin gene are necessary for normalizing gene expression when all studied samples are considered. However, only 2 TEFs are required for normalizing expression in stem tissues. In contrast, NormFinder identified glyceraldehyde-3-phosphate dehydrogenase (GADPH) as the most stably expressed gene when all samples were grouped together, as well as when samples were classed into different sub-groups.qRT-PCR was then used to investigate the relative expression levels of two splice variants of the flax LuMYB1 gene (homologue of AtMYB59). LuMYB1-1 and LuMYB1-2 were highly expressed in the internal stem tissues as compared to outer stem tissues and other samples. This result was confirmed with both geNorm-designated- and Norm

Study on construction of pEgr-hPTEN expression vector induced by irradiation and its anti-tumor effect in vitro

International Nuclear Information System (INIS)

Tian Mei; Jin Guanghui; Piao Chunji; Li Xiuyi; Liu Linlin

2003-01-01

Objective: To clone the cDNA of human tumor suppressor gene-PTEN, construct pEgr-hPTEN expression vector induced by irradiation and study its inhibitory effect on proliferation of malignant glioma cell line SHG-44 transfected steadily with pEgr-hPTEN after different doses of X-ray irradiation. Methods: A DNA fragment about 1200 bp, PTEN, was amplified from human placenta tissues by using RT-nested PCR and was cloned into pUCm-T vector after automatic sequencing, then the fragment was inserted into a vector pcD-NA3.1-Egr to construct an expression vector pEgr-hPTEN. pEgr-hPTEN was transfected into SHG-44 cells in vitro. Stably transfected cell line SHG-44-sPTEN was selected through G418. The inhibitor effect on SHG-44-sPTEN was observed after different doses of X-ray irradiation in vitro. Results: The PTEN cDNA has been cloned correctly and its expression vector pEgr-hPTEN was also constructed. Growth of SHG-44 cells was inhibited significantly by stable pEgr-hPTEN transfection combined with X-ray irradiation. With the increase of dose, the inhibitory effect was enhanced within 5 Gy. Conclusion: Human tumor suppressor gene-PTEN cDNA has been cloned and its expression vector has been constructed. The tumor was inhibited significantly by gene-radiotherapy in vitro. The result provides the theoretical and experimental basis for improvement of clinical radiotherapeutic effect on tumors

A resampling-based meta-analysis for detection of differential gene expression in breast cancer

International Nuclear Information System (INIS)

Gur-Dedeoglu, Bala; Konu, Ozlen; Kir, Serkan; Ozturk, Ahmet Rasit; Bozkurt, Betul; Ergul, Gulusan; Yulug, Isik G

2008-01-01

Accuracy in the diagnosis of breast cancer and classification of cancer subtypes has improved over the years with the development of well-established immunohistopathological criteria. More recently, diagnostic gene-sets at the mRNA expression level have been tested as better predictors of disease state. However, breast cancer is heterogeneous in nature; thus extraction of differentially expressed gene-sets that stably distinguish normal tissue from various pathologies poses challenges. Meta-analysis of high-throughput expression data using a collection of statistical methodologies leads to the identification of robust tumor gene expression signatures. A resampling-based meta-analysis strategy, which involves the use of resampling and application of distribution statistics in combination to assess the degree of significance in differential expression between sample classes, was developed. Two independent microarray datasets that contain normal breast, invasive ductal carcinoma (IDC), and invasive lobular carcinoma (ILC) samples were used for the meta-analysis. Expression of the genes, selected from the gene list for classification of normal breast samples and breast tumors encompassing both the ILC and IDC subtypes were tested on 10 independent primary IDC samples and matched non-tumor controls by real-time qRT-PCR. Other existing breast cancer microarray datasets were used in support of the resampling-based meta-analysis. The two independent microarray studies were found to be comparable, although differing in their experimental methodologies (Pearson correlation coefficient, R = 0.9389 and R = 0.8465 for ductal and lobular samples, respectively). The resampling-based meta-analysis has led to the identification of a highly stable set of genes for classification of normal breast samples and breast tumors encompassing both the ILC and IDC subtypes. The expression results of the selected genes obtained through real-time qRT-PCR supported the meta-analysis results. The

A resampling-based meta-analysis for detection of differential gene expression in breast cancer

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Ergul Gulusan

2008-12-01

Full Text Available Abstract Background Accuracy in the diagnosis of breast cancer and classification of cancer subtypes has improved over the years with the development of well-established immunohistopathological criteria. More recently, diagnostic gene-sets at the mRNA expression level have been tested as better predictors of disease state. However, breast cancer is heterogeneous in nature; thus extraction of differentially expressed gene-sets that stably distinguish normal tissue from various pathologies poses challenges. Meta-analysis of high-throughput expression data using a collection of statistical methodologies leads to the identification of robust tumor gene expression signatures. Methods A resampling-based meta-analysis strategy, which involves the use of resampling and application of distribution statistics in combination to assess the degree of significance in differential expression between sample classes, was developed. Two independent microarray datasets that contain normal breast, invasive ductal carcinoma (IDC, and invasive lobular carcinoma (ILC samples were used for the meta-analysis. Expression of the genes, selected from the gene list for classification of normal breast samples and breast tumors encompassing both the ILC and IDC subtypes were tested on 10 independent primary IDC samples and matched non-tumor controls by real-time qRT-PCR. Other existing breast cancer microarray datasets were used in support of the resampling-based meta-analysis. Results The two independent microarray studies were found to be comparable, although differing in their experimental methodologies (Pearson correlation coefficient, R = 0.9389 and R = 0.8465 for ductal and lobular samples, respectively. The resampling-based meta-analysis has led to the identification of a highly stable set of genes for classification of normal breast samples and breast tumors encompassing both the ILC and IDC subtypes. The expression results of the selected genes obtained through real

Gelatinase (MMP-2 and -9 expression profiles during gestation in the bovine endometrium

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Sato Takashi

2008-12-01

Full Text Available Abstract Background Various molecules participate in implantation and maintaining endometrial function during gestation. The remodeling of endometrial matrices is a necessary process in the coordination of gestational progress. Matrix-metalloproteinases (MMPs like gelatinases (MMP-2 and -9 and collagenase (MMP-1 are considered to play important roles in this process. We examined MMP-2 and -9 expression using zymography, in situ hybridization, real-time PCR, and microarray analysis to clarify their roles in the bovine endometrium during gestation. Methods Endometria, placentomes, and fetal membranes were collected from Japanese black cows that were killed on day 15 to 252 of gestation or during their estrous cycle. The gene expression of MMP-related molecules (mainly MMP-2 and -9 was examined using a custom-made microarray, real-time RT-PCR, and in-situ hybridization. Gelatinase activity was detected by zymography and film in situ zymography. Results Both gelatinases were expressed in the endometrium and fetal tissues throughout gestation. MMP-2 gene expression declined with the progress of gestation, but its intensity was maintained at a high level during the peri-implantation period and increased in late gestation. The expression level of MMP-9 was stably maintained, but was relatively low compared to that of MMP-2. These gene expression patterns matched those detected by zymography for the proteins. Microarray analysis suggested that the functions of MMP-2 during implantation and the last part of gestation are closely related with those of other molecules such as tissue inhibitors of metalloproteinase (TIMP-2, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS 1, membrane type 1 (MT1-MMP, and extracellular matrix metalloproteinase inducer (EMMPRIN. Conclusion We detected MMP-2 and -9 gene expression in the bovine endometrium and placentome throughout gestation. These data suggest that MMP-2 is one of the main endometrial

Construction of a fusion gene containing hepatitis B virus L gene ...

African Journals Online (AJOL)

The results of SDS-PAGE and Western blot showed that the recombinant protein was induced by methanol and stably expressed in P. pastoris, while it has specific reaction with the serum containing anti-HbsAg or anti-Ag85B. However, the successful construction of a recombinant yeast expression vector containing gene ...

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Indian Academy of Sciences (India)

Unknown

The fibroin promoter can stably express foreign gene in lepidopteran cells. Total RNA was extracted from the gland of silkworm, Antheraea pernyi and the transcription initiation site of fibroin gene of A. pernyi was identi- fied by RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE). The expression vector ...

Mechanism of As2O3-Induced Action Potential Prolongation and Using hiPS-CMs to Evaluate the Rescue Efficacy of Drugs With Different Rescue Mechanism.

Science.gov (United States)

Yan, Meng; Feng, Lifang; Shi, Yanhui; Wang, Junnan; Liu, Yan; Li, Fengmei; Li, Baoxin

2017-08-01

Arsenic trioxide (As2O3) has been verified as a breakthrough in the management of acute promyelocytic leukemia in recent decades. However, cardiotoxicity, especially long QT syndrome (LQTS) has become the most important issue during As2O3 treatment. The characterized mechanisms behind this adverse effect are inhibition of cardiac hERG channel trafficking and increase of cardiac calcium currents. In our study, we found a new pathway underlying As2O3-induced cardiotoxicity that As2O3 accelerates lysosomal degradation of hERG on plasma membrane after using brefeldin A (BFA) to block protein trafficking. Then we explored pharmacological rescue strategies on As2O3-induced LQTS, and found that 4 therapeutic agents exert rescue efficacy via 3 different pathways: fexofenadine and astemizole facilitate hERG trafficking via promotion of channel-chaperone formation after As2O3 incubation; ranolazine slows hERG degradation in the presence of As2O3; and resveratrol shows significant attenuation on calcium current increase triggered by As2O3. Moreover, we used human-induced pluripotent stem cell derived cardiomyocytes (hiPS-CMs) to evaluate the rescue effects of the above agents on As2O3-induced prolongation of action potential duration (APD) and demonstrated that fexofenadine and resveratrol significantly ameliorate the prolonged APD. These observations suggested that pharmacological chaperone like fexofenadine and resveratrol might have the potential to protect against the cardiotoxicity of As2O3. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Neurotensin is an antagonist of the human neurotensin NT2 receptor expressed in Chinese hamster ovary cells.

Science.gov (United States)

Vita, N; Oury-Donat, F; Chalon, P; Guillemot, M; Kaghad, M; Bachy, A; Thurneyssen, O; Garcia, S; Poinot-Chazel, C; Casellas, P; Keane, P; Le Fur, G; Maffrand, J P; Soubrie, P; Caput, D; Ferrara, P

1998-11-06

The human levocabastine-sensitive neurotensin NT2 receptor was cloned from a cortex cDNA library and stably expressed in Chinese hamster ovary (CHO) cells in order to study its binding and signalling characteristics. The receptor binds neurotensin as well as several other ligands already described for neurotensin NT1 receptor. It also binds levocabastine, a histamine H1 receptor antagonist that is not recognised by neurotensin NT1 receptor. Neurotensin binding to recombinant neurotensin NT2 receptor expressed in CHO cells does not elicit a biological response as determined by second messenger measurements. Levocabastine, and the peptides neuromedin N and xenin were also ineffective on neurotensin NT2 receptor activation. Experiments with the neurotensin NT1 receptor antagonists SR48692 and SR142948A, resulted in the unanticipated discovery that both molecules are potent agonists on neurotensin NT2 receptor. Both compounds, following binding to neurotensin NT2 receptor, enhance inositol phosphates (IP) formation with a subsequent [Ca2+]i mobilisation; induce arachidonic acid release; and stimulate mitogen-activated protein kinase (MAPK) activity. Interestingly, these activities are antagonised by neurotensin and levocabastine in a concentration-dependent manner. These activities suggest that the human neurotensin NT2 receptor may be of physiological importance and that a natural agonist for the receptor may exist.

Expression of bovine non-classical major histocompatibility complex class I proteins in mouse P815 and human K562 cells.

Science.gov (United States)

Parasar, Parveen; Wilhelm, Amanda; Rutigliano, Heloisa M; Thomas, Aaron J; Teng, Lihong; Shi, Bi; Davis, William C; Suarez, Carlos E; New, Daniel D; White, Kenneth L; Davies, Christopher J

2016-08-01

Major histocompatibility complex class I (MHC-I) proteins can be expressed as cell surface or secreted proteins. To investigate whether bovine non-classical MHC-I proteins are expressed as cell surface or secreted proteins, and to assess the reactivity pattern of monoclonal antibodies with non-classical MHC-I isoforms, we expressed the MHC proteins in murine P815 and human K562 (MHC-I deficient) cells. Following antibiotic selection, stably transfected cell lines were stained with H1A or W6/32 antibodies to detect expression of the MHC-I proteins by flow cytometry. Two non-classical proteins (BoLA-NC1*00501 and BoLA-NC3*00101) were expressed on the cell surface in both cell lines. Surprisingly, the BoLA-NC4*00201 protein was expressed on the cell membrane of human K562 but not mouse P815 cells. Two non-classical proteins (BoLA-NC1*00401, which lacks a transmembrane domain, and BoLA-NC2*00102) did not exhibit cell surface expression. Nevertheless, Western blot analyses demonstrated expression of the MHC-I heavy chain in all transfected cell lines. Ammonium-sulfate precipitation of proteins from culture supernatants showed that BoLA-NC1*00401 was secreted and that all surface expressed proteins where shed from the cell membrane by the transfected cells. Interestingly, the surface expressed MHC-I proteins were present in culture supernatants at a much higher concentration than BoLA-NC1*00401. This comprehensive study shows that bovine non-classical MHC-I proteins BoLA-NC1*00501, BoLA-NC3*00101, and BoLA-NC4*00201 are expressed as surface isoforms with the latter reaching the cell membrane only in K562 cells. Furthermore, it demonstrated that BoLA-NC1*00401 is a secreted isoform and that significant quantities of membrane associated MHC-I proteins can be shed from the cell membrane. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

The Expression of Markers for Intratubular Germ Cell Neoplasia in Normal Infantile Testes

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Kolja Kvist

2018-06-01

Full Text Available BackgroundPositive immunohistochemical expression of testicular cancer markers is often reported beyond 12 months of age in cryptorchid testes, which is assumed to indicate delayed maturation of the fetal germ cells, or neoplastic changes. These findings allowed for questions as to the extent of positive reaction in normal testes. The aim of the study was to clarify the expression of these markers in a normal material up to 2 years.MethodsTesticular material from 69 boys aged 1–690 days, who died of causes with no association of testicular pathology. Histology sections were incubated with primary antibodies including anti-placental-like alkaline phosphatase (PLAP, anti-C-Kit, anti-D2–40, and anti-Oct3/4. The mean germ cell number per tubular transverse section (G/T was calculated based on the G/T of both testes of every boy.ResultsThe mean G/T declined through the 690 days. PLAP appeared stably expressed throughout the ages studied. The likelihood of a positive reaction for C-Kit waned with increasing age within the study period. Positive staining for D2–40 and Oct3/4 was demonstrated up to 6 and 9 months respectively.ConclusionUp to 1 or 2 years of age, normal infantile testes contain germ cells positive for the immunohistochemical markers commonly utilized to aid in the detection of testicular cancer. This finding supports the concept of germ cells undergoing a continuous maturational process in a heterogeneous fashion, and that this process is not complete by 2 years of age.

An ERG channel inhibitor from the scorpion Buthus eupeus

DEFF Research Database (Denmark)

Korolkova, Y.V.; Kozlov, S.A.; Lipkin, A.V.

2001-01-01

and the three mutants partly inhibited the native M-like current in NG108-15 at 100 nm. The effect of the recombinant BeKm-1 on different K(+) channels was also studied. BeKm-1 inhibited hERG1 channels with an IC(50) of 3.3 nm, but had no effect at 100 nm on hEAG, hSK1, rSK2, hIK, hBK, KCNQ1/KCNE1, KCNQ2/KCNQ3......, KCNQ4 channels, and minimal effect on rELK1. Thus, BeKm-1 was shown to be a novel specific blocker of hERG1 potassium channels....

Stable lentiviral transformation of CHO cells for the expression of the hemagglutinin H5 of avian influenza virus in suspension culture

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Alaín González Pose

2014-09-01

Full Text Available Avian influenza virus H5N1 has caused extensive damage worldwide among poultry and humans. Effective expression systems are needed for the production of viral proteins required for monitoring this devastating disease. The present study deals with the establishment of a stable expression system for the hemagglutinin H5 (HAH5 of avian influenza virus using CHO cells in suspension culture transduced with a recombinant lentiviral vector. The synthetic gene coding the HAH5 protein was inserted in a lentiviral vector with the aim of performing a stable transduction of CHO cells. After the selection of recombinant clones, the one with the highest expression level was adapted to suspension culture and the HAH5 protein was purified by immunoaffinity chromatography from the culture supernatant. There were no significant differences when this protein, purified or direct from the culture supernatant of CHO or SiHa cells, was utilized in an immunologic assay using positive and negative sera as reference. It was also demonstrated that the HAH5 protein in its purified form is able to bind anti-HAH5 antibodies generated with proper and non-proper folded proteins. The results demonstrate that the CHO cell line stably transduced with a lentiviral vector coding the sequence of the HAH5 protein and cultured in suspension can be a suitable expression system to obtain this protein for diagnostic purpose in a consistent and reliable manner.

Potential biological process of X-linked inhibitor of apoptosis protein in renal cell carcinoma based upon differential protein expression analysis.

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Chen, Chao; Zhao, Si Cong; Yang, Wen Zheng; Chen, Zong Ping; Yan, Yong

2018-01-01

The X-linked inhibitor of apoptosis protein (XIAP) is the best characterized member of the IAP family and is a potent inhibitor of the caspase/apoptosis pathway. It has also been revealed that XIAP has additional biological functions that rely on its direct inhibition of apoptosis. In the present study, stably transfected Caki-1 cells with XIAP-knockdown were generated, and an isobaric tag for relative and absolute quantitation-based proteomics approach was employed to investigate the regulatory mechanism of XIAP in renal cell carcinoma (RCC). The results demonstrate that the sensitivity of the RCC cell line to apoptotic stimulation increased markedly with XIAP-knockdown. A number of differentially expressed proteins were detected between the original Caki-1 cell line and the XIAP-knockdown Caki-1 cell line; 87 at 0 h (prior to etoposide treatment), 178 at 0.5 h and 169 at 3 h, while no differentially expressed proteins were detected (ratio >1.5 or <0.5; P<0.05) at 12 h after etoposide treatment. Through analysis of the differentially expressed proteins, it was revealed that XIAP may participate in the tumor protein p53 pathway, the Wnt signaling pathway, glucose metabolism, endoplasmic reticulum stress, cytoskeletal regulation and DNA repair. These results indicate that XIAP may have a number of biological functions and may provide an insight into the biomedical significance of XIAP overexpression in RCC.

Presenilin expression during induced differentiation of the human neuroblastoma SH-SY5Y cell line.

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Flood, Fiona; Sundström, Erik; Samuelsson, Eva-Britt; Wiehager, Birgitta; Seiger, Ake; Johnston, Janet A; Cowburn, Richard F

2004-06-01

Human neuroblastoma SH-SY5Y cells stably transfected with both wild-type and exon-9 deleted (deltaE9) presenilin constructs were used to study the role of the presenilin proteins during differentiation. Cells transfected with either wild-type or deltaE9 PS1, of which the latter abolishes normal endoproteolytic cleavage of the protein, showed no obvious differences in their ability to differentiate to a neuronal-like phenotype upon treatment with retinoic acid (RA). A defined pattern of PS1 expression was observed during differentiation with both RA and the phorbol ester TPA. Full-length PS1 was shown to increase dramatically within 5-24 h of RA treatment. TPA gave an earlier and longer lasting increase in full-length PS1 levels. The intracellular distribution pattern of PS1 was markedly altered following RA treatment. Within 24h PS1 was highly up-regulated throughout the cell body around the nucleus. Between 2 and 4 weeks PS1 staining appeared punctate and also localised to the nucleus. Increases in PS1 expression upon treatment with RA and TPA were blocked by treatment with cycloheximide, indicating a role of de-novo protein synthesis in this effect. PS2 expression remained unchanged during differentiation. Levels of full-length PS1 were also seen to increase during neurogenesis and neuronal differentiation in the forebrain of first trimester human foetuses between 6.5 and 11 weeks. These combined observations support the idea that PS1 is involved in neuronal differentiation by a mechanism likely independent of endoproteolysis of the protein.

The changing brain--insights into the mechanisms of neural and behavioral adaptation to the environment

DEFF Research Database (Denmark)

Bergersen, L H; Bramham, C R; Hugdahl, K

2013-01-01

level and behavior. Thus a single amino acid change in a transcriptional repressor can disrupt gene regulation through neural activity (Greenberg). Deep sequencing analysis of the neuropil transcriptome indicates that a large fraction of the synaptic proteome is synthesized in situ in axons...... and dendrites, permitting local regulation (Schuman). The nature of the 'reset' function that makes animals dependent of sleep is being revealed (Cirelli). Maternal behavior can cause changes in gene expression that stably modify behavior in the offspring (Meaney). Removal of a single sensory channel protein...... in the vomero-nasal organ can switch off male-specific and switch on female-specific innate behavior of mice in response to environmental stimulation (Dulac). Innate behaviors can be stably transmitted from parent to offspring through generations even when those behaviors cannot be expressed, as illustrated...

In vitro transfection of the hepatitis B virus PreS2 gene into the human hepatocarcinoma cell line HepG2 induces upregulation of human telomerase reverse transcriptase

International Nuclear Information System (INIS)

Liu Hua; Luan Fang; Ju Ying; Shen Hongyu; Gao Lifen; Wang Xiaoyan; Liu Suxia; Zhang Lining; Sun Wensheng; Ma Chunhong

2007-01-01

The preS2 domain is the minimal functional unit of transcription activators that is encoded by the Hepatitis B virus (HBV) surface (S) gene. It is present in more than one-third of the HBV-integrates in HBV induced hepatocarcinoma (HCC). To further understand the functional role of PreS2 in hepatocytes, a PreS2 expression plasmid, pcS2, was constructed and stably transfected into HepG2 cells. We conducted growth curve and colony-forming assays to study the impact of PreS2 expression on cell proliferation. Cells transfected with PreS2 proliferated more rapidly and formed colonies in soft agar. PreS2 expressing cells also induced upregulation of human telomerase reverse transcriptase (hTERT) and telomerase activation by RT-PCR and the modified TRAP assay. Blocking expression of hTERT with antisense oligonuleotide reversed the growth rate in cells stably transfected with PreS2. Our data suggest that PreS2 may increase the malignant transformation of human HCC cell line HepG2 by upregulating hTERT and inducing telomerase activation

In vitro transfection of the hepatitis B virus PreS2 gene into the human hepatocarcinoma cell line HepG2 induces upregulation of human telomerase reverse transcriptase

Energy Technology Data Exchange (ETDEWEB)

Hua, Liu [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Fang, Luan [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Ying, Ju [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Hongyu, Shen [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Lifen, Gao [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Xiaoyan, Wang [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Suxia, Liu [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Lining, Zhang [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Wensheng, Sun [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Chunhong, Ma [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Key Laboratory for Experimental Teratology, Ministry of Education (China)]. E-mail: machunhong@sdu.edu.cn

2007-04-06

The preS2 domain is the minimal functional unit of transcription activators that is encoded by the Hepatitis B virus (HBV) surface (S) gene. It is present in more than one-third of the HBV-integrates in HBV induced hepatocarcinoma (HCC). To further understand the functional role of PreS2 in hepatocytes, a PreS2 expression plasmid, pcS2, was constructed and stably transfected into HepG2 cells. We conducted growth curve and colony-forming assays to study the impact of PreS2 expression on cell proliferation. Cells transfected with PreS2 proliferated more rapidly and formed colonies in soft agar. PreS2 expressing cells also induced upregulation of human telomerase reverse transcriptase (hTERT) and telomerase activation by RT-PCR and the modified TRAP assay. Blocking expression of hTERT with antisense oligonuleotide reversed the growth rate in cells stably transfected with PreS2. Our data suggest that PreS2 may increase the malignant transformation of human HCC cell line HepG2 by upregulating hTERT and inducing telomerase activation.

Overexpression of heterogeneous nuclear ribonucleoprotein F stimulates renal Ace-2 gene expression and prevents TGF-β1-induced kidney injury in a mouse model of diabetes.

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Lo, Chao-Sheng; Shi, Yixuan; Chang, Shiao-Ying; Abdo, Shaaban; Chenier, Isabelle; Filep, Janos G; Ingelfinger, Julie R; Zhang, Shao-Ling; Chan, John S D

2015-10-01

We investigated whether heterogeneous nuclear ribonucleoprotein F (hnRNP F) stimulates renal ACE-2 expression and prevents TGF-β1 signalling, TGF-β1 inhibition of Ace-2 gene expression and induction of tubulo-fibrosis in an Akita mouse model of type 1 diabetes. Adult male Akita transgenic (Tg) mice overexpressing specifically hnRNP F in their renal proximal tubular cells (RPTCs) were studied. Non-Akita littermates and Akita mice served as controls. Immortalised rat RPTCs stably transfected with plasmid containing either rat Hnrnpf cDNA or rat Ace-2 gene promoter were also studied. Overexpression of hnRNP F attenuated systemic hypertension, glomerular filtration rate, albumin/creatinine ratio, urinary angiotensinogen (AGT) and angiotensin (Ang) II levels, renal fibrosis and profibrotic gene (Agt, Tgf-β1, TGF-β receptor II [Tgf-βrII]) expression, stimulated anti-profibrotic gene (Ace-2 and Ang 1-7 receptor [MasR]) expression, and normalised urinary Ang 1-7 level in Akita Hnrnpf-Tg mice as compared with Akita mice. In vitro, hnRNP F overexpression stimulated Ace-2 gene promoter activity, mRNA and protein expression, and attenuated Agt, Tgf-β1 and Tgf-βrII gene expression. Furthermore, hnRNP F overexpression prevented TGF-β1 signalling and TGF-β1 inhibition of Ace-2 gene expression. These data demonstrate that hnRNP F stimulates Ace-2 gene transcription, prevents TGF-β1 inhibition of Ace-2 gene transcription and induction of kidney injury in diabetes. HnRNP F may be a potential target for treating hypertension and renal fibrosis in diabetes.

c-MYC expression sensitizes medulloblastoma cells to radio- and chemotherapy and has no impact on response in medulloblastoma patients

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Stearns Duncan

2011-02-01

Full Text Available Abstract Background To study whether and how c-MYC expression determines response to radio- and chemotherapy in childhood medulloblastoma (MB. Methods We used DAOY and UW228 human MB cells engineered to stably express different levels of c-MYC, and tested whether c-MYC expression has an effect on radio- and chemosensitivity using the colorimetric 3-(4,5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl-2H-tetrazolium inner salt (MTS assay, clonogenic survival, apoptosis assays, cell cycle analysis, and western blot assessment. In an effort to validate our results, we analyzed c-MYC mRNA expression in formalin-fixed paraffin-embedded tumor samples from well-documented patients with postoperative residual tumor and compared c-MYC mRNA expression with response to radio- and chemotherapy as examined by neuroradiological imaging. Results In DAOY - and to a lesser extent in UW228 - cells expressing high levels of c-MYC, the cytotoxicity of cisplatin, and etoposide was significantly higher when compared with DAOY/UW228 cells expressing low levels of c-MYC. Irradiation- and chemotherapy-induced apoptotic cell death was enhanced in DAOY cells expressing high levels of c-MYC. The response of 62 of 66 residual tumors was evaluable and response to postoperative radio- (14 responders (CR, PR vs. 5 non-responders (SD, PD or chemotherapy (23 CR/PR vs. 20 SD/PD was assessed. c-MYC mRNA expression was similar in primary MB samples of responders and non-responders (Mann-Whitney U test, p = 0.50, ratio 0.49, 95% CI 0.008-30.0 and p = 0.67, ratio 1.8, 95% CI 0.14-23.5, respectively. Conclusions c-MYC sensitizes MB cells to some anti-cancer treatments in vitro. As we failed to show evidence for such an effect on postoperative residual tumors when analyzed by imaging, additional investigations in xenografts and larger MB cohorts may help to define the exact function of c-MYC in modulating response to treatment.

In search of suitable reference genes for gene expression studies of human renal cell carcinoma by real-time PCR

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Kristiansen Glen

2007-06-01

Full Text Available Abstract Background Housekeeping genes are commonly used as endogenous reference genes for the relative quantification of target genes in gene expression studies. No conclusive systematic study comparing the suitability of different candidate reference genes in clear cell renal cell carcinoma has been published to date. To remedy this situation, 10 housekeeping genes for normalizing purposes of RT-PCR measurements already recommended in various studies were examined with regard to their usefulness as reference genes. Results The expression of the potential reference genes was examined in matched malignant and non-malignant tissue specimens from 25 patients with clear cell renal cell carcinoma. Quality assessment of isolated RNA performed with a 2100 Agilent Bioanalyzer showed a mean RNA integrity number of 8.7 for all samples. The between-run variations related to the crossing points of PCR reactions of a control material ranged from 0.17% to 0.38%. The expression of all genes did not depend on age, sex, and tumour stage. Except the genes TATA box binding protein (TBP and peptidylprolyl isomerase A (PPIA, all genes showed significant differences in expression between malignant and non-malignant pairs. The expression stability of the candidate reference genes was additionally controlled using the software programs geNorm and NormFinder. TBP and PPIA were validated as suitable reference genes by normalizing the target gene ADAM9 using these two most stably expressed genes in comparison with up- and down-regulated housekeeping genes of the panel. Conclusion Our study demonstrated the suitability of the two housekeeping genes PPIA and TBP as endogenous reference genes when comparing malignant tissue samples with adjacent normal tissue samples from clear cell renal cell carcinoma. Both genes are recommended as reference genes for relative gene quantification in gene profiling studies either as single gene or preferably in combination.

Aspirin Has Antitumor Effects via Expression of Calpain Gene in Cervical Cancer Cells

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Sang Koo Lee

2008-01-01

Full Text Available Aspirin and other nonsteroidal anti-inflammatory drugs show efficacy in the prevention of cancers. It is known that they can inhibit cyclooxygenases, and some studies have shown that they can induce apoptosis. Our objective in this study was to investigate the mechanism by which aspirin exerts its apoptosis effects in human cervical cancer HeLa cells. The effect of aspirin on the gene expression was studied by differential mRNA display RT-PCR. Among the isolated genes, mu-type calpain gene was upregulated by aspirin treatment. To examine whether calpain mediates the antitumor effects, HeLa cells were stably transfected with the mammalian expression vector pCR3.1 containing mu-type calpain cDNA (pCRCAL/HeLa, and tumor formations were measured in nude mice. When tumor burden was measured by day 49, HeLa cells and pCR/HeLa cells (vector control produced tumors of 2126 mm3 and 1638 mm3, respectively, while pCRCAL/HeLa cells produced markedly smaller tumor of 434 mm3 in volume. The caspase-3 activity was markedly elevated in pCRCAL/HeLa cells. The increased activity levels of caspase-3 in pCRCAL/HeLa cells, in parallel with the decreased tumor formation, suggest a correlation between caspase-3 activity and calpain protein. Therefore, we conclude that aspirin-induced calpain mediates an antitumor effect via caspase-3 in cervical cancer cells.

UMG Lenti: novel lentiviral vectors for efficient transgene- and reporter gene expression in human early hematopoietic progenitors.

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Emanuela Chiarella

Full Text Available Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and -LV6 where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG-LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in

UMG Lenti: novel lentiviral vectors for efficient transgene- and reporter gene expression in human early hematopoietic progenitors.

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Chiarella, Emanuela; Carrà, Giovanna; Scicchitano, Stefania; Codispoti, Bruna; Mega, Tiziana; Lupia, Michela; Pelaggi, Daniela; Marafioti, Maria G; Aloisio, Annamaria; Giordano, Marco; Nappo, Giovanna; Spoleti, Cristina B; Grillone, Teresa; Giovannone, Emilia D; Spina, Raffaella; Bernaudo, Francesca; Moore, Malcolm A S; Bond, Heather M; Mesuraca, Maria; Morrone, Giovanni

2014-01-01

Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein) is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and -LV6) where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG-LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in hematopoietic stem and

CCR5 Gene Disruption via Lentiviral Vectors Expressing Cas9 and Single Guided RNA Renders Cells Resistant to HIV-1 Infection

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Liu, Jingjing; Zhang, Di; Kimata, Jason T.; Zhou, Paul

2014-01-01

CCR5, a coreceptor for HIV-1 entry, is a major target for drug and genetic intervention against HIV-1. Genetic intervention strategies have knocked down CCR5 expression levels by shRNA or disrupted the CCR5 gene using zinc finger nucleases (ZFN) or Transcription activator-like effector nuclease (TALEN). In the present study, we silenced CCR5 via CRISPR associated protein 9 (Cas9) and single guided RNAs (sgRNAs). We constructed lentiviral vectors expressing Cas9 and CCR5 sgRNAs. We show that a single round transduction of lentiviral vectors expressing Cas9 and CCR5 sgRNAs into HIV-1 susceptible human CD4+ cells yields high frequencies of CCR5 gene disruption. CCR5 gene-disrupted cells are not only resistant to R5-tropic HIV-1, including transmitted/founder (T/F) HIV-1 isolates, but also have selective advantage over CCR5 gene-undisrupted cells during R5-tropic HIV-1 infection. Importantly, using T7 endonuclease I assay we did not detect genome mutations at potential off-target sites that are highly homologous to these CCR5 sgRNAs in stably transduced cells even at 84 days post transduction. Thus we conclude that silencing of CCR5 via Cas9 and CCR5-specific sgRNAs could be a viable alternative strategy for engineering resistance against HIV-1. PMID:25541967

Expression and fast preparation of biologically active recombinant human coagulation factor VII in CHO-K1 cells.

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Xiao, W; Li, C Q; Xiao, X P; Lin, F Z

2013-12-16

Human coagulation factor VII (FVII) plays an important role in the blood coagulation process and exists in micro amounts in human plasma; therefore, any attempt at the large-scale production of FVII in significant quantities is challenging. The purpose of this study was to express and obtain biologically active recombinant FVII (rFVII) from Chinese hamster ovary K1 (CHO-K1) cells. The full-length FVII cDNA was isolated from a HepG2 cell line and then subcloned in pcDNA3.1 to construct an expression vector, pcDNA-FVII. CHO-K1 cells were transfected with 1 µg pcDNA-FVII. The cell line that stably expressed secretory FVII was screened using 900 µg/mL G418. The FVII copy number in CHO-K1 cells was detected by quantitative polymerase chain reaction (qPCR). The rFVII was purified in ligand affinity chromatography medium. The purified protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The biological activity of the purified FVII protein was determined by a prothrombin time assay. Three cell lines that permanently expressed rFVII were screened. The qPCR results demonstrated that each CHO-K1 cell harbored two FVII DNA copies. The SDS-PAGE and Western blot analysis showed that the purified protein was about 50 kDa. The purity of the target protein was 95%. The prothrombin time assay indicated that the FVII-specific activity of rFVII was 2573 ± 75 IU/mg. This method enabled the fast preparation of high-purity rFVII from CHO-K1 cells, and the purified protein had good biological activity.

Behavior of a cloned murine interferon alpha/beta receptor expressed in homospecific or heterospecific background.

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Uzé, G; Lutfalla, G; Bandu, M T; Proudhon, D; Mogensen, K E

1992-05-15

A murine interferon (IFN) alpha/beta receptor was cloned from the IFN-sensitive L1210 cell line on the basis of its homology with the human receptor. A combination of methods that includes the screening of random-primed and oligo(dT)-primed cDNA libraries and polymerase chain reactions with a single-side specificity was used. At the amino acid level, the murine IFN-alpha/beta shows 46% identity with its human counterpart. Both human WISH cells presenting a low sensitivity to mouse IFN and a murine L1210 mutant subline that does not express the receptor have been stably transfected with the murine IFN-alpha/beta receptor. Whereas transfected human cells became sensitive to a limited number of mouse IFN-alpha/beta subtypes, the transfected murine L1210 mutant was found to be fully complemented and became sensitive to all mouse IFN-alpha/beta subtypes tested, including those that were not active on transfected human cells. These results strongly suggest that the receptor described here is implicated in the mediation of the activities of all murine IFN-alpha/beta subtypes.

Identification of reference genes for quantitative expression analysis of microRNAs and mRNAs in barley under various stress conditions.

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Jannatul Ferdous

Full Text Available For accurate and reliable gene expression analysis using quantitative real-time reverse transcription PCR (qPCR, the selection of appropriate reference genes as an internal control for normalization is crucial. We hypothesized that non-coding, small nucleolar RNAs (snoRNAswould be stably expressed in different barley varieties and under different experimental treatments,in different tissues and at different developmental stages of plant growth and therefore might prove to be suitable reference genes for expression analysis of both microRNAs (miRNAsand mRNAs. In this study, we examined the expression stability of ten candidate reference genes in six barley genotypes under five experimental stresses, drought, fungal infection,boron toxicity, nutrient deficiency and salinity. We compared four commonly used housekeeping genes; Actin (ACT, alpha-Tubulin (α-TUB, Glycolytic glyceraldehyde-3-phosphate dehydrogenase(GAPDH, ADP-ribosylation factor 1-like protein (ADP, four snoRNAs; (U18,U61, snoR14 and snoR23 and two microRNAs (miR168, miR159 as candidate reference genes. We found that ADP, snoR14 and snoR23 were ranked as the best of these candidates across diverse samples. Additionally, we found that miR168 was a suitable reference gene for expression analysis in barley. Finally, we validated the performance of our stable and unstable candidate reference genes for both mRNA and miRNA qPCR data normalization under different stress conditions and demonstrated the superiority of the stable candidates. Our data demonstrate the suitability of barley snoRNAs and miRNAs as potential reference genes form iRNA and mRNA qPCR data normalization under different stress treatments [corrected].

Differentially expressed androgen-regulated genes in androgen-sensitive tissues reveal potential biomarkers of early prostate cancer.

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Dogus Murat Altintas

Full Text Available BACKGROUND: Several data favor androgen receptor implication in prostate cancer initiation through the induction of several gene activation programs. The aim of the study is to identify potential biomarkers for early diagnosis of prostate cancer (PCa among androgen-regulated genes (ARG and to evaluate comparative expression of these genes in normal prostate and normal prostate-related androgen-sensitive tissues that do not (or rarely give rise to cancer. METHODS: ARG were selected in non-neoplastic adult human prostatic epithelial RWPE-1 cells stably expressing an exogenous human androgen receptor, using RNA-microarrays and validation by qRT-PCR. Expression of 48 preselected genes was quantified in tissue samples (seminal vesicles, prostate transitional zones and prostate cancers, benign prostatic hypertrophy obtained from surgical specimens using TaqMan® low-density arrays. The diagnostic performances of these potential biomarkers were compared to that of genes known to be associated with PCa (i.e. PCA3 and DLX1. RESULTS AND DISCUSSION: By crossing expression studies in 26 matched PCa and normal prostate transitional zone samples, and 35 matched seminal vesicle and PCa samples, 14 genes were identified. Similarly, 9 genes were overexpressed in 15 benign prostatic hypertrophy samples, as compared to PCa samples. Overall, we selected 8 genes of interest to evaluate their diagnostic performances in comparison with that of PCA3 and DLX1. Among them, 3 genes: CRYAB, KCNMA1 and SDPR, were overexpressed in all 3 reference non-cancerous tissues. The areas under ROC curves of these genes reached those of PCA3 (0.91 and DLX1 (0.94. CONCLUSIONS: We identified ARG with reduced expression in PCa and with significant diagnostic values for discriminating between cancerous and non-cancerous prostatic tissues, similar that of PCA3. Given their expression pattern, they could be considered as potentially protective against prostate cancer. Moreover, they could

Molecular Basis of Cardiac Delayed Rectifier Potassium Channel Function and Pharmacology.

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Wu, Wei; Sanguinetti, Michael C

2016-06-01

Human cardiomyocytes express 3 distinct types of delayed rectifier potassium channels. Human ether-a-go-go-related gene (hERG) channels conduct the rapidly activating current IKr; KCNQ1/KCNE1 channels conduct the slowly activating current IKs; and Kv1.5 channels conduct an ultrarapid activating current IKur. Here the authors provide a general overview of the mechanistic and structural basis of ion selectivity, gating, and pharmacology of the 3 types of cardiac delayed rectifier potassium ion channels. Most blockers bind to S6 residues that line the central cavity of the channel, whereas activators interact with the channel at 4 symmetric binding sites outside the cavity. Copyright © 2016 Elsevier Inc. All rights reserved.

Different protein kinase C isoenzymes mediate inhibition of cardiac rapidly activating delayed rectifier K+ current by different G-protein coupled receptors.

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Liu, Xueli; Wang, Yuhong; Zhang, Hua; Shen, Li; Xu, Yanfang

2017-12-01

Elevated angiotensin II (Ang II) and sympathetic activity contributes to a high risk of ventricular arrhythmias in heart disease. The rapidly activating delayed rectifier K + current (I Kr ) carried by the hERG channels plays a critical role in cardiac repolarization, and decreased I Kr is involved in increased cardiac arrhythmogenicity. Stimulation of α 1A -adrenoreceptors or angiotensin II AT 1 receptors is known to inhibit I Kr via PKC. Here, we have identified the PKC isoenzymes mediating the inhibition of I Kr by activation of these two different GPCRs. The whole-cell patch-clamp technique was used to record I Kr in guinea pig cardiomyocytes and HEK293 cells co-transfected with hERG and α 1A -adrenoreceptor or AT 1 receptor genes. A broad spectrum PKC inhibitor Gö6983 (not inhibiting PKCε), a selective cPKC inhibitor Gö6976 and a PKCα-specific inhibitor peptide, blocked the inhibition of I Kr by the α 1A -adrenoreceptor agonist A61603. However, these inhibitors did not affect the reduction of I Kr by activation of AT 1 receptors, whereas the PKCε-selective inhibitor peptide did block the effect. The effects of angiotensin II and the PKCε activator peptide were inhibited in mutant hERG channels in which 17 of the 18 PKC phosphorylation sites were deleted, whereas a deletion of the N-terminus of the hERG channels selectively prevented the inhibition elicited by A61603 and the cPKC activator peptide. Our results indicated that inhibition of I Kr by activation of α 1A -adrenoreceptors or AT 1 receptors were mediated by PKCα and PKCε isoforms respectively, through different molecular mechanisms. © 2017 The British Pharmacological Society.

Evolving regulatory paradigm for proarrhythmic risk assessment for new drugs.

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Vicente, Jose; Stockbridge, Norman; Strauss, David G

Fourteen drugs were removed from the market worldwide because their potential to cause torsade de pointes (torsade), a potentially fatal ventricular arrhythmia. The observation that most drugs that cause torsade block the potassium channel encoded by the human ether-à-go-go related gene (hERG) and prolong the heart rate corrected QT interval (QTc) on the ECG, led to a focus on screening new drugs for their potential to block the hERG potassium channel and prolong QTc. This has been a successful strategy keeping torsadogenic drugs off the market, but has resulted in drugs being dropped from development, sometimes inappropriately. This is because not all drugs that block the hERG potassium channel and prolong QTc cause torsade, sometimes because they block other channels. The regulatory paradigm is evolving to improve proarrhythmic risk prediction. ECG studies can now use exposure-response modeling for assessing the effect of a drug on the QTc in small sample size first-in-human studies. Furthermore, the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative is developing and validating a new in vitro paradigm for cardiac safety evaluation of new drugs that provides a more accurate and comprehensive mechanistic-based assessment of proarrhythmic potential. Under CiPA, the prediction of proarrhythmic potential will come from in vitro ion channel assessments coupled with an in silico model of the human ventricular myocyte. The preclinical assessment will be checked with an assessment of human phase 1 ECG data to determine if there are unexpected ion channel effects in humans compared to preclinical ion channel data. While there is ongoing validation work, the heart rate corrected J-T peak interval is likely to be assessed under CiPA to detect inward current block in presence of hERG potassium channel block. Copyright © 2016 Elsevier Inc. All rights reserved.

Expression of the Native Cholera Toxin B Subunit Gene and Assembly as Functional Oligomers in Transgenic Tobacco Chloroplasts

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Daniell, Henry; Lee, Seung-Bum; Panchal, Tanvi; Wiebe, Peter O.

2012-01-01

The B subunits of enterotoxigenic Escherichia coli (LTB) and cholera toxin of Vibrio cholerae (CTB) are candidate vaccine antigens. Integration of an unmodified CTB-coding sequence into chloroplast genomes (up to 10,000 copies per cell), resulted in the accumulation of up to 4.1% of total soluble tobacco leaf protein as functional oligomers (410-fold higher expression levels than that of the unmodified LTB gene expressed via the nuclear genome). However, expresssion levels reported are an underestimation of actual accumulation of CTB in transgenic chloroplasts, due to aggregation of the oligomeric forms in unboiled samples similar to the aggregation observed for purified bacterial antigen. PCR and Southern blot analyses confirmed stable integration of the CTB gene into the chloroplast genome. Western blot analysis showed that the chloroplast-synthesized CTB assembled into oligomers and were antigenically identical with purified native CTB. Also, binding assays confirmed that chloroplast- synthesized CTB binds to the intestinal membrane GM1-ganglioside receptor, indicating correct folding and disulfide bond formation of CTB pentamers within transgenic chloroplasts. In contrast to stunted nuclear transgenic plants, chloroplast transgenic plants were morphologically indistinguishable from untransformed plants, when CTB was constitutively expressed in chloroplasts. Introduced genes were inherited stably in subsequent generations, as confirmed by PCR and Southern blot analyses. Increased production of an efficient transmucosal carrier molecule and delivery system, like CTB, in transgenic chloroplasts makes plant-based oral vaccines and fusion proteins with CTB needing oral administration commercially feasible. Successful expression of foreign genes in transgenic chromoplasts and availability of marker-free chloroplast transformation techniques augurs well for development of vaccines in edible parts of transgenic plants. Furthermore, since the quaternary structure of

Evaluation of candidate reference genes for gene expression normalization in Brassica juncea using real time quantitative RT-PCR.

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Ruby Chandna

Full Text Available The real time quantitative reverse transcription PCR (qRT-PCR is becoming increasingly important to gain insight into function of genes. Given the increased sensitivity, ease and reproducibility of qRT-PCR, the requirement of suitable reference genes for normalization has become important and stringent. It is now known that the expression of internal control genes in living organism vary considerably during developmental stages and under different experimental conditions. For economically important Brassica crops, only a couple of reference genes are reported till date. In this study, expression stability of 12 candidate reference genes including ACT2, ELFA, GAPDH, TUA, UBQ9 (traditional housekeeping genes, ACP, CAC, SNF, TIPS-41, TMD, TSB and ZNF (new candidate reference genes, in a diverse set of 49 tissue samples representing different developmental stages, stress and hormone treated conditions and cultivars of Brassica juncea has been validated. For the normalization of vegetative stages the ELFA, ACT2, CAC and TIPS-41 combination would be appropriate whereas TIPS-41 along with CAC would be suitable for normalization of reproductive stages. A combination of GAPDH, TUA, TIPS-41 and CAC were identified as the most suitable reference genes for total developmental stages. In various stress and hormone treated samples, UBQ9 and TIPS-41 had the most stable expression. Across five cultivars of B. juncea, the expression of CAC and TIPS-41 did not vary significantly and were identified as the most stably expressed reference genes. This study provides comprehensive information that the new reference genes selected herein performed better than the traditional housekeeping genes. The selection of most suitable reference genes depends on the experimental conditions, and is tissue and cultivar-specific. Further, to attain accuracy in the results more than one reference genes are necessary for normalization.

Catalase overexpression prevents nuclear factor erythroid 2-related factor 2 stimulation of renal angiotensinogen gene expression, hypertension, and kidney injury in diabetic mice.

Science.gov (United States)

Abdo, Shaaban; Shi, Yixuan; Otoukesh, Abouzar; Ghosh, Anindya; Lo, Chao-Sheng; Chenier, Isabelle; Filep, Janos G; Ingelfinger, Julie R; Zhang, Shao Ling; Chan, John S D

2014-10-01

This study investigated the impact of catalase (Cat) overexpression in renal proximal tubule cells (RPTCs) on nuclear factor erythroid 2-related factor 2 (Nrf2) stimulation of angiotensinogen (Agt) gene expression and the development of hypertension and renal injury in diabetic Akita transgenic mice. Additionally, adult male mice were treated with the Nrf2 activator oltipraz with or without the inhibitor trigonelline. Rat RPTCs, stably transfected with plasmid containing either rat Agt or Nrf2 gene promoter, were also studied. Cat overexpression normalized systolic BP, attenuated renal injury, and inhibited RPTC Nrf2, Agt, and heme oxygenase-1 (HO-1) gene expression in Akita Cat transgenic mice compared with Akita mice. In vitro, high glucose level, hydrogen peroxide, and oltipraz stimulated Nrf2 and Agt gene expression; these changes were blocked by trigonelline, small interfering RNAs of Nrf2, antioxidants, or pharmacological inhibitors of nuclear factor-κB and p38 mitogen-activated protein kinase. The deletion of Nrf2-responsive elements in the rat Agt gene promoter abolished the stimulatory effect of oltipraz. Oltipraz administration also augmented Agt, HO-1, and Nrf2 gene expression in mouse RPTCs and was reversed by trigonelline. These data identify a novel mechanism, Nrf2-mediated stimulation of intrarenal Agt gene expression and activation of the renin-angiotensin system, by which hyperglycemia induces hypertension and renal injury in diabetic mice. © 2014 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

T cell receptor zeta allows stable expression of receptors containing the CD3gamma leucine-based receptor-sorting motif

DEFF Research Database (Denmark)

Dietrich, J; Geisler, C

1998-01-01

The leucine-based motif in the T cell receptor (TCR) subunit CD3gamma constitutes a strong internalization signal. In fully assembled TCR this motif is inactive unless phosphorylated. In contrast, the motif is constitutively active in CD4/CD3gamma and Tac/CD3gamma chimeras independently of phosph......The leucine-based motif in the T cell receptor (TCR) subunit CD3gamma constitutes a strong internalization signal. In fully assembled TCR this motif is inactive unless phosphorylated. In contrast, the motif is constitutively active in CD4/CD3gamma and Tac/CD3gamma chimeras independently...... of phosphorylation and leads to rapid internalization and sorting of these chimeras to lysosomal degradation. Because the TCRzeta chain rescues incomplete TCR complexes from lysosomal degradation and allows stable surface expression of fully assembled TCR, we addressed the question whether TCRzeta has the potential...... to mask the CD3gamma leucine-based motif. By studying CD4/CD3gamma and CD16/CD3gamma chimeras, we found that CD16/CD3gamma chimeras associated with TCRzeta. The CD16/CD3gamma-TCRzeta complexes were stably expressed at the cell surface and had a low spontaneous internalization rate, indicating...

Identification of the first nonsense CDSN mutation with expression of a truncated protein causing peeling skin syndrome type B.

Science.gov (United States)

Mallet, A; Kypriotou, M; George, K; Leclerc, E; Rivero, D; Mazereeuw-Hautier, J; Serre, G; Huber, M; Jonca, N; Hohl, D

2013-12-01

Peeling skin disease (PSD), a generalized inflammatory form of peeling skin syndrome, is caused by autosomal recessive nonsense mutations in the corneodesmosin gene (CDSN). To investigate a novel mutation in CDSN. A 50-year-old white woman showed widespread peeling with erythema and elevated serum IgE. DNA sequencing, immunohistochemistry, Western blot and real-time polymerase chain reaction analyses of skin biopsies were performed in order to study the genetics and to characterize the molecular profile of the disease. Histology showed hyperkeratosis and acanthosis of the epidermis, and inflammatory infiltrates in the dermis. DNA sequencing revealed a homozygous mutation leading to a premature termination codon in CDSN: p.Gly142*. Protein analyses showed reduced expression of a 16-kDa corneodesmosin mutant in the upper epidermal layers, whereas the full-length protein was absent. These results are interesting regarding the genotype-phenotype correlations in diseases caused by CDSN mutations. The PSD-causing CDSN mutations identified heretofore result in total corneodesmosin loss, suggesting that PSD is due to full corneodesmosin deficiency. Here, we show for the first time that a mutant corneodesmosin can be stably expressed in some patients with PSD, and that this truncated protein is very probably nonfunctional. © 2013 British Association of Dermatologists.

Development of a cell-based reporter assay for screening of inhibitors of hypoxia-inducible factor 2-induced gene expression.

Science.gov (United States)

Woldemichael, Girma M; Vasselli, James R; Gardella, Roberta S; McKee, Tawnya C; Linehan, W Marston; McMahon, James B

2006-09-01

Reporter cell lines have been developed for the identification of inhibitors of gene expression enhanced by hypoxia-inducible factor 2, which has been implicated as a transcription factor involved in the tumorigenesis of clear cell renal carcinoma. Stably transformed reporter clones of the human renal clear cell carcinoma cell line 786-O were generated by transfection or retroviral infection. Luciferase reporter expression in the vectors used was driven by either the natural human vascular endothelial growth factor (VEGF) promoter-enhancer or by the VEGF and the human endothelial nitric oxide synthase enhancers modulating minimal human cytomegalovirus promoter. Utility of the generated reporter cell lines was validated by introducing the von Hippel-Lindau protein complex and testing for reporter inducibility by hypoxia. The dynamic range in reporter activity under hypoxic stress was found to be at least 30- to 40-fold, with a signal-to-noise ratio of 60:1. Properties of the cell lines such as tolerance to up to 3% DMSO, signal stability with multiple in vitro passages, and utility in both 96- and 384-well plate formats indicated their suitability for use in a high-throughput screen. In addition, the potential use of these reporter lines in the evaluation of high-throughput screening hits in vivo in various mice models has been demonstrated.

MicroRNA-17-5p post-transcriptionally regulates p21 expression in irradiated betel quid chewing-related oral squamous cell carcinoma cells

International Nuclear Information System (INIS)

Wu, S.Y.; HungKuang Univ., Taichung; Lin, K.C.; Chiou, J.F.; Taipei Medical Univ. Hospital, Taipei; Taipei Medical Univ. Hospital, Taipei; Taipei Medical Univ. Hospital, Taipei; Jeng, S.C.; Cheng, W.H.; Chang, C.I.; Lin, W.C.; Wu, L.L.; Lee, H.L.; Chen, R.J.

2013-01-01

Background and purpose: Betel nut chewing is associated with oral cavity cancer in Taiwan. OC3 is an oral carcinoma cell line that was established from cells collected from a long-term betel nut chewer who does not smoke. After we found that microRNA-17-5p (miR-17-5p) is induced in OC3 cells, we used this cell line to examine the biological role(s) of this microRNA in response to exposure to ionizing radiation. Materials and methods: A combined SYBR green-based real-time PCR and oligonucleotide ligation assay was used to examine the expression of the miR-17 polycistron in irradiated OC3 cells. The roles of miR-17-5p and p21 were evaluated with specific antisense oligonucleotides (ODN) that were designed and used to inhibit their expression. Expression of the p21 protein was evaluated by Western blotting. The clonogenic assay and annexin V staining were used to evaluate cell survival and apoptosis, respectively. Cells in which miR-17-5p was stably knocked down were used to create ectopic xenografts to evaluate in vivo the role of miR-17-5p. Results: A radiation dose of 5 Gy significantly increased miR-17-5p expression in irradiated OC3 cells. Inhibition of miR-17-5p expression enhanced the radiosensitivity of the OC3 cells. We found that miR-17-5p downregulates radiation-induced p21 expression in OC3 cells and, by using a tumor xenograft model, it was found that p21 plays a critical role in increasing the radiosensitivity of OC3 cells in vitro and in vivo. Conclusion: miR-17-5p is induced in irradiated OC3 cells and it downregulates p21 protein expression, contributing to the radioresistance of OC3 cells. (orig.)

MicroRNA-17-5p post-transcriptionally regulates p21 expression in irradiated betel quid chewing-related oral squamous cell carcinoma cells

Energy Technology Data Exchange (ETDEWEB)

Wu, S.Y. [Taipei Medical Univ., Wan Fang Hospital, Taipei (China). Dept. of Radiation-Oncology; HungKuang Univ., Taichung (China). Dept. of Biotechnology; Lin, K.C. [Taipei Medical Univ., Wan Fang Hospital, Taipei (China). Dept. of Oral and Maxillofacial Surgery; Chiou, J.F. [Taipei Medical Univ., Taipei (China). Dept. of Radiology; Taipei Medical Univ. Hospital, Taipei (China). Dept. of Radiation Oncology; Taipei Medical Univ. Hospital, Taipei (China). Dept. of Hospice and Palliative Center; Taipei Medical Univ. Hospital, Taipei (China). Cancer Center; Jeng, S.C. [Taipei Medical Univ. Hospital, Taipei (China). Dept. of Radiation Oncology; Cheng, W.H.; Chang, C.I. [Taipei Medical Univ., Wan Fang Hospital, Taipei (China). Dept. of Hemato-Ongology; Lin, W.C. [Taipei Medical Univ., Wan Fang Hospital, Taipei (China). Div. of Thoracic Surgery; Wu, L.L. [National Taiwan Univ. Hospital, Taipei (China). Dept. of Ophthalmology; Lee, H.L. [Taipei Medical Univ., Wan Fang Hospital, Taipei (China). Dept. of Radiation-Oncology; Chen, R.J. [National Taiwan Univ. Hospital and National Taiwan Univ., Taipei (China). Dept. of Obstetrics and Gynecology

2013-08-15

Background and purpose: Betel nut chewing is associated with oral cavity cancer in Taiwan. OC3 is an oral carcinoma cell line that was established from cells collected from a long-term betel nut chewer who does not smoke. After we found that microRNA-17-5p (miR-17-5p) is induced in OC3 cells, we used this cell line to examine the biological role(s) of this microRNA in response to exposure to ionizing radiation. Materials and methods: A combined SYBR green-based real-time PCR and oligonucleotide ligation assay was used to examine the expression of the miR-17 polycistron in irradiated OC3 cells. The roles of miR-17-5p and p21 were evaluated with specific antisense oligonucleotides (ODN) that were designed and used to inhibit their expression. Expression of the p21 protein was evaluated by Western blotting. The clonogenic assay and annexin V staining were used to evaluate cell survival and apoptosis, respectively. Cells in which miR-17-5p was stably knocked down were used to create ectopic xenografts to evaluate in vivo the role of miR-17-5p. Results: A radiation dose of 5 Gy significantly increased miR-17-5p expression in irradiated OC3 cells. Inhibition of miR-17-5p expression enhanced the radiosensitivity of the OC3 cells. We found that miR-17-5p downregulates radiation-induced p21 expression in OC3 cells and, by using a tumor xenograft model, it was found that p21 plays a critical role in increasing the radiosensitivity of OC3 cells in vitro and in vivo. Conclusion: miR-17-5p is induced in irradiated OC3 cells and it downregulates p21 protein expression, contributing to the radioresistance of OC3 cells. (orig.)

Lysophosphatidic acid activates peroxisome proliferator activated receptor-γ in CHO cells that over-express glycerol 3-phosphate acyltransferase-1.

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Cliona M Stapleton

2011-04-01

Full Text Available Lysophosphatidic acid (LPA is an agonist for peroxisome proliferator activated receptor-γ (PPARγ. Although glycerol-3-phosphate acyltransferase-1 (GPAT1 esterifies glycerol-3-phosphate to form LPA, an intermediate in the de novo synthesis of glycerolipids, it has been assumed that LPA synthesized by this route does not have a signaling role. The availability of Chinese Hamster Ovary (CHO cells that stably overexpress GPAT1, allowed us to analyze PPARγ activation in the presence of LPA produced as an intracellular intermediate. LPA levels in CHO-GPAT1 cells were 6-fold higher than in wild-type CHO cells, and the mRNA abundance of CD36, a PPARγ target, was 2-fold higher. Transactivation assays showed that PPARγ activity was higher in the cells that overexpressed GPAT1. PPARγ activity was enhanced further in CHO-GPAT1 cells treated with the PPARγ ligand troglitazone. Extracellular LPA, phosphatidic acid (PA or a membrane-permeable diacylglycerol had no effect, showing that PPARγ had been activated by LPA generated intracellularly. Transient transfection of a vector expressing 1-acylglycerol-3-phosphate acyltransferase-2, which converts endogenous LPA to PA, markedly reduced PPARγ activity, as did over-expressing diacylglycerol kinase, which converts DAG to PA, indicating that PA could be a potent inhibitor of PPARγ. These data suggest that LPA synthesized via the glycerol-3-phosphate pathway can activate PPARγ and that intermediates of de novo glycerolipid synthesis regulate gene expression.

Identification of genes for normalization of real-time RT-PCR data in breast carcinomas

DEFF Research Database (Denmark)

Lyng, Maria B; Laenkholm, Anne-Vibeke; Pallisgaard, Niels

2008-01-01

BACKGROUND: Quantitative real-time RT-PCR (RT-qPCR) has become a valuable molecular technique in basic and translational biomedical research, and is emerging as an equally valuable clinical tool. Correlation of inter-sample values requires data normalization, which can be accomplished by various...... means, the most common of which is normalization to internal, stably expressed, reference genes. Recently, such traditionally utilized reference genes as GAPDH and B2M have been found to be regulated in various circumstances in different tissues, emphasizing the need to identify genes independent...... of factors influencing the tissue, and that are stably expressed within the experimental milieu. In this study, we identified genes for normalization of RT-qPCR data for invasive breast cancer (IBC), with special emphasis on estrogen receptor positive (ER+) IBC, but also examined their applicability to ER...

Biomarker-based classification of bacterial and fungal whole-blood infections in a genome-wide expression study

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Andreas eDix

2015-03-01

Full Text Available Sepsis is a clinical syndrome that can be caused by bacteria or fungi. Early knowledge on the nature of the causative agent is a prerequisite for targeted anti-microbial therapy. Besides currently used detection methods like blood culture and PCR-based assays, the analysis of the transcriptional response of the host to infecting organisms holds great promise. In this study, we aim to examine the transcriptional footprint of infections caused by the bacterial pathogens Staphylococcus aureus and Escherichia coli and the fungal pathogens Candida albicans and Aspergillus fumigatus in a human whole-blood model. Moreover, we use the expression information to build a random forest classifier to classify if a sample contains a bacterial, fungal, or mock-infection. After normalizing the transcription intensities using stably expressed reference genes, we filtered the gene set for biomarkers of bacterial or fungal blood infections. This selection is based on differential expression and an additional gene relevance measure. In this way, we identified 38 biomarker genes, including IL6, SOCS3, and IRG1 which were already associated to sepsis by other studies. Using these genes, we trained the classifier and assessed its performance. It yielded a 96% accuracy (sensitivities >93%, specificities >97% for a 10-fold stratified cross-validation and a 92% accuracy (sensitivities and specificities >83% for an additional test dataset comprising Cryptococcus neoformans infections. Furthermore, the classifier is robust to Gaussian noise, indicating correct class predictions on datasets of new species. In conclusion, this genome-wide approach demonstrates an effective feature selection process in combination with the construction of a well-performing classification model. Further analyses of genes with pathogen-dependent expression patterns can provide insights into the systemic host responses, which may lead to new anti-microbial therapeutic advances.

Neural Crest Cells Isolated from the Bone Marrow of Transgenic Mice Express JCV T-Antigen.

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Jennifer Gordon

Full Text Available JC virus (JCV, a common human polyomavirus, is the etiological agent of the demyelinating disease, progressive multifocal leukoencephalopathy (PML. In addition to its role in PML, studies have demonstrated the transforming ability of the JCV early protein, T-antigen, and its association with some human cancers. JCV infection occurs in childhood and latent virus is thought to be maintained within the bone marrow, which harbors cells of hematopoietic and non-hematopoietic lineages. Here we show that non-hematopoietic mesenchymal stem cells (MSCs isolated from the bone marrow of JCV T-antigen transgenic mice give rise to JCV T-antigen positive cells when cultured under neural conditions. JCV T-antigen positive cells exhibited neural crest characteristics and demonstrated p75, SOX-10 and nestin positivity. When cultured in conditions typical for mesenchymal cells, a population of T-antigen negative cells, which did not express neural crest markers arose from the MSCs. JCV T-antigen positive cells could be cultured long-term while maintaining their neural crest characteristics. When these cells were induced to differentiate into neural crest derivatives, JCV T-antigen was downregulated in cells differentiating into bone and maintained in glial cells expressing GFAP and S100. We conclude that JCV T-antigen can be stably expressed within a fraction of bone marrow cells differentiating along the neural crest/glial lineage when cultured in vitro. These findings identify a cell population within the bone marrow permissible for JCV early gene expression suggesting the possibility that these cells could support persistent viral infection and thus provide clues toward understanding the role of the bone marrow in JCV latency and reactivation. Further, our data provides an excellent experimental model system for studying the cell-type specificity of JCV T-antigen expression, the role of bone marrow-derived stem cells in the pathogenesis of JCV-related diseases

APETx4, a Novel Sea Anemone Toxin and a Modulator of the Cancer-Relevant Potassium Channel KV10.1

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Lien Moreels

2017-09-01

Full Text Available The human ether-à-go-go channel (hEag1 or KV10.1 is a cancer-relevant voltage-gated potassium channel that is overexpressed in a majority of human tumors. Peptides that are able to selectively inhibit this channel can be lead compounds in the search for new anticancer drugs. Here, we report the activity-guided purification and electrophysiological characterization of a novel KV10.1 inhibitor from the sea anemone Anthopleura elegantissima. Purified sea anemone fractions were screened for inhibitory activity on KV10.1 by measuring whole-cell currents as expressed in Xenopus laevis oocytes using the two-microelectrode voltage clamp technique. Fractions that showed activity on Kv10.1 were further purified by RP-HPLC. The amino acid sequence of the peptide was determined by a combination of MALDI- LIFT-TOF/TOF MS/MS and CID-ESI-FT-ICR MS/MS and showed a high similarity with APETx1 and APETx3 and was therefore named APETx4. Subsequently, the peptide was electrophysiologically characterized on KV10.1. The selectivity of the toxin was investigated on an array of voltage-gated ion channels, including the cardiac human ether-à-go-go-related gene potassium channel (hERG or Kv11.1. The toxin inhibits KV10.1 with an IC50 value of 1.1 μM. In the presence of a similar toxin concentration, a shift of the activation curve towards more positive potentials was observed. Similar to the effect of the gating modifier toxin APETx1 on hERG, the inhibition of Kv10.1 by the isolated toxin is reduced at more positive voltages and the peptide seems to keep the channel in a closed state. Although the peptide also induces inhibitory effects on other KV and NaV channels, it exhibits no significant effect on hERG. Moreover, APETx4 induces a concentration-dependent cytotoxic and proapoptotic effect in various cancerous and noncancerous cell lines. This newly identified KV10.1 inhibitor can be used as a tool to further characterize the oncogenic channel KV10.1 or as a

Expression of the A56 and K2 proteins is sufficient to inhibit vaccinia virus entry and cell fusion.

Science.gov (United States)

Wagenaar, Timothy R; Moss, Bernard

2009-02-01

Many animal viruses induce cells to fuse and form syncytia. For vaccinia virus, this phenomenon is associated with mutations affecting the A56 and K2 proteins, which form a multimer (A56/K2) on the surface of infected cells. Recent evidence that A56/K2 interacts with the entry/fusion complex (EFC) and that the EFC is necessary for syncytium formation furnishes a strong connection between virus entry and cell fusion. Among the important remaining questions are whether A56/K2 can prevent virus entry as well as cell-cell fusion and whether these two viral proteins are sufficient as well as necessary for this. To answer these questions, we transiently and stably expressed A56 and K2 in uninfected cells. Uninfected cells expressing A56 and K2 exhibited resistance to fusing with A56 mutant virus-infected cells, whereas expression of A56 or K2 alone induced little or no resistance, which fits with the need for both proteins to bind the EFC. Furthermore, transient or stable expression of A56/K2 interfered with virus entry and replication as determined by inhibition of early expression of a luciferase reporter gene, virus production, and plaque formation. The specificity of this effect was demonstrated by restoring entry after enzymatically removing a chimeric glycophosphatidylinositol-anchored A56/K2 or by binding a monoclonal antibody to A56. Importantly, the antibody disrupted the interaction between A56/K2 and the EFC without disrupting the A56-K2 interaction itself. Thus, we have shown that A56/K2 is sufficient to prevent virus entry and fusion as well as formation of syncytia through interaction with the EFC.

Reference genes for normalization: A study of rat brain tissue

DEFF Research Database (Denmark)

Bonefeld, Birgit; Elfving, Betina; Wegener, Gregers

2008-01-01

are warranted. With the overall aim to inspect the gene expression of three target genes, NMDAR1, SORT, and CREB, in rat hippocampus, we tested a panel of eight HKGs, 18s rRNA, ActB, CycA, Gapd, Hmbs, Hprt1, Rpl13A, and Ywhaz in order to select the most stably expressed gene, using the NormFinder and ge...

Evaluation of reference gene suitability for quantitative expression analysis by quantitative polymerase chain reaction in the mandibular condyle of sheep.

Science.gov (United States)

Jiang, Xin; Xue, Yang; Zhou, Hongzhi; Li, Shouhong; Zhang, Zongmin; Hou, Rui; Ding, Yuxiang; Hu, Kaijin

2015-10-01

Reference genes are commonly used as a reliable approach to normalize the results of quantitative polymerase chain reaction (qPCR), and to reduce errors in the relative quantification of gene expression. Suitable reference genes belonging to numerous functional classes have been identified for various types of species and tissue. However, little is currently known regarding the most suitable reference genes for bone, specifically for the sheep mandibular condyle. Sheep are important for the study of human bone diseases, particularly for temporomandibular diseases. The present study aimed to identify a set of reference genes suitable for the normalization of qPCR data from the mandibular condyle of sheep. A total of 12 reference genes belonging to various functional classes were selected, and the expression stability of the reference genes was determined in both the normal and fractured area of the sheep mandibular condyle. RefFinder, which integrates the following currently available computational algorithms: geNorm, NormFinder, BestKeeper, and the comparative ΔCt method, was used to compare and rank the candidate reference genes. The results obtained from the four methods demonstrated a similar trend: RPL19, ACTB, and PGK1 were the most stably expressed reference genes in the sheep mandibular condyle. As determined by RefFinder comprehensive analysis, the results of the present study suggested that RPL19 is the most suitable reference gene for studies associated with the sheep mandibular condyle. In addition, ACTB and PGK1 may be considered suitable alternatives.

Identification of normalization factors for quantitative real-time RT-PCR analysis of gene expression in Pacific abalone Haliotis discus hannai

Science.gov (United States)

Qiu, Reng; Sun, Boguang; Fang, Shasha; Sun, Li; Liu, Xiao

2013-03-01

Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is widely used in studies of gene expression. In most of these studies, housekeeping genes are used as internal references without validation. To identify appropriate reference genes for qRT-PCR in Pacific abalone Haliotis discus hannai, we examined the transcription stability of six housekeeping genes in abalone tissues in the presence and absence of bacterial infection. For this purpose, abalone were infected with the bacterial pathogen Vibrio anguillarum for 12 h and 48 h. The mRNA levels of the housekeeping genes in five tissues (digestive glands, foot muscle, gill, hemocyte, and mantle) were determined by qRT-PCR. The PCR data was subsequently analyzed with the geNorm and NormFinder algorithms. The results show that in the absence of bacterial infection, elongation factor-1-alpha and beta-actin were the most stably expressed genes in all tissues, and thus are suitable as cross-tissue type normalization factors. However, we did not identify any universal reference genes post infection because the most stable genes varied between tissue types. Furthermore, for most tissues, the optimal reference genes identified by both algorithms at 12 h and 48 h post-infection differed. These results indicate that bacterial infection induced significant changes in the expression of abalone housekeeping genes in a manner that is dependent on tissue type and duration of infection. As a result, different normalization factors must be used for different tissues at different infection points.

Antiproliferative activity of recombinant human interferon-λ2 ...

African Journals Online (AJOL)

Antiproliferative activity of recombinant human interferon-λ2 expressed in stably ... The representing 26 kDa protein band of IFN-λ2 was detected by SDS-PAGE and ... The antiproliferative activity of hIFN-λ2 was determined by MTT assay.

Seminal Plasma Proteins as Androgen Receptor Corregulators Promote Prostate Cancer Growth

Science.gov (United States)

2016-12-01

protease activity of PSA [14]. Semenogelins are expressed in other male genital organs, such as the vas def - erens, epididymis, and prostate, as well...expression and secretion in prostate cancer lines stably expressing SgI. Cell extracts (A) or acetone-precipitated proteins in con - ditioned media... con - firmed this by demonstrating the failure to detect SgI signals in the conditioned medium after culturing control LNCaP with endogenous SgI and

Identification, Expression Analysis, and Target Prediction of Flax Genotroph MicroRNAs Under Normal and Nutrient Stress Conditions

Science.gov (United States)

Melnikova, Nataliya V.; Dmitriev, Alexey A.; Belenikin, Maxim S.; Koroban, Nadezhda V.; Speranskaya, Anna S.; Krinitsina, Anastasia A.; Krasnov, George S.; Lakunina, Valentina A.; Snezhkina, Anastasiya V.; Sadritdinova, Asiya F.; Kishlyan, Natalya V.; Rozhmina, Tatiana A.; Klimina, Kseniya M.; Amosova, Alexandra V.; Zelenin, Alexander V.; Muravenko, Olga V.; Bolsheva, Nadezhda L.; Kudryavtseva, Anna V.

2016-01-01

Cultivated flax (Linum usitatissimum L.) is an important plant valuable for industry. Some flax lines can undergo heritable phenotypic and genotypic changes (LIS-1 insertion being the most common) in response to nutrient stress and are called plastic lines. Offspring of plastic lines, which stably inherit the changes, are called genotrophs. MicroRNAs (miRNAs) are involved in a crucial regulatory mechanism of gene expression. They have previously been assumed to take part in nutrient stress response and can, therefore, participate in genotroph formation. In the present study, we performed high-throughput sequencing of small RNAs (sRNAs) extracted from flax plants grown under normal, phosphate deficient and nutrient excess conditions to identify miRNAs and evaluate their expression. Our analysis revealed expression of 96 conserved miRNAs from 21 families in flax. Moreover, 475 novel potential miRNAs were identified for the first time, and their targets were predicted. However, none of the identified miRNAs were transcribed from LIS-1. Expression of seven miRNAs (miR168, miR169, miR395, miR398, miR399, miR408, and lus-miR-N1) with up- or down-regulation under nutrient stress (on the basis of high-throughput sequencing data) was evaluated on extended sampling using qPCR. Reference gene search identified ETIF3H and ETIF3E genes as most suitable for this purpose. Down-regulation of novel potential lus-miR-N1 and up-regulation of conserved miR399 were revealed under the phosphate deficient conditions. In addition, the negative correlation of expression of lus-miR-N1 and its predicted target, ubiquitin-activating enzyme E1 gene, as well as, miR399 and its predicted target, ubiquitin-conjugating enzyme E2 gene, was observed. Thus, in our study, miRNAs expressed in flax plastic lines and genotrophs were identified and their expression and expression of their targets was evaluated using high-throughput sequencing and qPCR for the first time. These data provide new insights

Comparison of human sodium iodide symporter (hNIS) gene expression between lentiviral and adenoviral vectors in rat mesenchymal stem cell

International Nuclear Information System (INIS)

Park, So Yeon; Lee, Won Woo; Kim, Sung Jin; Lee, Heui Ran; Kim, Hyun Joo; Chung, June Key; Kim, Sang Eun

2007-01-01

Quantitative comparison of transgene expression within stem cells between lentivirus and adenovirus-mediated delivery systems has not been done. Here, we evaluated the human sodium iodide symporter (hNIS) gene expression in rat mesenchymal stem cell (rMSC) transduced by lentivirus or adenovirus, and compared the hNIS expression quantitatively between the two delivery systems. Lentiviral-mediated stably hNIS expressing rMSC (lenti-hNIS-rMSC) was constructed by cloning the hNIS gene into pLenti6/UbC/V5-DEST (Invitrogen) to obtain pLenti-hNIS, transducing rMSC with the pLenti-hNIS, and selecting with blasticidin for 3 weeks. Recombinant adenovirus expressing hNIS gene (Rad-hNIS) was produced by homologous recombination and Rad-hNIS transduced rMSC (adeno-hNIS-rMSC) was evaluated for the hNIS expression 48 hours post infection at MOI 1, 5, 20, 50, and 100. The hNIS expression in lenti-hNIS-rMSC or adeno-hNIS-rMSC was assessed by immunocytochemistry, western blot, and I-125 uptake. Immunocytochemistry using mono-clonal anti-hNIS antibody revealed that intensity of hNIS immunoreactivity in lenti-hNIS-rMSC was greater than that in adeno-hNIS-rMSC at MOl 20 but lower than that at MOl 50. Western blot analysis also showed that lenti-hNIS-rMSC was intermediate between adeno-hNIS-rMSCs at MOl 20 and 50 in hNIS expression. However in vitro I-125 uptake test demonstrated that iodide uptake in lenti-hNIS-rMSC (297046659 picomole/106 cells) was greater than that in adeno-hNIS-rMSC at MOI 100 (61682134 picomole/106 cells). These results suggest that lentivirus mediated hNIS expression is greater in terms of hNIS function but lower in terms of hNIS protein amount than adenovirus mediated hNIS expression 48 hours post infection. Stem cell tracking using hNIS as a reporter gene should be conducted in consideration of relative viral efficiency of transgene expression

Suppression of progranulin expression inhibits bladder cancer growth and sensitizes cancer cells to cisplatin.

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Buraschi, Simone; Xu, Shi-Qiong; Stefanello, Manuela; Moskalev, Igor; Morcavallo, Alaide; Genua, Marco; Tanimoto, Ryuta; Birbe, Ruth; Peiper, Stephen C; Gomella, Leonard G; Belfiore, Antonino; Black, Peter C; Iozzo, Renato V; Morrione, Andrea

2016-06-28

We have recently demonstrated a critical role for progranulin in bladder cancer. Progranulin contributes, as an autocrine growth factor, to the transformed phenotype by modulating Akt-and MAPK-driven motility, invasion and anchorage-independent growth. Progranulin also induces F-actin remodeling by interacting with the F-actin binding protein drebrin. In addition, progranulin is overexpressed in invasive bladder cancer compared to normal tissue controls, suggesting that progranulin might play a key role in driving the transition to the invasive phenotype of urothelial cancer. However, it is not established whether targeting progranulin could have therapeutic effects on bladder cancer. In this study, we stably depleted urothelial cancer cells of endogenous progranulin by shRNA approaches and determined that progranulin depletion severely inhibited the ability of tumorigenic urothelial cancer cells to migrate, invade and grow in anchorage-independency. We further demonstrate that progranulin expression is critical for tumor growth in vivo, in both xenograft and orthotopic tumor models. Notably, progranulin levels correlated with response to cisplatin treatment and were upregulated in bladder tumors. Our data indicate that progranulin may constitute a novel target for therapeutic intervention in bladder tumors. In addition, progranulin may serve as a novel biomarker for bladder cancer.

Reprogramming of enteroendocrine K cells to pancreatic β-cells through the combined expression of Nkx6.1 and Neurogenin3, and reaggregation in suspension culture

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Lee, Esder; Ryu, Gyeong Ryul; Moon, Sung-Dae; Ko, Seung-Hyun; Ahn, Yu-Bae; Song, Ki-Ho, E-mail: kihos@catholic.ac.kr

2014-01-17

Highlights: •K cells were selected from STC-1 cells, a heterogeneous enteroendocrine cell line. •K cells did not express Nkx6.1 and Neurogenin3. •Combined expression of Nkx6.1 and Neurogenin3 reprogrammed K cells to β-cells. •Reprogramming of K cells to β-cells was not complete. -- Abstract: Recent studies have demonstrated that adult cells such as pancreatic exocrine cells can be converted to pancreatic β-cells in a process called cell reprogramming. Enteroendocrine cells and β-cells share similar pathways of differentiation during embryonic development. Notably, enteroendocrine K cells express many of the key proteins found in β-cells. Thus, K cells could be reprogrammed to β-cells under certain conditions. However, there is no clear evidence on whether these cells convert to β-cells. K cells were selected from STC-1 cells, an enteroendocrine cell line expressing multiple hormones. K cells were found to express many genes of transcription factors crucial for islet development and differentiation except for Nkx6.1 and Neurogenin3. A K cell clone stably expressing Nkx6.1 (Nkx6.1{sup +}-K cells) was established. Induction of Neurogenin3 expression in Nkx6.1{sup +}-K cells, by either treatment with a γ-secretase inhibitor or infection with a recombinant adenovirus expressing Neurogenin3, led to a significant increase in Insulin1 mRNA expression. After infection with the adenovirus expressing Neurogenin3 and reaggregation in suspension culture, about 50% of Nkx6.1{sup +}-K cells expressed insulin as determined by immunostaining. The intracellular insulin content was increased markedly. Electron microscopy revealed the presence of insulin granules. However, glucose-stimulated insulin secretion was defective, and there was no glucose lowering effect after transplantation of these cells in diabetic mice. In conclusion, we demonstrated that K cells could be reprogrammed partially to β-cells through the combined expression of Nkx6.1 and Neurogenin3, and

Reprogramming of enteroendocrine K cells to pancreatic β-cells through the combined expression of Nkx6.1 and Neurogenin3, and reaggregation in suspension culture

International Nuclear Information System (INIS)

Lee, Esder; Ryu, Gyeong Ryul; Moon, Sung-Dae; Ko, Seung-Hyun; Ahn, Yu-Bae; Song, Ki-Ho

2014-01-01

Highlights: •K cells were selected from STC-1 cells, a heterogeneous enteroendocrine cell line. •K cells did not express Nkx6.1 and Neurogenin3. •Combined expression of Nkx6.1 and Neurogenin3 reprogrammed K cells to β-cells. •Reprogramming of K cells to β-cells was not complete. -- Abstract: Recent studies have demonstrated that adult cells such as pancreatic exocrine cells can be converted to pancreatic β-cells in a process called cell reprogramming. Enteroendocrine cells and β-cells share similar pathways of differentiation during embryonic development. Notably, enteroendocrine K cells express many of the key proteins found in β-cells. Thus, K cells could be reprogrammed to β-cells under certain conditions. However, there is no clear evidence on whether these cells convert to β-cells. K cells were selected from STC-1 cells, an enteroendocrine cell line expressing multiple hormones. K cells were found to express many genes of transcription factors crucial for islet development and differentiation except for Nkx6.1 and Neurogenin3. A K cell clone stably expressing Nkx6.1 (Nkx6.1 + -K cells) was established. Induction of Neurogenin3 expression in Nkx6.1 + -K cells, by either treatment with a γ-secretase inhibitor or infection with a recombinant adenovirus expressing Neurogenin3, led to a significant increase in Insulin1 mRNA expression. After infection with the adenovirus expressing Neurogenin3 and reaggregation in suspension culture, about 50% of Nkx6.1 + -K cells expressed insulin as determined by immunostaining. The intracellular insulin content was increased markedly. Electron microscopy revealed the presence of insulin granules. However, glucose-stimulated insulin secretion was defective, and there was no glucose lowering effect after transplantation of these cells in diabetic mice. In conclusion, we demonstrated that K cells could be reprogrammed partially to β-cells through the combined expression of Nkx6.1 and Neurogenin3, and reaggregation

Breast cancer cell cyclooxygenase-2 expression alters extracellular matrix structure and function and numbers of cancer associated fibroblasts.

Science.gov (United States)

Krishnamachary, Balaji; Stasinopoulos, Ioannis; Kakkad, Samata; Penet, Marie-France; Jacob, Desmond; Wildes, Flonne; Mironchik, Yelena; Pathak, Arvind P; Solaiyappan, Meiyappan; Bhujwalla, Zaver M

2017-03-14

Cyclooxygenase-2 (COX-2) is a critically important mediator of inflammation that significantly influences tumor angiogenesis, invasion, and metastasis. We investigated the role of COX-2 expressed by triple negative breast cancer cells in altering the structure and function of the extracellular matrix (ECM). COX-2 downregulation effects on ECM structure and function were investigated using magnetic resonance imaging (MRI) and second harmonic generation (SHG) microscopy of tumors derived from triple negative MDA-MB-231 breast cancer cells, and a derived clone stably expressing a short hairpin (shRNA) molecule downregulating COX-2. MRI of albumin-GdDTPA was used to characterize macromolecular fluid transport in vivo and SHG microscopy was used to quantify collagen 1 (Col1) fiber morphology. COX-2 downregulation decreased Col1 fiber density and altered macromolecular fluid transport. Immunohistochemistry identified significantly fewer activated cancer associated fibroblasts (CAFs) in low COX-2 expressing tumors. Metastatic lung nodules established by COX-2 downregulated cells were infrequent, smaller, and contained fewer Col1 fibers.COX-2 overexpression studies were performed with tumors derived from triple negative SUM-149 breast cancer cells lentivirally transduced to overexpress COX-2. SHG microscopy identified significantly higher Col1 fiber density in COX-2 overexpressing tumors with an increase of CAFs. These data expand upon the roles of COX-2 in shaping the structure and function of the ECM in primary and metastatic tumors, and identify the potential role of COX-2 in modifying the number of CAFs in tumors that may have contributed to the altered ECM.

Gene expression profiling reveals novel regulation by bisphenol-A in estrogen receptor-α-positive human cells

International Nuclear Information System (INIS)

Singleton, David W.; Feng, Yuxin; Yang, Jun; Puga, Alvaro; Lee, Adrian V.; Khan, Sohaib A.

2006-01-01

Bisphenol-A (BPA) shows proliferative actions in uterus and mammary glands and may influence the development of male and female reproductive tracts in utero or during early postnatal life. Because of its ability to function as an estrogen receptor (ER) agonist, BPA has the potential to disrupt normal endocrine signaling through regulation of ER target genes. Some genes are regulated by both estradiol (E2) and BPA, but those exclusive to either agent have not been described. Using a yeast strain incorporating a vitellogenin A2 ERE-LacZ reporter gene into the genome, we found that BPA induced expression of the reporter in colonies transformed with the ERα expression plasmid, illustrating BPA-mediated regulation within a chromatin context. Additionally, a reporter gene transiently transfected into the endometrial cancer (Ishikawa) cell line also showed BPA activity, although at 100-fold less potency than E2. To compare global gene expression in response to BPA and E2, we used a variant of the MCF-7 breast cancer cell line stably expressing HA-tagged ERα. Cultures were treated for 3 h with an ethanol vehicle, E2 (10 -8 M), or BPA (10 -6 M), followed by isolation of RNA and microarray analysis with the human U95A probe array (Affymetrix, Santa Clara, CA, USA). More than 300 genes were changed 2-fold or more by either or both agents, with roughly half being up-regulated and half down-regulated. A number of growth- and development-related genes, such as HOXC1 and C6, Wnt5A, Frizzled, TGFβ-2, and STAT inhibitor 2, were found to be affected exclusively by BPA. We used quantitative real-time PCR to verify regulation of the HOXC6 gene, which showed decreased expression of approximately 2.5-fold by BPA. These results reveal novel effects by BPA and E2, raising interesting possibilities regarding the role of endocrine disruptors in sexual development

Identification of a secreted casein kinase 1 in Leishmania donovani: effect of protein over expression on parasite growth and virulence.

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Mary Dan-Goor

Full Text Available Casein kinase 1 (CK1 plays an important role in eukaryotic signaling pathways, and their substrates include key regulatory proteins involved in cell differentiation, proliferation and chromosome segregation. The Leishmania genome encodes six potential CK1 isoforms, of which five have orthologs in other trypanosomatidae. Leishmania donovani CK1 isoform 4 (Ldck1.4, orthologous to LmjF27.1780 is unique to Leishmania and contains a putative secretion signal peptide. The full-length gene and three shorter constructs were cloned and expressed in E. coli as His-tag proteins. Only the full-length 62.3 kDa protein showed protein kinase activity indicating that the N-terminal and C-terminal domains are essential for protein activity. LdCK1.4-FLAG was stably over expressed in L. donovani, and shown by immunofluorescence to be localized primarily in the cytosol. Western blotting using anti-FLAG and anti-CK1.4 antibodies showed that this CK1 isoform is expressed and secreted by promastigotes. Over expression of LdCK1.4 had a significant effect on promastigote growth in culture with these parasites growing to higher cell densities than the control parasites (wild-type or Ld:luciferase, P<0.001. Analysis by flow cytometry showed a higher percentage, ∼4-5-fold, of virulent metacyclic promastigotes on day 3 among the LdCK1.4 parasites. Finally, parasites over expressing LdCK1.4 gave significantly higher infections of mouse peritoneal macrophages compared to wild-type parasites, 28.6% versus 6.3%, respectively (p = 0.0005. These results suggest that LdCK1.4 plays an important role in parasite survival and virulence. Further studies are needed to validate CK1.4 as a therapeutic target in Leishmania.

The radiosensitivity of glioblastoma cell lines after hypoxia-induced Bax expression

International Nuclear Information System (INIS)

Chen, J.K.; Hu, L.J.; Kong, E.L.; Lamborn, K.R.; Deen, D.F.

2003-01-01

Full text: Radiation therapy is the most effective treatment after surgery for patients with malignant gliomas. However, the hypoxic cells exclusive to tumor tissue have proven resistant to both radiotherapy and many forms of chemotherapy. In order to specifically target these hypoxic cells, U-251 MG and U-87 MG human glioblastoma cells were stably transfected with constructs containing the suicide gene Bax under the regulation of nine copies of hypoxia-responsive elements (HREs). During hypoxia, the transcriptional complex hypoxia-inducible-factor 1 (HIF-1) binds to HRE and facilitates the transcription of downstream genes. Previously, hypoxia-induced Bax expression in transfected U-251 and U-87 clone cells has been shown to increase cell killing. The benefits of the gene therapy could be further expanded if Bax also acted to increase the sensitivity of these clone cells to radiation. To determine whether this was the case, parent and clone cells were irradiated with graded doses of X-rays under hypoxic conditions. These cells were then left hypoxic for varying durations of time, after which they were incubated for two weeks under aerated conditions to assay for clonogenic cell survival. After less than an hour under hypoxia, both U-251 and U-87 clone cells appeared significantly more sensitive to radiation than their respective parent cells. However, after longer amounts of time under anoxia, higher surviving fractions were found in each clone that were consistent with those of their respective parent cell line, showing that potentially lethal damage repair (PLDR) had occurred in the clone cells. Parent cells did not exhibit PLDR. Results are inconclusive at this point in time. Western blot analyses detailing the amount of Bax expression at each time point as well as further research exploring different durations of hypoxia will be necessary to reveal the nature of the correlation between Bax expression and radiosensitivity. Supported by NS-42927 and CA-85356

Identification and quantification of N alpha-acetylated Y. pestis fusion protein F1-V expressed in Escherichia coli using LCMS E.

Science.gov (United States)

Bariola, Pauline A; Russell, Brett A; Monahan, Steven J; Stroop, Steven D

2007-05-31

N-terminal acetylation in E coli is a rare event catalyzed by three known N-acetyl-transferases (NATs), each having a specific ribosomal protein substrate. Multiple, gram-scale lots of recombinant F1-V, a fusion protein constructed from Y. Pestis antigens, were expressed and purified from a single stably transformed E. coli cell bank. A variant form of F1-V with mass increased by 42-43 Da was detected in all purified lots by electrospray orthogonal acceleration time-of-flight mass spectrometry (MS). Peptide mapping LCMS localized the increased mass to an N-terminal Lys-C peptide, residues 1-24, and defined it as +42.0308+/-0.0231 Da using a LockSpray exact mass feature and a leucine enkaphalin mass standard. Sequencing of the variant 1-24 peptide by LCMS and high-energy collision induced dissociation (LCMS(E)) further localized the modification to the amino terminal tri-peptide ADL and identified the modification as N(alpha)-acetylation. The average content of N(alpha)-acetylated F1-V in five lots was 24.7+/-2.6% indicating that a stable acetylation activity for F1-V was established in the E. coli expression system. Alignment of the F1-V N-terminal sequence with those of other known N(alpha)-acetylated ectopic proteins expressed in E. coli reveals a substrate motif analogous to the eukaryote NatA' acetylation pathway and distinct from endogenous E. coli NAT substrates.

Differential regulation by AMP and ADP of AMPK complexes containing different γ subunit isoforms

DEFF Research Database (Denmark)

Ross, Fiona A; Jensen, Thomas Elbenhardt; Hardie, D Grahame

2016-01-01

The g subunits of heterotrimeric AMPK complexes contain the binding sites for the regulatory adenine nucleotides AMP, ADP and ATP. We addressed whether complexes containing different g isoforms display different responses to adenine nucleotides by generating cells stably expressing FLAG-tagged ve...

Selection of suitable reference genes for normalization of genes of interest in canine soft tissue sarcomas using quantitative real-time polymerase chain reaction.

Science.gov (United States)

Zornhagen, K W; Kristensen, A T; Hansen, A E; Oxboel, J; Kjaer, A

2015-12-01

Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is a sensitive technique for quantifying gene expression. Stably expressed reference genes are necessary for normalization of RT-qPCR data. Only a few articles have been published on reference genes in canine tumours. The objective of this study was to demonstrate how to identify suitable reference genes for normalization of genes of interest in canine soft tissue sarcomas using RT-qPCR. Primer pairs for 17 potential reference genes were designed and tested in archival tumour biopsies from six dogs. The geNorm algorithm was used to analyse the most suitable reference genes. Eight potential reference genes were excluded from this final analysis because of their dissociation curves. β-Glucuronidase (GUSB) and proteasome subunit, beta type, 6 (PSMB6) were most stably expressed with an M value of 0.154 and a CV of 0.053 describing their average stability. We suggest that choice of reference genes should be based on specific testing in every new experimental set-up. © 2014 John Wiley & Sons Ltd.

Efficient transformation and expression of gfp gene in Valsa mali var. mali.

Science.gov (United States)

Chen, Liang; Sun, Gengwu; Wu, Shujing; Liu, Huixiang; Wang, Hongkai

2015-01-01

Valsa mali var. mali, the causal agent of valsa canker of apple, causes great loss of apple production in apple producing regions. The pathogenic mechanism of the pathogen has not been studied extensively, thus a suitable gene marker for pathogenic invasion analysis and a random insertion of T-DNA for mutants are desirable. In this paper, we reported the construction of a binary vector pKO1-HPH containing a positive selective gene hygromycin phosphotransferase (hph), a reporter gene gfp conferring green fluorescent protein, and an efficient protocol for V. mali var. mali transformation mediated by Agrobacterium tumefaciens. A transformation efficiency up to about 75 transformants per 10(5) conidia was achieved when co-cultivation of V. mali var. mali and A. tumefaciens for 48 h in A. tumefaciens inductive medium agar plates. The insertions of hph gene and gfp gene into V. mali var. mali genome verified by polymerase chain reaction and southern blot analysis showed that 10 randomly-selected transformants exhibited a single, unique hybridization pattern. This is the first report of A. tumefaciens-mediated transformation of V. mali var mali carrying a 'reporter' gfp gene that stably and efficiently expressed in the transformed V. mali var. mali species.

Imaging of gene expression in live pancreatic islet cell lines using dual-isotope SPECT.

Science.gov (United States)

Tai, Joo Ho; Nguyen, Binh; Wells, R Glenn; Kovacs, Michael S; McGirr, Rebecca; Prato, Frank S; Morgan, Timothy G; Dhanvantari, Savita

2008-01-01

We are combining nuclear medicine with molecular biology to establish a sensitive, quantitative, and tomographic method with which to detect gene expression in pancreatic islet cells in vivo. Dual-isotope SPECT can be used to image multiple molecular events simultaneously, and coregistration of SPECT and CT images enables visualization of reporter gene expression in the correct anatomic context. We have engineered pancreatic islet cell lines for imaging with SPECT/CT after transplantation under the kidney capsule. INS-1 832/13 and alphaTC1-6 cells were stably transfected with a herpes simplex virus type 1-thymidine kinase-green fluorescent protein (HSV1-thymidine kinase-GFP) fusion construct (tkgfp). After clonal selection, radiolabel uptake was determined by incubation with 5-(131)I-iodo-1-(2-deoxy-2-fluoro-beta-d-arabinofuranosyl)uracil ((131)I-FIAU) (alphaTC1-6 cells) or (123)I-FIAU (INS-1 832/13 cells). For the first set of in vivo experiments, SPECT was conducted after alphaTC1-6/tkgfp cells had been labeled with either (131)I-FIAU or (111)In-tropolone and transplanted under the left kidney capsule of CD1 mice. Reconstructed SPECT images were coregistered to CT. In a second study using simultaneous acquisition dual-isotope SPECT, INS-1 832/13 clone 9 cells were labeled with (111)In-tropolone before transplantation. Mice were then systemically administered (123)I-FIAU and data for both (131)I and (111)In were acquired simultaneously. alphaTC1-6/tkgfp cells showed a 15-fold greater uptake of (131)I-FIAU, and INS-1/tkgfp cells showed a 12-fold greater uptake of (123)I-FIAU, compared with that of wild-type cells. After transplantation under the kidney capsule, both reporter gene expression and location of cells could be visualized in vivo with dual-isotope SPECT. Immunohistochemistry confirmed the presence of glucagon- and insulin-positive cells at the site of transplantation. Dual-isotope SPECT is a promising method to detect gene expression in and location of

In vivo effects of the IKr agonist NS3623 on cardiac electrophysiology of the guinea pig

DEFF Research Database (Denmark)

Hansen, Rie Schultz; Olesen, Søren-Peter; Rønn, Lars Christian B

2008-01-01

to examining the in vivo effects of NS3623. Injection of 30 mg/kg NS3623 shortened the corrected QT interval by 25 +/- 4% in anaesthetized guinea pigs. Accordingly, 50 mg/kg of NS3623 shortened the QT interval by 30 +/- 6% in conscious guinea pigs. Finally, pharmacologically induced QT prolongation by a h......ERG channel antagonist (0.15 mg/kg E-4031) could be reverted by injection of NS3623 (50 mg/kg) in conscious guinea pigs. In conclusion, the present in vivo study demonstrates that injection of the hERG channel agonist NS3623 results in shortening of the QTc interval as well as reversal of a pharmacologically...... induced QT prolongation in both anaesthetized and conscious guinea pigs....

The oligodendroglial precursor cell line Oli-neu represents a cell culture system to examine functional expression of the mouse gap junction gene connexin29 (Cx29

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Goran Christoph Söhl

2013-06-01

Full Text Available The potential gap junction forming mouse connexin29 (Cx29 protein is concomitantly expressed with connexin32 (Cx32 in peripheral myelin forming Schwann cells and together with both Cx32 and connexin47 (Cx47 in oligodendrocytes of the CNS. To study the genomic structure and functional expression of Cx29, either primary cells or cell culture systems might be selected, from which the latter are easier to cultivate. Both structure and expression of Cx29 is still not fully understood. In the mouse sciatic nerve, brain and the oligodendroglial precursor cell line Oli-neu the Cx29 gene is processed in two transcript isoforms both harbouring a unique reading frame. In contrast to Cx32 and Cx47, only Cx29 protein is abundantly expressed in undifferentiated as well as differentiated Oli-neu cells but the absence of Etbr dye transfer after microinjection concealed the function of Cx29 mediated gap junction communication between those cells. Although HeLa cells stably transfected with Cx29 or Cx29-eGFP neither demonstrated any permeability for Lucifer yellow nor for neurobiotin, blocking of Etbr uptake from the media by gap junction blockers does suppose a role of Cx29 in hemi-channel function. Thus, we conclude that, due to its high abundance of Cx29 expression and its reproducible culture conditions, the oligodendroglial precursor cell line Oli-neu might constitute an appropriate cell culture system to study molecular mechanisms or putative extracellular stimuli to functionally open Cx29 channels or hemi-channels.

Reconstruction of Cell Surface Densities of Ion Pumps, Exchangers, and Channels from mRNA Expression, Conductance Kinetics, Whole-Cell Calcium, and Current-Clamp Voltage Recordings, with an Application to Human Uterine Smooth Muscle Cells.

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Jolene Atia

2016-04-01

Full Text Available Uterine smooth muscle cells remain quiescent throughout most of gestation, only generating spontaneous action potentials immediately prior to, and during, labor. This study presents a method that combines transcriptomics with biophysical recordings to characterise the conductance repertoire of these cells, the 'conductance repertoire' being the total complement of ion channels and transporters expressed by an electrically active cell. Transcriptomic analysis provides a set of potential electrogenic entities, of which the conductance repertoire is a subset. Each entity within the conductance repertoire was modeled independently and its gating parameter values were fixed using the available biophysical data. The only remaining free parameters were the surface densities for each entity. We characterise the space of combinations of surface densities (density vectors consistent with experimentally observed membrane potential and calcium waveforms. This yields insights on the functional redundancy of the system as well as its behavioral versatility. Our approach couples high-throughput transcriptomic data with physiological behaviors in health and disease, and provides a formal method to link genotype to phenotype in excitable systems. We accurately predict current densities and chart functional redundancy. For example, we find that to evoke the observed voltage waveform, the BK channel is functionally redundant whereas hERG is essential. Furthermore, our analysis suggests that activation of calcium-activated chloride conductances by intracellular calcium release is the key factor underlying spontaneous depolarisations.

Expression of intracellular interferon-alpha confers antiviral properties in transfected bovine fetal fibroblasts and does not affect the full development of SCNT embryos.

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Dawei Yu

Full Text Available Foot-and-mouth disease, one of the most significant diseases of dairy herds, has substantial effects on farm economics, and currently, disease control measures are limited. In this study, we constructed a vector with a human interferon-α (hIFN-α (without secretory signal sequence gene cassette containing the immediate early promoter of human cytomegalovirus. Stably transfected bovine fetal fibroblasts were obtained by G418 selection, and hIFN-α transgenic embryos were produced by somatic cell nuclear transfer (SCNT. Forty-six transgenic embryos were transplanted into surrogate cows, and five cows (10.9% became pregnant. Two male cloned calves were born. Expression of hIFN-α was detected in transfected bovine fetal fibroblasts, transgenic SCNT embryos, and different tissues from a transgenic SCNT calf at two days old. In transfected bovine fetal fibroblasts, expression of intracellular IFN-α induced resistance to vesicular stomatitis virus infection, increased apoptosis, and induced the expression of double-stranded RNA-activated protein kinase gene (PKR and the 2'-5'-oligoadenylate synthetase gene (2'-5' OAS, which are IFN-inducible genes with antiviral activity. Analysis by qRT-PCR showed that the mRNA expression levels of PKR, 2'-5' OAS, and P53 were significantly increased in wild-type bovine fetal fibroblasts stimulated with extracellular recombinant human IFN-α-2b, showing that intracellular IFN-α induces biological functions similar to extracellular IFN-α. In conclusion, expression of intracellular hIFN-α conferred antiviral properties in transfected bovine fetal fibroblasts and did not significantly affect the full development of SCNT embryos. Thus, IFN-α transgenic technology may provide a revolutionary way to achieve elite breeding of livestock.

Validation of reference genes for normalization of qPCR mRNA expression levels in Staphylococcus aureus exposed to osmotic and lactic acid stress conditions encountered during food production and preservation.

Science.gov (United States)

Sihto, Henna-Maria; Tasara, Taurai; Stephan, Roger; Johler, Sophia

2014-07-01

Staphylococcus aureus represents the most prevalent cause of food-borne intoxications worldwide. While being repressed by competing bacteria in most matrices, this pathogen exhibits crucial competitive advantages during growth at high salt concentrations or low pH, conditions frequently encountered in food production and preservation. We aimed to identify reference genes that could be used to normalize qPCR mRNA expression levels during growth of S. aureus in food-related osmotic (NaCl) and acidic (lactic acid) stress adaptation models. Expression stability of nine housekeeping genes was evaluated in full (LB) and nutrient-deficient (CYGP w/o glucose) medium under conditions of osmotic (4.5% NaCl) and acidic stress (lactic acid, pH 6.0) after 2-h exposure. Among the set of candidate reference genes investigated, rplD, rpoB,gyrB, and rho were most stably expressed in LB and thus represent the most suitable reference genes for normalization of qPCR data in osmotic or lactic acid stress models in a rich medium. Under nutrient-deficient conditions, expression of rho and rpoB was highly stable across all tested conditions. The presented comprehensive data on changes in expression of various S. aureus housekeeping genes under conditions of osmotic and lactic acid stress facilitate selection of reference genes for qPCR-based stress response models. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Stable expression of lipocalin-type prostaglandin D synthase in cultured preadipocytes impairs adipogenesis program independently of endogenous prostanoids

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Hossain, Mohammad Salim; Chowdhury, Abu Asad; Rahman, Mohammad Sharifur [Department of Life Science and Biotechnology, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane 690-8504 (Japan); Nishimura, Kohji [Department of Molecular and Functional Genomics, Center for Integrated Research in Science, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane 690-8504 (Japan); Jisaka, Mitsuo; Nagaya, Tsutomu [Department of Life Science and Biotechnology, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane 690-8504 (Japan); Shono, Fumiaki [Department of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Yamashiro-cho, Tokushima-shi, Tokushima 770-8514 (Japan); Yokota, Kazushige, E-mail: yokotaka@life.shimane-u.ac.jp [Department of Life Science and Biotechnology, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane 690-8504 (Japan)

2012-02-15

Lipocalin-type prostaglandin D synthase (L-PGDS) expressed preferentially in adipocytes is responsible for the synthesis of PGD{sub 2} and its non-enzymatic dehydration products, PGJ{sub 2} series, serving as pro-adipogenic factors. However, the role of L-PGDS in the regulation of adipogenesis is complex because of the occurrence of several derivatives from PGD{sub 2} and their distinct receptor subtypes as well as other functions such as a transporter of lipophilic molecules. To manipulate the expression levels of L-PGDS in cultured adipocytes, cultured preadipogenic 3T3-L1 cells were transfected stably with a mammalian expression vector having cDNA encoding murine L-PGDS oriented in the sense direction. The isolated cloned stable transfectants with L-PGDS expressed higher levels of the transcript and protein levels of L-PGDS, and synthesized PGD{sub 2} from exogenous arachidonic acid at significantly higher levels. By contrast, the synthesis of PGE{sub 2} remained unchanged, indicating no influence on the reactions of cyclooxygenase (COX) and PGE synthase. Furthermore, the ability of those transfectants to synthesize {Delta}{sup 12}-PGJ{sub 2} increased more greatly during the maturation phase. The sustained expression of L-PGDS in cultured stable transfectants hampered the storage of fats during the maturation phase of adipocytes, which was accompanied by the reduced gene expression of adipocyte-specific markers reflecting the down-regulation of the adipogenesis program. The suppressed adipogenesis was not rescued by either exogenous aspirin or peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonists including troglitazone and {Delta}{sup 12}-PGJ{sub 2}. Taken together, the results indicate the negative regulation of the adipogenesis program by the enhanced expression of L-PGDS through a cellular mechanism involving the interference of the PPAR{gamma} signaling pathway without the contribution of endogenous pro-adipogenic prostanoids

Thrombospondin-2 promotes prostate cancer bone metastasis by the up-regulation of matrix metalloproteinase-2 through down-regulating miR-376c expression

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Po-Chun Chen

2017-01-01

Full Text Available Abstract Background Thrombospondin-2 (TSP-2 is a secreted matricellular glycoprotein that is found to mediate cell-to-extracellular matrix attachment and participates in many physiological and pathological processes. The expression profile of TSP-2 on tumors is controversial, and it up-regulates in some cancers, whereas it down-regulates in others, suggesting that the functional role of TSP-2 on tumors is still uncertain. Methods The expression of TSP-2 on prostate cancer progression was determined in the tissue array by the immunohistochemistry. The molecular mechanism of TSP-2 on prostate cancer (PCa metastasis was investigated through pharmaceutical inhibitors, siRNAs, and miRNAs analyses. The role of TSP-2 on PCa metastasis in vivo was verified through xenograft in vivo imaging system. Results Based on the gene expression omnibus database and immunohistochemistry, we found that TSP-2 increased with the progression of PCa, especially in metastatic PCa and is correlated with the matrix metalloproteinase-2 (MMP-2 expression. Additionally, through binding to CD36 and integrin ανβ3, TSP-2 increased cell migration and MMP-2 expression. With inhibition of p38, ERK, and JNK, the TSP-2-induced cell migration and MMP-2 expression were abolished, indicating that the TSP-2’s effect on PCa is MAPK dependent. Moreover, the microRNA-376c (miR-376c was significantly decreased by the TSP-2 treatment. Furthermore, the TSP-2-induced MMP-2 expression and the subsequent cell motility were suppressed upon miR-376c mimic stimulation. On the other hand, the animal studies revealed that the bone metastasis was abolished when TSP-2 was stably knocked down in PCa cells. Conclusions Taken together, our results indicate that TSP-2 enhances the migration of PCa cells by increasing MMP-2 expression through down-regulation of miR-376c expression. Therefore, TSP-2 may represent a promising new target for treating PCa.

Ectopic expression of AID in a non-B cell line triggers A:T and G:C point mutations in non-replicating episomal vectors.

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Tihana Jovanic

Full Text Available Somatic hypermutation (SHM of immunoglobulin genes is currently viewed as a two step process initiated by the deamination of deoxycytidine (C to deoxyuridine (U, catalysed by the activation induced deaminase (AID. Phase 1 mutations arise from DNA replication across the uracil residue or the abasic site, generated by the uracil-DNA glycosylase, yielding transitions or transversions at G:C pairs. Phase 2 mutations result from the recognition of the U:G mismatch by the Msh2/Msh6 complex (MutS Homologue, followed by the excision of the mismatched nucleotide and the repair, by the low fidelity DNA polymerase eta, of the gap generated by the exonuclease I. These mutations are mainly focused at A:T pairs. Whereas in activated B cells both G:C and A:T pairs are equally targeted, ectopic expression of AID was shown to trigger only G:C mutations on a stably integrated reporter gene. Here we show that when using non-replicative episomal vectors containing a GFP gene, inactivated by the introduction of stop codons at various positions, a high level of EGFP positive cells was obtained after transient expression in Jurkat cells constitutively expressing AID. We show that mutations at G:C and A:T pairs are produced. EGFP positive cells are obtained in the absence of vector replication demonstrating that the mutations are dependent only on the mismatch repair (MMR pathway. This implies that the generation of phase 1 mutations is not a prerequisite for the expression of phase 2 mutations.

Podoplanin increases migration and angiogenesis in malignant glioma

OpenAIRE

Grau, Stefan J; Trillsch, Fabian; Tonn, Joerg-Christian; Goldbrunner, Roland H; Noessner, Elfriede; Nelson, Peter J; von Luettichau, Irene

2015-01-01

Expression of podoplanin in glial brain tumors is grade dependent. While serving as a marker for tumor progression and modulating invasion in various neoplasms, little is known about podoplanin function in gliomas. Therefore we stably transfected two human glioma cell lines (U373MG and U87MG) with expression plasmids encoding podoplanin. The efficacy of transfection was confirmed by FACS analysis, PCR and immunocytochemistry. Cells were then sorted for highly podoplanin expressing cells (U373...

Identification of valid reference genes for the normalization of RT-qPCR expression studies in human breast cancer cell lines treated with and without transient transfection.

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Lin-Lin Liu

Full Text Available Reverse transcription-quantitative polymerase chain reaction (RT-qPCR is a powerful technique for examining gene expression changes during tumorigenesis. Target gene expression is generally normalized by a stably expressed endogenous reference gene; however, reference gene expression may differ among tissues under various circumstances. Because no valid reference genes have been documented for human breast cancer cell lines containing different cancer subtypes treated with transient transfection, we identified appropriate and reliable reference genes from thirteen candidates in a panel of 10 normal and cancerous human breast cell lines under experimental conditions with/without transfection treatments with two transfection reagents. Reference gene expression stability was calculated using four algorithms (geNorm, NormFinder, BestKeeper and comparative delta Ct, and the recommended comprehensive ranking was provided using geometric means of the ranking values using the RefFinder tool. GeNorm analysis revealed that two reference genes should be sufficient for all cases in this study. A stability analysis suggests that 18S rRNA-ACTB is the best reference gene combination across all cell lines; ACTB-GAPDH is best for basal breast cancer cell lines; and HSPCB-ACTB is best for ER+ breast cancer cells. After transfection, the stability ranking of the reference gene fluctuated, especially with Lipofectamine 2000 transfection reagent in two subtypes of basal and ER+ breast cell lines. Comparisons of relative target gene (HER2 expression revealed different expressional patterns depending on the reference genes used for normalization. We suggest that identifying the most stable and suitable reference genes is critical for studying specific cell lines under certain circumstances.

Stably Integrated luxCDABE for Assessment of Salmonella Invasion Kinetics

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Kelly N. Flentie

2008-09-01

Full Text Available Salmonella Typhimurium is a common cause of gastroenteritis in humans and also localizes to neoplastic tumors in animals. Invasion of specific eukaryotic cells is a key mechanism of Salmonella interactions with host tissues. Early stages of gastrointestinal cell invasion are mediated by a Salmonella type III secretion system, powered by the adenosine triphosphatase invC. The aim of this work was to characterize the invC dependence of invasion kinetics into disparate eukaryotic cells traditionally used as models of gut epithelium or neoplasms. Thus, a nondestructive real-time assay was developed to report eukaryotic cell invasion kinetics using lux+ Salmonella that contain chromosomally integrated luxCDABE genes. Bioluminescence-based invasion assays using lux+ Salmonella exhibited inoculum dose-response correlation, distinguished invasion-competent from invasion-incompetent Salmonella, and discriminated relative Salmonella invasiveness in accordance with environmental conditions that induce invasion gene expression. In standard gentamicin protection assays, bioluminescence from lux+ Salmonella correlated with recovery of colony-forming units of internalized bacteria and could be visualized by bioluminescence microscopy. Furthermore, this assay distinguished invasion-competent from invasion-incompetent bacteria independent of gentamicin treatment in real time. Bioluminescence reported Salmonella invasion of disparate eukaryotic cell lines, including neoplastic melanoma, colon adenocarcinoma, and glioma cell lines used in animal models of malignancy. In each case, Salmonella invasion of eukaryotic cells was invC dependent.

Inhibitory effects of two G protein-coupled receptor kinases on the cell surface expression and signaling of the human adrenomedullin receptor

Energy Technology Data Exchange (ETDEWEB)

Kuwasako, Kenji, E-mail: kuwasako@med.miyazaki-u.ac.jp [Frontier Science Research Center, University of Miyazaki, Miyazaki, 889-1692 (Japan); Sekiguchi, Toshio [Noto Marine Laboratory, Division of Marine Environmental Studies, Institute of Nature and Environmental Technology, Kanazawa University, Ishikawa, 927-0553 (Japan); Nagata, Sayaka [Division of Circulatory and Body Fluid Regulation, Faculty of Medicine, University of Miyazaki, Miyazaki, 889-1692 (Japan); Jiang, Danfeng; Hayashi, Hidetaka [Frontier Science Research Center, University of Miyazaki, Miyazaki, 889-1692 (Japan); Murakami, Manabu [Department of Pharmacology, Hirosaki University, Graduate School of Medicine, Hirosaki, 036-8562 (Japan); Hattori, Yuichi [Department of Molecular and Medical Pharmacology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, 930-0194 (Japan); Kitamura, Kazuo [Division of Circulatory and Body Fluid Regulation, Faculty of Medicine, University of Miyazaki, Miyazaki, 889-1692 (Japan); Kato, Johji [Frontier Science Research Center, University of Miyazaki, Miyazaki, 889-1692 (Japan)

2016-02-19

Receptor activity-modifying protein 2 (RAMP2) enables the calcitonin receptor-like receptor (CLR, a family B GPCR) to form the type 1 adrenomedullin receptor (AM{sub 1} receptor). Here, we investigated the effects of the five non-visual GPCR kinases (GRKs 2 through 6) on the cell surface expression of the human (h)AM{sub 1} receptor by cotransfecting each of these GRKs into HEK-293 cells that stably expressed hRAMP2. Flow cytometric analysis revealed that when coexpressed with GRK4 or GRK5, the cell surface expression of the AM{sub 1} receptor was markedly decreased prior to stimulation with AM, thereby attenuating both the specific [{sup 125}I]AM binding and AM-induced cAMP production. These inhibitory effects of both GRKs were abolished by the replacement of the cytoplasmic C-terminal tail (C-tail) of CLR with that of the calcitonin receptor (a family B GPCR) or β{sub 2}-adrenergic receptor (a family A GPCR). Among the sequentially truncated CLR C-tail mutants, those lacking the five residues 449–453 (Ser-Phe-Ser-Asn-Ser) abolished the inhibition of the cell surface expression of CLR via the overexpression of GRK4 or GRK5. Thus, we provided new insight into the function of GRKs in agonist-unstimulated GPCR trafficking using a recombinant AM{sub 1} receptor and further determined the region of the CLR C-tail responsible for this GRK function. - Highlights: • We discovered a novel function of GRKs in GPCR trafficking using human CLR/RAMP2. • GRKs 4 and 5 markedly inhibited the cell surface expression of human CLR/RAMP2. • Both GRKs exhibited highly significant receptor signaling inhibition. • Five residues of the C-terminal tail of CLR govern this function of GRKs.

Inhibitory effects of two G protein-coupled receptor kinases on the cell surface expression and signaling of the human adrenomedullin receptor

International Nuclear Information System (INIS)

Kuwasako, Kenji; Sekiguchi, Toshio; Nagata, Sayaka; Jiang, Danfeng; Hayashi, Hidetaka; Murakami, Manabu; Hattori, Yuichi; Kitamura, Kazuo; Kato, Johji

2016-01-01

Receptor activity-modifying protein 2 (RAMP2) enables the calcitonin receptor-like receptor (CLR, a family B GPCR) to form the type 1 adrenomedullin receptor (AM_1 receptor). Here, we investigated the effects of the five non-visual GPCR kinases (GRKs 2 through 6) on the cell surface expression of the human (h)AM_1 receptor by cotransfecting each of these GRKs into HEK-293 cells that stably expressed hRAMP2. Flow cytometric analysis revealed that when coexpressed with GRK4 or GRK5, the cell surface expression of the AM_1 receptor was markedly decreased prior to stimulation with AM, thereby attenuating both the specific ["1"2"5I]AM binding and AM-induced cAMP production. These inhibitory effects of both GRKs were abolished by the replacement of the cytoplasmic C-terminal tail (C-tail) of CLR with that of the calcitonin receptor (a family B GPCR) or β_2-adrenergic receptor (a family A GPCR). Among the sequentially truncated CLR C-tail mutants, those lacking the five residues 449–453 (Ser-Phe-Ser-Asn-Ser) abolished the inhibition of the cell surface expression of CLR via the overexpression of GRK4 or GRK5. Thus, we provided new insight into the function of GRKs in agonist-unstimulated GPCR trafficking using a recombinant AM_1 receptor and further determined the region of the CLR C-tail responsible for this GRK function. - Highlights: • We discovered a novel function of GRKs in GPCR trafficking using human CLR/RAMP2. • GRKs 4 and 5 markedly inhibited the cell surface expression of human CLR/RAMP2. • Both GRKs exhibited highly significant receptor signaling inhibition. • Five residues of the C-terminal tail of CLR govern this function of GRKs.

Experiment list: SRX054578 [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available P-seq rep1b source_name=breast adenocarcinoma cell line || cell type=MCF7 cell line stably expressing HA ER ...of Extraction=Pleura|Tissue Diagnosis=Adenocarcinoma 12033159,72.5,1.7,159 GSM699991: Input MCF7 stables ChI

Expression, purification, and functional characterization of the insulin-responsive facilitative glucose transporter GLUT4.

Science.gov (United States)

Kraft, Thomas E; Hresko, Richard C; Hruz, Paul W

2015-12-01

The insulin-responsive facilitative glucose transporter GLUT4 is of fundamental importance for maintenance of glucose homeostasis. Despite intensive effort, the ability to express and purify sufficient quantities of structurally and functionally intact protein for biophysical analysis has previously been exceedingly difficult. We report here the development of novel methods to express, purify, and functionally reconstitute GLUT4 into detergent micelles and proteoliposomes. Rat GLUT4 containing FLAG and His tags at the amino and carboxy termini, respectively, was engineered and stably transfected into HEK-293 cells. Overexpression in suspension culture yielded over 1.5 mg of protein per liter of culture. Systematic screening of detergent solubilized GLUT4-GFP fusion protein via fluorescent-detection size exclusion chromatography identified lauryl maltose neopentyl glycol (LMNG) as highly effective for isolating monomeric GLUT4 micelles. Preservation of structural integrity and ligand binding was demonstrated via quenching of tryptophan fluorescence and competition of ATB-BMPA photolabeling by cytochalasin B. GLUT4 was reconstituted into lipid nanodiscs and proper folding was confirmed. Reconstitution of purified GLUT4 with amphipol A8-35 stabilized the transporter at elevated temperatures for extended periods of time. Functional activity of purified GLUT4 was confirmed by reconstitution of LMNG-purified GLUT4 into proteoliposomes and measurement of saturable uptake of D-glucose over L-glucose. Taken together, these data validate the development of an efficient means to generate milligram quantities of stable and functionally intact GLUT4 that is suitable for a wide array of biochemical and biophysical analyses. © 2015 The Protein Society.

The HPV-16 E7 oncoprotein induces centriole multiplication through deregulation of Polo-like kinase 4 expression

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Duensing Stefan

2011-05-01

Full Text Available Abstract Background Infection with high-risk human papillomaviruses (HPVs such as HPV-16 is intimately associated with squamous cell carcinomas (SCCs of the anogenital tract and a subset of oropharyngeal carcinomas. Such lesions, including pre-invasive precursors, frequently show multipolar mitoses and aneuploidy. The high-risk HPV-16-encoded E7 oncoprotein has been shown to rapidly induce centrosome abnormalities thereby causing the formation of supernumerary mitotic spindle poles and increasing the risk for chromosome missegregation. HPV-16 E7 has been found to rapidly induce centriole overduplication, in part, through the simultaneous formation of more than one daughter centriole at single maternal centrioles (centriole multiplication. The precise molecular mechanism that underlies HPV-16 E7-induced centriole multiplication, however, remains poorly understood. Findings Here, we show that human keratinocytes engineered to stably express the HPV-16 E7 oncoprotein exhibit aberrant Polo-like kinase 4 (PLK4 protein expression at maternal centrioles. Real-time quantitative reverse transcriptase (qRT-PCR analysis of these cells revealed an increase of PLK4 mRNA levels compared to control cells. Importantly, the ability of the HPV-16 E7 oncoprotein to induce centriole multiplication was found to correlate with its ability to activate the PLK4 promoter and to up-regulate PLK4 mRNA. Conclusions These results highlight the critical role of PLK4 transcriptional deregulation in centriole multiplication in HPV-16 E7-expressing cells. Our findings encourage further experiments to test transcriptional inhibitors or small molecules targeting PLK4 to prevent centriole abnormalities, mitotic infidelity and malignant progression in HPV-associated neoplasms and other tumors in which PLK4 regulation is disrupted.

Lopinavir up-regulates expression of the antiviral protein ribonuclease L in human papillomavirus-positive cervical carcinoma cells.

Science.gov (United States)

Batman, Gavin; Oliver, Anthony W; Zehbe, Ingeborg; Richard, Christina; Hampson, Lynne; Hampson, Ian N

2011-01-01

We have previously shown that the HIV protease inhibitor lopinavir has selective toxicity against human papillomavirus (HPV)-positive cervical carcinoma cells via an unknown mechanism. SiHa cervical carcinoma cells were stably transfected with the proteasome sensor vector pZsProSensor-1 to confirm lopinavir inhibits the proteasome in these cells. The Panorama Xpress profiler 725 antibody array was then used to analyse specific changes in protein expression in lopinavir-treated versus control untreated SiHa cells followed by PCR and western blotting. Colorimetric growth assays of lopinavir-treated E6/E7 immortalised versus control human keratinocytes were performed. Targeted small interfering RNA gene silencing followed by growth assay comparison of lopinavir-treated/untreated SiHa cells was also used. Lopinavir induced an increase in the fluorescence of pZsProSensor-1 transfected SiHa cells, indicative of proteasomal inhibition. Ribonuclease L (RNASEL) protein was shown to be up-regulated in lopinavir-treated SiHa cells, which was confirmed by PCR and western blot. Targeted silencing of RNASEL reduced the sensitivity of SiHa cells to lopinavir. Selective toxicity against E6/E7 immortalised keratinocytes versus control cells was also seen with lopinavir and was associated with up-regulated RNASEL expression. These data are consistent with the toxicity of lopinavir against HPV-positive cervical carcinoma cells being related to its ability to block viral proteasome activation and induce an up-regulation of the antiviral protein RNASEL. This is supported by the drug's selective toxicity and up-regulation of RNASEL in E6/E7 immortalised keratinocytes combined with the increased resistance to lopinavir observed in SiHa cells following silencing of RNASEL gene expression.

Osmotic stress changes the expression and subcellular localization of the Batten disease protein CLN3.

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Amanda Getty

Full Text Available Juvenile CLN3 disease (formerly known as juvenile neuronal ceroid lipofuscinosis is a fatal childhood neurodegenerative disorder caused by mutations in the CLN3 gene. CLN3 encodes a putative lysosomal transmembrane protein with unknown function. Previous cell culture studies using CLN3-overexpressing vectors and/or anti-CLN3 antibodies with questionable specificity have also localized CLN3 in cellular structures other than lysosomes. Osmoregulation of the mouse Cln3 mRNA level in kidney cells was recently reported. To clarify the subcellular localization of the CLN3 protein and to investigate if human CLN3 expression and localization is affected by osmotic changes we generated a stably transfected BHK (baby hamster kidney cell line that expresses a moderate level of myc-tagged human CLN3 under the control of the human ubiquitin C promoter. Hyperosmolarity (800 mOsm, achieved by either NaCl/urea or sucrose, dramatically increased the mRNA and protein levels of CLN3 as determined by quantitative real-time PCR and Western blotting. Under isotonic conditions (300 mOsm, human CLN3 was found in a punctate vesicular pattern surrounding the nucleus with prominent Golgi and lysosomal localizations. CLN3-positive early endosomes, late endosomes and cholesterol/sphingolipid-enriched plasma membrane microdomain caveolae were also observed. Increasing the osmolarity of the culture medium to 800 mOsm extended CLN3 distribution away from the perinuclear region and enhanced the lysosomal localization of CLN3. Our results reveal that CLN3 has multiple subcellular localizations within the cell, which, together with its expression, prominently change following osmotic stress. These data suggest that CLN3 is involved in the response and adaptation to cellular stress.

Production of a Marfan cellular phenotype by expressing a mutant human fibrillin allele on a normal human or murine genetic background

Energy Technology Data Exchange (ETDEWEB)

Eldadah, Z.A.; Dietz, H.C. [Johns Hopkins Univ. School of Medicine, Baltimore, MD (United States); Brenn, T. [Stanford Univ. Medical Center, CA (United States)] [and others

1994-09-01

The Marfan Syndrome (MFS) is a heritable disorder of connective tissue caused by defects in fibrillin (FBN1), a 350 kD glycoprotein and principal component of the extracellular microfibril. Previous correlations of mutant transcript level and disease severity suggested a dominant negative model of MFS pathogenesis. To address this hypothesis we assembled an expression construct containing the mutant allele from a patient with severe MFS. This mutation causes skipping of FBN1 exon 2 and a frame shift, leading to a premature termination codon in exon 4. The predicted peptide would thus consist of 55 wild type and 45 missense amino acids. The construct was stably transfected into cultured human and mouse fibroblasts, and several clonal cell populations were established. Human and mouse cells expressing the truncated peptide exhibited markedly diminished fibrillin deposition and disorganized microfibrillar architecture by immunofluorescence. Pulse-chase analysis of these cells demonstrated normal levels of fibrillin synthesis but substantially decreased fibrillin deposition into the extracellular matrix. These data illustrate that expression of a mutant FBN1 allele, on a background of two normal alleles, is sufficient to disrupt normal fibrillin aggregation and reproduce the MFS cellular phenotype. This provides confirmation of a dominant negative model of MFS pathogenesis and may offer mutant allele knockout as a strategy for gene therapy. In addition, these data underscore the importance of the FBN1 amino-terminus in normal multimer formation and suggest that expression of the human extreme 5{prime} FBN1 coding sequence may be sufficient, in isolation, to produce an animal model of MFS. Indeed, transgenic mice harboring this mutant allele have been produced, and phenotype analysis is currently in progress.

Virally and physically transgenized equine adipose-derived stromal cells as a cargo for paracrine secreted factors

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Cavirani Sandro

2010-09-01

Full Text Available Abstract Background Adipose-Derived Stromal Cells have been shown to have multiple lineage differentiation properties and to be suitable for tissues regeneration in many degenerative processes. Their use has been proposed for the therapy of joint diseases and tendon injuries in the horse. In the present report the genetic manipulation of Equine Adipose-Derived Stromal Cells has been investigated. Results Equine Adipose-Derived Stromal Cells were successfully virally transduced as well as transiently and stably transfected with appropriate parameters, without detrimental effect on their differentiation properties. Moreover, green fluorescent protein alone, fused to neo gene, or co-expressed as bi-cistronic reporter constructs, driven by viral and house-keeping gene promoters, were tested. The better expressed cassette was employed to stably transfect Adipose-Derived Stromal Cells for cell therapy purposes. Stably transfected Equine Adipose-Derived Stromal Cells with a heterologous secreted viral antigen were able to immunize horses upon injection into the lateral wall of the neck. Conclusion This study provides the methods to successfully transgenize Adipose-Derived Stromal Cells both by lentiviral vector and by transfection using optimized constructs with suitable promoters and reporter genes. In conclusion these findings provide a working platform for the delivery of potentially therapeutic proteins to the site of cells injection via transgenized Equine Adipose-Derived Stromal Cells.

Gravity Waves in the Martian Atmosphere detected by the Radio Science Experiment MaRS on Mars Express

Science.gov (United States)

Tellmann, S.; Pätzold, M.; Häusler, B.; Tyler, G. L.; Hinson, D. P.

2013-09-01

Gravity waves are an ubiquitous feature in all stably stratified planetary atmospheres. They are known to play a significant role in the energy and momentum budget of the Earth, and they are assumed to be of importance for the redistribution of energy and momentum throughout the Martian atmosphere.

Cosmosiin Increases ADAM10 Expression via Mechanisms Involving 5’UTR and PI3K Signaling

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Zhuo Min

2018-06-01

Full Text Available The α-secretase “a disintegrin and metalloproteinase domain-containing protein” (ADAM10 is involved in the processing of amyloid precursor protein (APP. Upregulation of ADAM10 precludes the generation of neurotoxic β-amyloid protein (Aβ and represents a plausible therapeutic strategy for Alzheimer’s disease (AD. In this study, we explored compounds that can potentially promote the expression of ADAM10. Therefore, we performed high-throughput small-molecule screening in SH-SY5Y (human neuroblastoma cells that stably express a luciferase reporter gene driven by the ADAM10 promoter, including a portion of its 5’-untranslated region (5’UTR. This has led to the discovery of cosmosiin (apigenin 7-O-β-glucoside. Here, we report that in human cell lines (SH-SY5Y and HEK293, cosmosiin proportionally increased the levels of the immature and mature forms of the ADAM10 protein without altering its mRNA level. This effect was attenuated by translation inhibitors or by deleting the 5’UTR of ADAM10, suggesting that a translational mechanism was responsible for the increased levels of ADAM10. Luciferase deletion assays revealed that the first 144 nucleotides of the 5’UTR were necessary for mediating the cosmosiin-induced enhancement of ADAM10 expression in SH-SY5Y cells. Cosmosiin failed to increase the levels of the ADAM10 protein in murine cells, which lack native expression of the ADAM10 transcript containing the identified 5’UTR element. The potential signaling pathway may involve phosphatidylinositide 3-kinase (PI3K because pharmacological inhibition of PI3K attenuated the effect of cosmosiin on the expression of the ADAM10 protein. Finally, cosmosiin attenuated Aβ generation because the levels of Aβ40/42 in HEK-APP cells were significantly reduced after cosmosiin treatment. Collectively, we found that the first 144 nucleotides of the ADAM10 5’UTR, and PI3K signaling, are involved in cosmosiin-induced enhancement of the expression

PTEN overexpression improves cisplatin-resistance of human ovarian cancer cells through upregulating KRT10 expression

International Nuclear Information System (INIS)

Wu, Huijuan; Wang, Ke; Liu, Wenxin; Hao, Quan

2014-01-01

Highlights: • Overexpression of PTEN enhanced the sensitivity of C13K cells to cisplatin. • KRT10 is a downstream molecule of PTEN involved in the resistance-reversing effect. • Overexpression of KRT10 enhanced the chemosensitivity of C13K cells to cisplatin. - Abstract: Multi-drug resistance (MDR) is a common cause of the failure of chemotherapy in ovarian cancer. PTEN, a tumor suppressor gene, has been demonstrated to be able to reverse cisplatin-resistance in ovarian cancer cell line C13K. However, the downstream molecules of PTEN involved in the resistance-reversing effect have not been completely clarified. Therefore, we screened the downstream molecules of PTEN and studied their interactions in C13K ovarian cancer cells using a 3D culture model. Firstly, we constructed an ovarian cancer cell line stably expressing PTEN, C13K/PTEN. MTT assay showed that overexpression of PTEN enhanced the sensitivity of C13K cells to cisplatin, but not to paclitaxel. Then we examined the differently expressed proteins that interacted with PTEN in C13K/PTEN cells with or without cisplatin treatment by co-immunoprecipitation. KRT10 was identified as a differently expressed protein in cisplatin-treated C13K/PTEN cells. Further study confirmed that cisplatin could induce upregulation of KRT10 mRNA and protein in C13K/PTEN cells and there was a directly interaction between KRT10 and PTEN. Forced expression of KRT10 in C13K cells also enhanced cisplatin-induced proliferation inhibition and apoptosis of C13K cells. In addition, KRT10 siRNA blocked cisplatin-induced proliferation inhibition of C13K/PTEN cells. In conclusion, our data demonstrate that KRT10 is a downstream molecule of PTEN which improves cisplatin-resistance of ovarian cancer and forced KRT10 overexpression may also act as a therapeutic method for overcoming MDR in ovarian cancer

Glutamine deprivation induces interleukin-8 expression in ataxia telangiectasia fibroblasts.

Science.gov (United States)

Kim, Min-Hyun; Kim, Aryung; Yu, Ji Hoon; Lim, Joo Weon; Kim, Hyeyoung

2014-05-01

To investigate whether glutamine deprivation induces expression of inflammatory cytokine interleukin-8 (IL-8) by determining NF-κB activity and levels of oxidative indices (ROS, reactive oxygen species; hydrogen peroxide; GSH, glutathione) in fibroblasts isolated from patients with ataxia telangiectasia (A-T). We used A-T fibroblasts stably transfected with empty vector (Mock) or with human full-length ataxia telangiectasia mutated (ATM) cDNA (YZ5) and mouse embryonic fibroblasts (MEFs) transiently transfected with ATM small interfering RNA (siRNA) or with non-specific control siRNA. The cells were cultured with or without glutamine or GSH. ROS levels were determined using a fluorescence reader and confocal microscopy. IL-8 or murine IL-8 homolog, keratinocyte chemoattractant (KC), and hydrogen peroxide levels in the medium were determined by enzyme-linked immunosorbent assay and colorimetric assay. GSH level was assessed by enzymatic assay, while IL-8 (KC) mRNA level was measured by reverse transcription-polymerase chain reaction (RT-PCR) and/or quantitative real-time PCR. NF-κB DNA-binding activity was determined by electrophoretic mobility shift assay. Catalase activity and ATM protein levels were determined by O2 generation and Western blotting. While glutamine deprivation induced IL-8 expression and increased NF-κB DNA-binding activity in Mock cells, both processes were decreased by treatment of cells with glutamine or GSH or both glutamine and GSH. Glutamine deprivation had no effect on IL-8 expression or NF-κB DNA-binding activity in YZ5 cells. Glutamine-deprived Mock cells had higher oxidative stress indices (increases in ROS and hydrogen peroxide, reduction in GSH) than glutamine-deprived YZ5 cells. In Mock cells, glutamine deprivation-induced oxidative stress indices were suppressed by treatment with glutamine or GSH or both glutamine and GSH. GSH levels and catalase activity were lower in Mock cells than YZ5 cells. MEFs transfected with ATM siRNA and

Effects of gamma rays, ultraviolet radiation, sunlight, microwaves and electromagnetic fields on gene expression mediated by human immunodeficiency virus promoter

International Nuclear Information System (INIS)

Libertin, C.R.; Woloschak, G.E.; Panozzo, J.; Groh, K.R.; Chang-Liu, Chin-Mei; Schreck, S.

1994-01-01

Previous work by our group and others has shown the modulation of human immunodeficiency virus (HIV) promoter or long terminal repeat (LTR) after exposure to neutrons and ultraviolet radiations. Using HeLa cells stably transfected with a construct containing the chloramphenicol acetyl transferase (CAT) gene, the transcription of which is mediated by the HIV-LTR, we designed experiments to examine the effects of exposure to different types of radiation (such as γ rays, ultraviolet and sunlight irradiations, electromagnetic fields and microwaves) in HIV-LTR-driven expression of CAT. These results demonstrated ultraviolet-light-induced transcription from the HIV promoter, as has been shown by others. Exposure to other DNA-damaging agents such as γ rays and sunlight (with limited exposures) had no significant effect on transcription mediated by HIV-LTR, suggesting that induction of HIV is not mediated by just any type of DNA damage but rather may require specific types of DNA damage. Microwaves did not cause cell killing when cells in culture were exposed in high volumes of medium, and the same cells showed no changes in expression. When microwave exposure was carried out in low volumes of medium (so that excessive heat was generated) induction of HIV-LTR transcription (as assayed by CAT activity) was evident. Electromagnetic field exposures had no effect on expression of HIV-LTR. These results demonstrate that not all types of radiation and not all DNA-damaging agents are capable of inducing HIV. We hypothesize that induction of HIV transcription may be mediated by several different signals exposure to radiation. 22 refs., 8 figs

Functional characterisation of human glycine receptors in a fluorescence-based high throughput screening assay

DEFF Research Database (Denmark)

Jensen, Anders A.

2005-01-01

The human glycine receptor subtypes alpha1beta and alpha2 have been expressed stably in HEK293 cells, and the functional characteristics of the receptors have been characterised in the FLIPR Membrane Potential Assay. The pharmacological properties obtained for nine standard ligands at the two rec...

Experiment list: SRX054577 [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available ast adenocarcinoma cell line || cell type=MCF7 cell line stably expressing HA ER E2...of Extraction=Pleura|Tissue Diagnosis=Adenocarcinoma 6681805,90.4,2.4,90 GSM699990: Input MCF7 stables ChIP-seq rep1a source_name=bre

Trichostatin A induced histone acetylation causes decondensation of interphase chromatin.

NARCIS (Netherlands)

T.A. Knoch (Tobias); M. Wachsmuth (Malte); M. Frank-Stöhr (Monika); M. Stöhr (Michael); C.P. Bacher (Christian); K. Rippe (Karsten)

2004-01-01

textabstractThe effect of trichostatin A (TSA)-induced histone acetylation on the interphase chromatin structure was visualized in vivo with a HeLa cell line stably expressing histone H2A, which was fused to enhanced yellow fluorescent protein. The globally increased histone acetylation caused a

Tlx3 exerts context-dependent transcriptional regulation and promotes neuronal differentiation from embryonic stem cells

OpenAIRE

Kondo, Takako; Sheets, Patrick L.; Zopf, David A.; Aloor, Heather L.; Cummins, Theodore R.; Chan, Rebecca J.; Hashino, Eri

2008-01-01

The T cell leukemia 3 (Tlx3) gene has been implicated in specification of glutamatergic sensory neurons in the spinal cord. In cranial sensory ganglia, Tlx3 is highly expressed in differentiating neurons during early embryogenesis. To study a role of Tlx3 during neural differentiation, mouse embryonic stem (ES) cells were transfected with a Tlx3 expression vector. ES cells stably expressing Tlx3 were grown in the presence or absence of a neural induction medium. In undifferentiated ES cells, ...